Edgar Engleman

All Publications

• The acid-sensing receptor GPR65 on tumor macrophages drives tumor growth in obesity. Science immunology Bagchi, S., Yuan, R., Huang, H. L., Zhang, W., Chiu, D. K., Kim, H., Cha, S. L., Tolentino, L., Lowitz, J., Liu, Y., Moshnikova, A., Andreev, O., Plevritis, S., Engleman, E. G. 2024; 9 (100): eadg6453

  Abstract

  Multiple cancers, including colorectal cancer (CRC), are more frequent and often more aggressive in individuals with obesity. Here, we showed that macrophages accumulated within tumors of patients with obesity and CRC and in obese CRC mice and that they promoted accelerated tumor growth. These changes were initiated by oleic acid accumulation and subsequent tumor cell-derived acid production and were driven by macrophage signaling through the acid-sensing receptor GPR65. We found a similar role for GPR65 in hepatocellular carcinoma (HCC) in obese mice. Tumors in patients with obesity and CRC or HCC also exhibited increased GPR65 expression, suggesting that the mechanism revealed here may contribute to tumor growth in a range of obesity-associated cancers and represent a potential therapeutic target.

  View details for DOI 10.1126/sciimmunol.adg6453

  View details for PubMedID 39423285

• Metabolic regulator LKB1 controls adipose tissue ILC2 PD-1 expression and mitochondrial homeostasis to prevent insulin resistance. Immunity Sun, J., Zhang, Y., Zhang, Q., Hu, L., Zhao, L., Wang, H., Yuan, Y., Niu, H., Wang, D., Zhang, H., Liu, J., Feng, X., Su, X., Qiu, J., Sun, J., Xu, H., Zhang, C., Wang, K., Bi, Y., Engleman, E. G., Shen, L. 2024

  Abstract

  Adipose tissue group 2 innate lymphoid cells (ILC2s) help maintain metabolic homeostasis by sustaining type 2 immunity and promoting adipose beiging. Although impairment of the ILC2 compartment contributes to obesity-associated insulin resistance, the underlying mechanisms have not been elucidated. Here, we found that ILC2s in obese mice and humans exhibited impaired liver kinase B1 (LKB1) activation. Genetic ablation of LKB1 disrupted ILC2 mitochondrial metabolism and suppressed ILC2 responses, resulting in exacerbated insulin resistance. Mechanistically, LKB1 deficiency induced aberrant PD-1 expression through activation of NFAT, which in turn enhanced mitophagy by suppressing Bcl-xL expression. Blockade of PD-1 restored the normal functions of ILC2s and reversed obesity-induced insulin resistance in mice. Collectively, these data present the LKB1-PD-1 axis as a promising therapeutic target for the treatment of metabolic disease.

  View details for DOI 10.1016/j.immuni.2024.04.024

  View details for PubMedID 38772366

• Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver. Nature Fan, W., Adebowale, K., Váncza, L., Li, Y., Rabbi, M. F., Kunimoto, K., Chen, D., Mozes, G., Chiu, D. K., Li, Y., Tao, J., Wei, Y., Adeniji, N., Brunsing, R. L., Dhanasekaran, R., Singhi, A., Geller, D., Lo, S. H., Hodgson, L., Engleman, E. G., Charville, G. W., Charu, V., Monga, S. P., Kim, T., Wells, R. G., Chaudhuri, O., Török, N. J. 2024

  Abstract

  Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic β-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-β1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

  View details for DOI 10.1038/s41586-023-06991-9

  View details for PubMedID 38297127

  View details for PubMedCentralID 7733542

• Galectin-1 mediates chronic STING activation in tumors to promote metastasis through MDSC recruitment. Cancer research Nambiar, D. K., Viswanathan, V., Cao, H., Zhang, W., Guan, L., Chamoli, M., Holmes, B., Kong, C., Hildebrand, R., Koong, A. J., von Eyben, R., Plevritis, S., Li, L., Giaccia, A., Engleman, E., Le, Q. T. 2023

  Abstract

  The immune system plays a crucial role in the regulation of metastasis. Tumor cells systemically change immune functions to facilitate metastatic progression. Through this study, we deciphered how tumoral Galectin-1 (Gal1) expression shapes the systemic immune environment to promote metastasis in head and neck cancer (HNC). In multiple preclinical models of HNC and lung cancer in immunogenic mice, Gal1 fostered the establishment of a pre-metastatic niche through polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), which altered the local microenvironment to support metastatic spread. RNA sequencing of MDSCs from pre-metastatic lungs in these models demonstrated the role of PMN-MDSCs in collagen and extracellular matrix remodeling in the pre-metastatic compartment. Gal1 promoted MDSC accumulation in the pre-metastatic niche through the NF-κB signaling axis, triggering enhanced CXCL2-mediated MDSC migration. Mechanistically, Gal1 sustained NF-κB activation in tumor cells by enhancing STING protein stability, leading to prolonged inflammation-driven MDSC expansion. These findings suggest an unexpected pro-tumoral role of STING activation in metastatic progression and establish Gal1 as an endogenous positive regulator of STING in advanced-stage cancers.

  View details for DOI 10.1158/0008-5472.CAN-23-0046

  View details for PubMedID 37409887

• Lymph nodes: at the intersection of cancer treatment and progression. Trends in cell biology Reticker-Flynn, N. E., Engleman, E. G. 2023

  Abstract

  Metastasis to lymph nodes (LNs) is a common feature of disease progression in most solid organ malignancies. Consequently, LN biopsy and lymphadenectomy are common clinical practices, not only because of their diagnostic utility but also as a means of deterring further metastatic spread. LN metastases have the potential to seed additional tissues and can induce metastatic tolerance, a process by which tumor-specific immune tolerance in LNs promotes further disease progression. Nonetheless, phylogenetic studies have revealed that distant metastases are not necessarily derived from nodal metastases. Furthermore, immunotherapy efficacy is increasingly being attributed to initiation of systemic immune responses within LNs. We argue that lymphadenectomy and nodal irradiation should be approached with caution, particularly in patients receiving immunotherapy.

  View details for DOI 10.1016/j.tcb.2023.04.001

  View details for PubMedID 37149414

• Neutrophil-activating therapy for the treatment of cancer. Cancer cell Linde, I. L., Prestwood, T. R., Qiu, J., Pilarowski, G., Linde, M. H., Zhang, X., Shen, L., Reticker-Flynn, N. E., Chiu, D. K., Sheu, L. Y., Van Deursen, S., Tolentino, L. L., Song, W., Engleman, E. G. 2023

  Abstract

  Despite their cytotoxic capacity, neutrophils are often co-opted by cancers to promote immunosuppression, tumor growth, and metastasis. Consequently, these cells have received little attention as potential cancer immunotherapeutic agents. Here, we demonstrate in mouse models that neutrophils can be harnessed to induce eradication of tumors and reduce metastatic seeding through the combined actions of tumor necrosis factor, CD40 agonist, and tumor-binding antibody. The same combination activates human neutrophils invitro, enabling their lysis of human tumor cells. Mechanistically, this therapy induces rapid mobilization and tumor infiltration of neutrophils along with complement activation in tumors. Complement component C5a activates neutrophils to produce leukotriene B4, which stimulates reactive oxygen species production via xanthine oxidase, resulting in oxidative damage and Tcell-independent clearance of multiple tumor types. These data establish neutrophils as potent anti-tumor immune mediators and define an inflammatory pathway that can be harnessed to drive neutrophil-mediated eradication of cancer.

  View details for DOI 10.1016/j.ccell.2023.01.002

  View details for PubMedID 36706760

• Conditional Knockout of Hypoxia-Inducible Factor 1-Alpha in Tumor-Infiltrating Neutrophils Protects against Pancreatic Ductal Adenocarcinoma. International journal of molecular sciences Sieow, J. L., Penny, H. L., Gun, S. Y., Tan, L. Q., Duan, K., Yeong, J. P., Pang, A., Lim, D., Toh, H. C., Lim, T. K., Engleman, E., Rotzschke, O., Ng, L. G., Chen, J., Tan, S. M., Wong, S. C. 2023; 24 (1)

  Abstract

  Large numbers of neutrophils infiltrate tumors and comprise a notable component of the inflammatory tumor microenvironment. While it is established that tumor cells exhibit the Warburg effect for energy production, the contribution of the neutrophil metabolic state to tumorigenesis is unknown. Here, we investigated whether neutrophil infiltration and metabolic status promotes tumor progression in an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC). We observed a large increase in the proportion of neutrophils in the blood and tumor upon orthotopic transplantation. Intriguingly, these tumor-infiltrating neutrophils up-regulated glycolytic factors and hypoxia-inducible factor 1-alpha (HIF-1alpha) expression compared to neutrophils from the bone marrow and blood of the same mouse. This enhanced glycolytic signature was also observed in human PDAC tissue samples. Strikingly, neutrophil-specific deletion of HIF-1alpha (HIF-1alphaDeltaNphi) significantly reduced tumor burden and improved overall survival in orthotopic transplanted mice, by converting the pro-tumorigenic neutrophil phenotype to an anti-tumorigenic phenotype. This outcome was associated with elevated reactive oxygen species production and activated natural killer cells and CD8+ cytotoxic T cells compared to littermate control mice. These data suggest a role for HIF-1alpha in neutrophil metabolism, which could be exploited as a target for metabolic modulation in cancer.

  View details for DOI 10.3390/ijms24010753

  View details for PubMedID 36614196

• Identification of cell types in multiplexed in situ images by combining protein expression and spatial information using CELESTA. Nature methods Zhang, W., Li, I., Reticker-Flynn, N. E., Good, Z., Chang, S., Samusik, N., Saumyaa, S., Li, Y., Zhou, X., Liang, R., Kong, C. S., Le, Q., Gentles, A. J., Sunwoo, J. B., Nolan, G. P., Engleman, E. G., Plevritis, S. K. 2022

  Abstract

  Advances in multiplexed in situ imaging are revealing important insights in spatial biology. However, cell type identification remains a major challenge in imaging analysis, with most existing methods involving substantial manual assessment and subjective decisions for thousands of cells. We developed an unsupervised machine learning algorithm, CELESTA, which identifies the cell type of each cell, individually, using the cell's marker expression profile and, when needed, its spatial information. We demonstrate the performance of CELESTA on multiplexed immunofluorescence images of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Using the cell types identified by CELESTA, we identify tissue architecture associated with lymph node metastasis in HNSCC, and validate our findings in an independent cohort. By coupling our spatial analysis with single-cell RNA-sequencing data on proximal sections of the same specimens, we identify cell-cell crosstalk associated with lymph node metastasis, demonstrating the power of CELESTA to facilitate identification of clinically relevant interactions.

  View details for DOI 10.1038/s41592-022-01498-z

  View details for PubMedID 35654951

• Lymph node colonization induces tumor-immune tolerance to promote distant metastasis. Cell Reticker-Flynn, N. E., Zhang, W., Belk, J. A., Basto, P. A., Escalante, N. K., Pilarowski, G. O., Bejnood, A., Martins, M. M., Kenkel, J. A., Linde, I. L., Bagchi, S., Yuan, R., Chang, S., Spitzer, M. H., Carmi, Y., Cheng, J., Tolentino, L. L., Choi, O., Wu, N., Kong, C. S., Gentles, A. J., Sunwoo, J. B., Satpathy, A. T., Plevritis, S. K., Engleman, E. G. 2022

  Abstract

  For many solid malignancies, lymph node (LN) involvement represents a harbinger of distant metastatic disease and, therefore, an important prognostic factor. Beyond its utility as a biomarker, whether and how LN metastasis plays an active role in shaping distant metastasis remains an open question. Here, we develop a syngeneic melanoma mouse model of LN metastasis to investigate how tumors spread to LNs and whether LN colonization influences metastasis to distant tissues. We show that an epigenetically instilled tumor-intrinsic interferon response program confers enhanced LN metastatic potential by enabling the evasion of NK cells and promoting LN colonization. LN metastases resist T cell-mediated cytotoxicity, induce antigen-specific regulatory T cells, and generate tumor-specific immune tolerance that subsequently facilitates distant tumor colonization. These effects extend to human cancers and other murine cancer models, implicating a conserved systemic mechanism by which malignancies spread to distant organs.

  View details for DOI 10.1016/j.cell.2022.04.019

  View details for PubMedID 35525247

• Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer. Molecular cancer therapeutics Eggold, J. T., Chow, S., Melemenidis, S., Wang, J., Natarajan, S., Loo, P. E., Manjappa, R., Viswanathan, V., Kidd, E. A., Engleman, E., Dorigo, O., Loo, B. W., Rankin, E. B. 2021

  Abstract

  Treatment of advanced ovarian cancer using PD-1/PD-L1 immune checkpoint blockade shows promise, however current clinical trials are limited by modest response rates. Radiation therapy has been shown to synergize with PD-1/PD-L1 blockade in some cancers but has not been utilized in advanced ovarian cancer due to toxicity associated with conventional abdominopelvic irradiation. While ultra-high dose rate (FLASH) irradiation has emerged as a strategy to reduce radiation-induced toxicity, the immunomodulatory properties of FLASH irradiation remain unknown. Here we demonstrate that single high dose abdominopelvic FLASH irradiation promoted intestinal regeneration and maintained tumor control in a preclinical mouse model of ovarian cancer. Reduced tumor burden in conventional and FLASH treated mice was associated with an early decrease in intratumoral regulatory T cells and a late increase in cytolytic CD8+ T cells. Compared to conventional irradiation, FLASH irradiation increased intratumoral T cell infiltration at early timepoints. Moreover, FLASH irradiation maintained the ability to increase intratumoral CD8+ T cell infiltration and enhance the efficacy of alphaPD-1 therapy in preclinical models of ovarian cancer. These data highlight the potential for FLASH irradiation to improve the therapeutic efficacy of checkpoint inhibition in the treatment of ovarian cancer.

  View details for DOI 10.1158/1535-7163.MCT-21-0358

  View details for PubMedID 34866044

• DECTIN-2, A NOVEL TARGET FOR TUMOR MACROPHAGE REPROGRAMMING IN CANCER IMMUNOTHERAPY Kenkel, J., Ho, P., Kongara, S., Henning, K., Kreder, C., Nolin, J., Chapin, S., Kowanetz, M., Alonso, M., Ackerman, S., Engleman, E., Dornan, D. BMJ PUBLISHING GROUP. 2021: A903

  View details for DOI 10.1136/jitc-2021-SITC2021.862

  View details for Web of Science ID 000774877500831

• Brain profiling in murine colitis and human epilepsy reveals neutrophils and TNFalpha as mediators of neuronal hyperexcitability. Journal of neuroinflammation Barnes, S. E., Zera, K. A., Ivison, G. T., Buckwalter, M. S., Engleman, E. G. 2021; 18 (1): 199

  Abstract

  BACKGROUND: Patients with chronic inflammatory disorders such as inflammatory bowel disease frequently experience neurological complications including epilepsy, depression, attention deficit disorders, migraines, and dementia. However, the mechanistic basis for these associations is unknown. Given that many patients are unresponsive to existing medications or experience debilitating side effects, novel therapeutics that target the underlying pathophysiology of these conditions are urgently needed.METHODS: Because intestinal disorders such as inflammatory bowel disease are robustly associated with neurological symptoms, we used three different mouse models of colitis to investigate the impact of peripheral inflammatory disease on the brain. We assessed neuronal hyperexcitability, which is associated with many neurological symptoms, by measuring seizure threshold in healthy and colitic mice. We profiled the neuroinflammatory phenotype of colitic mice and used depletion and neutralization assays to identify the specific mediators responsible for colitis-induced neuronal hyperexcitability. To determine whether our findings in murine models overlapped with a human phenotype, we performed gene expression profiling, pathway analysis, and deconvolution on microarray data from hyperexcitable human brain tissue from patients with epilepsy.RESULTS: We observed that murine colitis induces neuroinflammation characterized by increased pro-inflammatory cytokine production, decreased tight junction protein expression, and infiltration of monocytes and neutrophils into the brain. We also observed sustained neuronal hyperexcitability in colitic mice. Colitis-induced neuronal hyperexcitability was ameliorated by neutrophil depletion or TNFalpha blockade. Gene expression profiling of hyperexcitable brain tissue resected from patients with epilepsy also revealed a remarkably similar pathology to that seen in the brains of colitic mice, including neutrophil infiltration and high TNFalpha expression.CONCLUSIONS: Our results reveal neutrophils and TNFalpha as central regulators of neuronal hyperexcitability of diverse etiology. Thus, there is a strong rationale for evaluating anti-inflammatory agents, including clinically approved TNFalpha inhibitors, for the treatment of neurological and psychiatric symptoms present in, and potentially independent of, a diagnosed inflammatory disorder.

  View details for DOI 10.1186/s12974-021-02262-4

  View details for PubMedID 34511110

• Development of immunosuppressive myeloid cells to induce tolerance in solid organ and hematopoietic cell transplant recipients. Blood advances Jensen, K. P., Hongo, D., Ji, X., Zheng, P., Pawar, R. D., Wu, H., Busque, S., Scandling, J. D., Shizuru, J. A., Lowsky, R., Shori, A., Dutt, S., Waters, J., Saraswathula, A., Baker, J., Tamaresis, J. S., Lavori, P., Negrin, R. S., Maecker, H. T., Engleman, E. G., Meyer, E., Strober, S. 2021

  Abstract

  Replacement of failed organs followed by safe withdrawal of immunosuppressive drugs have long been the goals of organ transplantation. We studied changes in the balance of T and myeloid cells in blood of HLA-matched and -mismatched patients given living donor kidney transplants (KTx) followed by total lymphoid irradiation (TLI), anti-thymocyte globulin (ATG) conditioning, and donor hematopoietic cell transplant (HCT) to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In pre-clinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive, polymorphonuclear (pmn), myeloid derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of the pmn-MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes including profound depletion of T cells and a marked increase in MDSCs in blood post-transplant. Post-transplant pmn-MDSCs transiently increased expression of lectin-type, oxidized LDL receptor-1 (LOX-1), a marker of immunosuppression, and production of the T cell inhibitor, arginase-1. These post-transplant pmn-MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous, TCR microbead-stimulated, pre-transplant T cells when co-cultured in vitro. In conclusion, we elucidated changes in receptors, and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to the that of MDSCs required for tolerance in mice. The clinical trials are registered in Clinicaltrials.gov under NCT #s 00319657 and 01165762.

  View details for DOI 10.1182/bloodadvances.2020003669

  View details for PubMedID 34432869

• Targeting Glycolysis in Macrophages Confers Protection Against Pancreatic Ductal Adenocarcinoma INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Penny, H., Sieow, J., Gun, S., Lau, M., Lee, B., Tan, J., Phua, C., Toh, F., Nga, Y., Yeap, W., Janela, B., Kumar, D., Chen, H., Yeong, J., Kenkel, J. A., Pang, A., Lim, D., Toh, H., Hon, T., Johnson, C., Khameneh, H., Mortellaro, A., Engleman, E. G., Rotzschke, O., Ginhoux, F., Abastado, J., Chen, J., Wong, S. 2021; 22 (12)

  Abstract

  Inflammation in the tumor microenvironment has been shown to promote disease progression in pancreatic ductal adenocarcinoma (PDAC); however, the role of macrophage metabolism in promoting inflammation is unclear. Using an orthotopic mouse model of PDAC, we demonstrate that macrophages from tumor-bearing mice exhibit elevated glycolysis. Macrophage-specific deletion of Glucose Transporter 1 (GLUT1) significantly reduced tumor burden, which was accompanied by increased Natural Killer and CD8+ T cell activity and suppression of the NLRP3-IL1β inflammasome axis. Administration of mice with a GLUT1-specific inhibitor reduced tumor burden, comparable with gemcitabine, the current standard-of-care. In addition, we observe that intra-tumoral macrophages from human PDAC patients exhibit a pronounced glycolytic signature, which reliably predicts poor survival. Our data support a key role for macrophage metabolism in tumor immunity, which could be exploited to improve patient outcomes.

  View details for DOI 10.3390/ijms22126350

  View details for Web of Science ID 000666535100001

  View details for PubMedID 34198548

• Antitumor effects of iPSC-based cancer vaccine in pancreatic cancer. Stem cell reports Ouyang, X., Liu, Y., Zhou, Y., Guo, J., Wei, T., Liu, C., Lee, B., Chen, B., Zhang, A., Casey, K. M., Wang, L., Kooreman, N. G., Habtezion, A., Engleman, E. G., Wu, J. C. 2021

  Abstract

  Induced pluripotent stem cells (iPSCs) and cancer cells share cellular similarities and transcriptomic profiles. Here, we show that an iPSC-based cancer vaccine, comprised of autologous iPSCs and CpG, stimulated cytotoxic antitumor CD8+ Tcell effector and memory responses, induced cancer-specific humoral immune responses, reduced immunosuppressive CD4+ T regulatory cells, and prevented tumor formation in 75% of pancreatic ductal adenocarcinoma (PDAC) mice. We demonstrate that shared gene expression profiles of "iPSC-cancer signature genes" and others are overexpressed in mouse and human iPSC lines, PDAC cells, and multiple human solid tumor types compared with normal tissues. These results support further studies of iPSC vaccination in PDAC in preclinical and clinical models and in other cancer types that have low mutational burdens.

  View details for DOI 10.1016/j.stemcr.2021.04.004

  View details for PubMedID 33961792

• Mechanical Stiffness Controls Dendritic Cell Metabolism and Function. Cell reports Chakraborty, M., Chu, K., Shrestha, A., Revelo, X. S., Zhang, X., Gold, M. J., Khan, S., Lee, M., Huang, C., Akbari, M., Barrow, F., Chan, Y. T., Lei, H., Kotoulas, N. K., Jovel, J., Pastrello, C., Kotlyar, M., Goh, C., Michelakis, E., Clemente-Casares, X., Ohashi, P. S., Engleman, E. G., Winer, S., Jurisica, I., Tsai, S., Winer, D. A. 2021; 34 (2): 108609

  Abstract

  Stiffness in the tissue microenvironment changes in most diseases and immunological conditions, but its direct influence on the immune system is poorly understood. Here, we show that static tension impacts immune cell function, maturation, and metabolism. Bone-marrow-derived and/or splenic dendritic cells (DCs) grown invitro at physiological resting stiffness have reduced proliferation, activation, and cytokine production compared with cells grown under higher stiffness, mimicking fibro-inflammatory disease. Consistently, DCs grown under higher stiffness show increased activation and flux of major glucose metabolic pathways. In DC models of autoimmune diabetes and tumor immunotherapy, tension primes DCs to elicit an adaptive immune response. Mechanistic workup identifies the Hippo-signaling molecule, TAZ, as well as Ca2+-related ion channels, including potentially PIEZO1, as important effectors impacting DC metabolism and function under tension. Tension also directs the phenotypes of monocyte-derived DCs in humans. Thus, mechanical stiffness is a critical environmental cue of DCs and innate immunity.

  View details for DOI 10.1016/j.celrep.2020.108609

  View details for PubMedID 33440149

• Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity NATURE CANCER Ackerman, S. E., Pearson, C. I., Gregorio, J. D., Gonzalez, J. C., Kenkel, J. A., Hartmann, F. J., Luo, A., Ho, P. Y., LeBlanc, H., Blum, L. K., Kimmey, S. C., Luo, A., Nguyen, M. L., Paik, J. C., Sheu, L. Y., Ackerman, B., Lee, A., Li, H., Melrose, J., Laura, R. P., Ramani, V. C., Henning, K. A., Jackson, D. Y., Safina, B. S., Yonehiro, G., Devens, B. H., Carmi, Y., Chapin, S. J., Bendall, S. C., Kowanetz, M., Dornan, D., Engleman, E. G., Alonso, M. N. 2021; 2 (1): 18-+

  View details for DOI 10.1038/s43018-020-00136-x

  View details for Web of Science ID 000610051100003

• Identification of Two Subsets of Murine DC1 Dendritic Cells That Differ by Surface Phenotype, Gene Expression, and Function. Frontiers in immunology Hongo, D., Zheng, P., Dutt, S., Pawar, R. D., Meyer, E., Engleman, E. G., Strober, S. 2021; 12: 746469

  Abstract

  Classical dendritic cells (cDCs) in mice have been divided into 2 major subsets based on the expression of nuclear transcription factors: a CD8+Irf8+Batf3 dependent (DC1) subset, and a CD8-Irf4+ (DC2) subset. We found that the CD8+DC1 subset can be further divided into CD8+DC1a and CD8+DC1b subsets by differences in surface receptors, gene expression, and function. Whereas all 3 DC subsets can act alone to induce potent Th1 cytokine responses to class I and II MHC restricted peptides derived from ovalbumin (OVA) by OT-I and OT-II transgenic T cells, only the DC1b subset could effectively present glycolipid antigens to natural killer T (NKT) cells. Vaccination with OVA protein pulsed DC1b and DC2 cells were more effective in reducing the growth of the B16-OVA melanoma as compared to pulsed DC1a cells in wild type mice. In conclusion, the Batf3-/- dependent DC1 cells can be further divided into two subsets with different immune functional profiles in vitro and in vivo.

  View details for DOI 10.3389/fimmu.2021.746469

  View details for PubMedID 34777358

  View details for PubMedCentralID PMC8589020

• Immune-stimulating antibody conjugates elicit robust myeloid activation and durable antitumor immunity. Nature cancer Ackerman, S. E., Pearson, C. I., Gregorio, J. D., Gonzalez, J. C., Kenkel, J. A., Hartmann, F. J., Luo, A., Ho, P. Y., LeBlanc, H., Blum, L. K., Kimmey, S. C., Luo, A., Nguyen, M. L., Paik, J. C., Sheu, L. Y., Ackerman, B., Lee, A., Li, H., Melrose, J., Laura, R. P., Ramani, V. C., Henning, K. A., Jackson, D. Y., Safina, B. S., Yonehiro, G., Devens, B. H., Carmi, Y., Chapin, S. J., Bendall, S. C., Kowanetz, M., Dornan, D., Engleman, E. G., Alonso, M. N. 2021; 2 (1): 18-33

  Abstract

  Innate pattern recognition receptor agonists, including Toll-like receptors (TLRs), alter the tumor microenvironment and prime adaptive antitumor immunity. However, TLR agonists present toxicities associated with widespread immune activation after systemic administration. To design a TLR-based therapeutic suitable for systemic delivery and capable of safely eliciting tumor-targeted responses, we developed immune-stimulating antibody conjugates (ISACs) comprising a TLR7/8 dual agonist conjugated to tumor-targeting antibodies. Systemically administered human epidermal growth factor receptor 2 (HER2)-targeted ISACs were well tolerated and triggered a localized immune response in the tumor microenvironment that resulted in tumor clearance and immunological memory. Mechanistically, ISACs required tumor antigen recognition, Fcγ-receptor-dependent phagocytosis and TLR-mediated activation to drive tumor killing by myeloid cells and subsequent T-cell-mediated antitumor immunity. ISAC-mediated immunological memory was not limited to the HER2 ISAC target antigen since ISAC-treated mice were protected from rechallenge with the HER2- parental tumor. These results provide a strong rationale for the clinical development of ISACs.

  View details for DOI 10.1038/s43018-020-00136-x

  View details for PubMedID 35121890

• Immune Checkpoint Inhibitors for the Treatment of Cancer: Clinical Impact and Mechanisms of Response and Resistance. Annual review of pathology Bagchi, S., Yuan, R., Engleman, E. G. 2020

  Abstract

  Immune checkpoint inhibitors (ICIs) have made an indelible mark in the field of cancer immunotherapy. Starting with the approval of anti-cytotoxic T lymphocyte-associated protein 4 (anti-CTLA-4) for advanced-stage melanoma in 2011, ICIs-which now also include antibodies against programmed cell death 1 (PD-1) and its ligand (PD-L1)-quickly gained US Food and Drug Administration approval for the treatment of a wide array of cancer types, demonstrating unprecedented extension of patient survival. However, despite the success of ICIs, resistance to these agents restricts the number of patients able to achieve durable responses, and immune-related adverse events complicate treatment. Thus, a better understanding of the requirements for an effective and safe antitumor immune response following ICI therapy is needed. Studies of both tumoral and systemic changes in the immune system following ICI therapy have yielded insight into the basis for both efficacy and resistance. Ultimately, by building on these insights, researchers should be able to combine ICIs with other agents, or design new immunotherapies, to achieve broader and more durable efficacy as well as greater safety. Here, we review the history and clinical utility of ICIs, the mechanisms of resistance to therapy, and local and systemic immune cell changes associated with outcome. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 16 is January 25, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-pathol-042020-042741

  View details for PubMedID 33197221

• COVALENT ATTACHMENT OF A TLR7/8 AGONIST TO TUMOR-TARGETING ANTIBODIES DRIVES POTENT ANTI-TUMOR EFFICACY BY SYNERGISTICALLY ACTIVATING FCGR- AND TLR- SIGNALING AND ENABLES SAFE SYSTEMIC ADMINISTRATION Ackerman, S., Hartmann, F., Pearson, C., Gonzalez, J., Ho, P., Kimmey, S., Luo, A., Ackerman, B., Lee, A., Laura, R., Paik, J., Henning, K., Jackson, D., Chapin, S., Devens, B., Dornan, D., Bendall, S., Engleman, E., Alonso, M. BMJ PUBLISHING GROUP. 2020: A360

  View details for DOI 10.1136/jitc-2020-SITC2020.0603

  View details for Web of Science ID 000616665301125

• A versatile system to record cell-cell interactions. eLife Tang, R., Murray, C. W., Linde, I. L., Kramer, N. J., Lyu, Z., Tsai, M. K., Chen, L. C., Cai, H., Gitler, A. D., Engleman, E., Lee, W., Winslow, M. M. 2020; 9

  Abstract

  Cell-cell interactions influence all aspects of development, homeostasis, and disease. In cancer, interactions between cancer cells and stromal cells play a major role in nearly every step of carcinogenesis. Thus, the ability to record cell-cell interactions would facilitate mechanistic delineation of the role of cancer microenvironment. Here, we describe GFP-based Touching Nexus (G-baToN) which relies upon nanobody-directed fluorescent protein transfer to enable sensitive and specific labeling of cells after cell-cell interactions. G-baToN is a generalizable system that enables physical contact-based labeling between various human and mouse cell types, including endothelial cell-pericyte, neuron-astrocyte, and diverse cancer-stromal cell pairs. A suite of orthogonal baToN tools enables reciprocal cell-cell labeling, interaction-dependent cargo transfer, and the identification of higher-order cell-cell interactions across a wide range of cell types. The ability to track physically interacting cells with these simple and sensitive systems will greatly accelerate our understanding of the outputs of cell-cell interactions in cancer as well as across many biological processes.

  View details for DOI 10.7554/eLife.61080

  View details for PubMedID 33025906

• Human Regulatory Dendritic Cells Develop From Monocytes in Response to Signals From Regulatory and Helper T Cells. Frontiers in immunology Zhang, X., Zheng, P., Prestwood, T. R., Zhang, H., Carmi, Y., Tolentino, L. L., Wu, N., Choi, O., Winer, D. A., Strober, S., Kang, E. S., Alonso, M. N., Engleman, E. G. 2020; 11: 1982

  Abstract

  Dendritic cells (DCs) are powerful antigen presenting cells, derived from bone marrow progenitors (cDCs) and monocytes (moDCs), that can shape the immune response by priming either proinflammatory or tolerogenic immune effector cells. The cellular mechanisms responsible for the generation of DCs that will prime a proinflammatory or tolerogenic response are poorly understood. Here we describe a novel mechanism by which tolerogenic DCs are formed from monocytes. When human monocytes were cultured with CD4+FoxP3+ natural regulatory T cells (Tregs) and T helper cells (Th) from healthy donor blood, they differentiated into regulatory DCs (DC Reg ), capable of generating induced Tregs from naïve T cells. DC Reg exhibited morphology, surface phenotype, cytokine secretion, and transcriptome that were distinct from other moDCs including those derived from monocytes cultured with Th or with GM-CSF/IL-4, as well as macrophages (MΦ). Direct cell contact between monocytes, Tregs and Th, along with Treg-derived CTLA-4, IL-10 and TGF-β, was required for the phenotypic differentiation of DC Reg , although only IL-10 was required for imprinting the Treg-inducing capacity of DC Reg . High ratios of Treg:Th, along with monocytes and DC Reg similar in function and phenotype to those induced in vitro, were present in situ in human colorectal cancer specimens. Thus, through the combined actions of Tregs and Th, monocytes differentiate into DCs with regulatory properties, forming a positive feedback loop to reinforce Treg initiated immune regulation. This mechanism may contribute to immune tolerance in tissues such as tumors, which contain an abundance of Tregs, Th and monocytes.

  View details for DOI 10.3389/fimmu.2020.01982

  View details for PubMedID 32973804

  View details for PubMedCentralID PMC7461788

• ImmunoGlobe: enabling systems immunology with a manually curated intercellular immune interaction network. BMC bioinformatics Atallah, M. B., Tandon, V., Hiam, K. J., Boyce, H., Hori, M., Atallah, W., Spitzer, M. H., Engleman, E., Mallick, P. 2020; 21 (1): 346

  Abstract

  BACKGROUND: While technological advances have made it possible to profile the immune system at high resolution, translating high-throughput data into knowledge of immune mechanisms has been challenged by the complexity of the interactions underlying immune processes. Tools to explore the immune network are critical for better understanding the multi-layered processes that underlie immune function and dysfunction, but require a standardized network map of immune interactions. To facilitate this we have developed ImmunoGlobe, a manually curated intercellular immune interaction network extracted from Janeway's Immunobiology textbook.RESULTS: ImmunoGlobe is the first graphical representation of the immune interactome, and is comprised of 253 immune system components and 1112 unique immune interactions with detailed functional and characteristic annotations. Analysis of this network shows that it recapitulates known features of the human immune system and can be used uncover novel multi-step immune pathways, examine species-specific differences in immune processes, and predict the response of immune cells to stimuli. ImmunoGlobe is publicly available through a user-friendly interface at www.immunoglobe.org and can be downloaded as a computable graph and network table.CONCLUSION: While the fields of proteomics and genomics have long benefited from network analysis tools, no such tool yet exists for immunology. ImmunoGlobe provides a ground truth immune interaction network upon which such tools can be built. These tools will allow us to predict the outcome of complex immune interactions, providing mechanistic insight that allows us to precisely modulate immune responses in health and disease.

  View details for DOI 10.1186/s12859-020-03702-3

  View details for PubMedID 32778050

• Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression Reticker-Flynn, N. E., Basto, P. A., Zhang, W., Martins, M. M., Chang, S., Gentles, A. J., Sunwoo, J. B., Plevritis, S. K., Engleman, E. G. AMER ASSOC CANCER RESEARCH. 2020

  View details for DOI 10.1158/1538-7445.AM2020-3419

  View details for Web of Science ID 000590059301099

• Cancer systems immunology. eLife Reticker-Flynn, N. E., Engleman, E. G. 2020; 9

  Abstract

  Tumor immunology is undergoing a renaissance due to the recent profound clinical successes of tumor immunotherapy. These advances have coincided with an exponential growth in the development of -omics technologies. Armed with these technologies and their associated computational and modeling toolsets, systems biologists have turned their attention to tumor immunology in an effort to understand the precise nature and consequences of interactions between tumors and the immune system. Such interactions are inherently multivariate, spanning multiple time and size scales, cell types, and organ systems, rendering systems biology approaches particularly amenable to their interrogation. While in its infancy, the field of 'Cancer Systems Immunology' has already influenced our understanding of tumor immunology and immunotherapy. As the field matures, studies will move beyond descriptive characterizations toward functional investigations of the emergent behavior that govern tumor-immune responses. Thus, Cancer Systems Immunology holds incredible promise to advance our ability to fight this disease.

  View details for DOI 10.7554/eLife.53839

  View details for PubMedID 32657757

• Lymph node colonization promotes distant tumor metastasis through the induction of tumor-specific immunosuppression. Reticker-Flynn, N. E., Basto, P. A., Zhang, W., Bejnood, A., Kenkel, J. A., Martins, M. M., Chang, S., Gentles, A. J., Sunwoo, J. B., Plevritis, S. K., Engleman, E. G. AMER ASSOC CANCER RESEARCH. 2020: 25–26

  View details for Web of Science ID 000537844900026

• Mixed chimerism and acceptance of kidney transplants after immunosuppressive drug withdrawal. Science translational medicine Busque, S. n., Scandling, J. D., Lowsky, R. n., Shizuru, J. n., Jensen, K. n., Waters, J. n., Wu, H. H., Sheehan, K. n., Shori, A. n., Choi, O. n., Pham, T. n., Fernandez Vina, M. A., Hoppe, R. n., Tamaresis, J. n., Lavori, P. n., Engleman, E. G., Meyer, E. n., Strober, S. n. 2020; 12 (528)

  Abstract

  Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)-matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype-matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

  View details for DOI 10.1126/scitranslmed.aax8863

  View details for PubMedID 31996467

• Enteric Glia Play a Critical Role in Promoting the Development of Colorectal Cancer Frontiers in Oncology Yuan, R., Bhattacharya, N., Kenkel, J. A., Shen, J., DiMaio, M. A., Bagchi, S., Prestwood, T. R., Habtezion, A., Engleman, E. G. 2020; 10: 595892

  Abstract

  Enteric glia are a distinct population of peripheral glial cells in the enteric nervous system that regulate intestinal homeostasis, epithelial barrier integrity, and gut defense. Given these unique attributes, we investigated the impact of enteric glia depletion on tumor development in azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice, a classical model of colorectal cancer (CRC). Depleting GFAP+ enteric glia resulted in a profoundly reduced tumor burden in AOM/DSS mice and additionally reduced adenomas in the ApcMin/+ mouse model of familial adenomatous polyposis, suggesting a tumor-promoting role for these cells at an early premalignant stage. This was confirmed in further studies of AOM/DSS mice, as enteric glia depletion did not affect the properties of established malignant tumors but did result in a marked reduction in the development of precancerous dysplastic lesions. Surprisingly, the protective effect of enteric glia depletion was not dependent on modulation of anti-tumor immunity or intestinal inflammation. These findings reveal that GFAP+ enteric glia play a critical pro-tumorigenic role during early CRC development and identify these cells as a potential target for CRC prevention.

  View details for DOI 10.3389/fonc.2020.595892

  View details for PubMedCentralID PMC7691584

• Human regulatory dendritic cells develop from monocytes in response to signals from regulatory and helper T cells Frontiers In Immunology Zhang, X., Zheng, P., Prestwood, T., Zhang, H., Cami, Y., Tolentino, L., Wu, N., Choi, O., Winer, D., Strober, S., Kang, E., Alonso, M., Engleman, E. 2020

  View details for DOI 10.3389/fimmu.2020.01982

• Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection. Matrix biology : journal of the International Society for Matrix Biology Marshall, P. L., Nagy, N. n., Kaber, G. n., Barlow, G. L., Ramesh, A. n., Xie, B. J., Linde, M. H., Haddock, N. L., Lester, C. A., Tran, Q. L., de Vries, C. n., Hargil, A. n., Malkovskiy, A. n., Gurevich, I. n., Martinez, H. A., Kuipers, H. F., Yadava, K. n., Zhang, X. n., Evanko, S. P., Gebe, J. A., Wang, X. n., Vernon, R. B., de la Motte, C. n., Wight, T. N., Engleman, E. G., Krams, S. M., Meyer, E. n., Bollyky, P. L. 2020

  Abstract

  A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

  View details for DOI 10.1016/j.matbio.2020.12.001

  View details for PubMedID 33290836

• HER2-targeting TLR7/8 immune-stimulating antibody conjugates elicit robust myeloid activation and anti-tumor immune responses in a TLR- and FcR- dependent manner Gonzalez, J., Pearson, C., Ackerman, S., Kenkel, J., Ho, P., Luo, A., Nguyen, M., Paik, J., Lee, A., Laura, R., Li, H., Kreder, C., Henning, K., Chapin, S., Devens, B., Safina, B., Jackson, D., Engleman, E., Dornan, D., Alonso, M. BMC. 2019

  View details for Web of Science ID 000496473200252

• Tumor-binding antibodies induce potent dendritic cell-mediated tumor immunity ONCOIMMUNOLOGY Carmi, Y., Prestwood, T., Engleman, E. G. 2019; 8 (10)

  View details for DOI 10.1080/2162402X.2015.1078063

  View details for Web of Science ID 000482222900015

• A Novel Type of Blood Biomarker: Distinct Changes of Cytokine-Induced STAT Phosphorylation in Blood T Cells Between Colorectal Cancer Patients and Healthy Individuals. Cancers Yun, J. W., Lee, S., Kim, H. M., Chun, S., Engleman, E. G., Kim, H. C., Kang, E. 2019; 11 (8)

  Abstract

  Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Although early diagnosis and treatment is the most successful strategy for improving patient survival, feasible and sensitive blood biomarkers for CRC screening remain elusive. Methods: Sixty-five CRC patients and thirty-three healthy individuals were enrolled. Peripheral blood (PB) and tumor tissues from CRC patients, and PB from healthy individuals were subjected to immunophenotyping and phospho-flow analysis of cytokine-induced phosphorylated STAT (CIPS). Logistic regression was used as a classifier that separates CRC patients from healthy individuals. Results: The proportion of regulatory T cells was increased in PB from CRC patients compared to PB from healthy individuals (p < 0.05). Interestingly, peripheral T cells share several cytokine-induced phosphorylated STAT (CIPS) signatures with T cells from CRC tumor-sites. Additionally, a classifier was made using two signatures distinct between T cells from CRC patients and T cells from healthy individuals. The AUCs (area under curves) of the classifier were 0.88 in initial cohort and 0.94 in the additional validation cohort. Overall AUC was 0.94 with sensitivity of 91% and specificity of 88%. Conclusion: This study highlights that immune cell signatures in peripheral blood could offer a new type of biomarker for CRC screening.

  View details for DOI 10.3390/cancers11081157

  View details for PubMedID 31409016

• Lymph node colonization promotes distant tumor metastasis through the induction of systemic immune tolerance Reticker-Flynn, N. E., Martins, M. M., Basto, P. A., Zhang, W., Bejnood, A., Gentles, A. J., Sunwoo, J. B., Plevritis, S. K., Engleman, E. G. AMER ASSOC CANCER RESEARCH. 2019

  View details for DOI 10.1158/1538-7445.AM2019-2703

  View details for Web of Science ID 000488279401153

• Tumor-binding antibodies induce potent dendritic cell-mediated tumor immunity. Oncoimmunology Carmi, Y., Prestwood, T., Engleman, E. G. 2019; 8 (10): e1078063

  Abstract

  The factors that determine the impact of antitumor antibodies on tumor progression are poorly defined. We found that the tumor microenvironment holds the key. In the absence of dendritic cell (DC) stimulation, such antibodies provide little benefit, but in a stimulatory context they can initiate potent antitumor immunity.

  View details for DOI 10.1080/2162402X.2015.1078063

  View details for PubMedID 31646066

  View details for PubMedCentralID PMC6791431

• Structure of the IFN gamma receptor complex guides design of biased agonists NATURE Mendoza, J. L., Escalante, N. K., Jude, K. M., Bellon, J., Su, L., Horton, T. M., Tsutsumi, N., Berardinelli, S. J., Haltiwanger, R. S., Piehler, J., Engleman, E. G., Garcia, K. 2019; 567 (7746): 56-+

  View details for DOI 10.1038/s41586-019-0988-7

  View details for Web of Science ID 000460426900040

• Structure of the IFNgamma receptor complex guides design of biased agonists. Nature Mendoza, J. L., Escalante, N. K., Jude, K. M., Sotolongo Bellon, J., Su, L., Horton, T. M., Tsutsumi, N., Berardinelli, S. J., Haltiwanger, R. S., Piehler, J., Engleman, E. G., Garcia, K. C. 2019

  Abstract

  The cytokine interferon-gamma (IFNgamma) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNgamma pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNgamma receptor IFNgammaR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNgamma-IFNgammaR1-IFNgammaR2 signalling complex at 3.25A resolution. The structure reveals the mechanism underlying deficits in IFNgamma responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNgammaR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNgamma variants to tune IFNgamma receptor signalling output. Unexpectedly, we found that several partial IFNgamma agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNgamma for therapeutic applications.

  View details for PubMedID 30814731

• A gut punch fights cancer and infection NATURE Reticker-Flynn, N. E., Engleman, E. G. 2019; 565 (7741): 573–74

  View details for Web of Science ID 000457404000031

  View details for PubMedID 30683936

• Myeloid sphingosine-1-phosphate receptor 1 is important for CNS autoimmunity and neuroinflammation. Journal of autoimmunity Tsai, H. C., Nguyen, K. n., Hashemi, E. n., Engleman, E. n., Hla, T. n., Han, M. H. 2019

  Abstract

  The critical role of sphingosine-1-phosphate (S1P) signaling in lymphocyte trafficking is well recognized, however, the contribution of myeloid cell-S1P signaling in neuroimmunity is less well understood. We previously reported that C57BL/6J mice harboring phosphorylation defective S1P receptor 1 (S1P1) (with mutated serines in the carboxyl terminus, leading to impaired receptor internalization) [S1P1(S5A)] developed severe, TH17-dominant experimental autoimmune encephalomyelitis. In this study, we demonstrate that S1P1-mediated TH17 polarization is not an intrinsic T cell effect, but dependent on sustained S1P1 signaling in myeloid cells. First, utilizing the S1P1(S5A) mice in the EAE model, we observed that S1P1 activated and enhanced antigen presentation function in myeloid cells. Second, sequential phosphorylation of STAT3 occurred in dendritic cells, monocytes, and macrophages/microglia during neuroinflammation. Third, we show that pro-inflammatory (CD45hiCD11b+Ly6Chi) monocytes contribute to TH17 differentiation and neuroinflammation by regulating IL-6 expression. Finally, results from experiments utilizing myeloid cell-specific S1P1 overexpression (S1pr1f/stop/f:LysMCre) mice demonstrate that myeloid cell S1P1 directly contributes to severity of neuroinflammation. These findings reveal the critical contribution of myeloid-S1P1 signaling in CNS autoimmunity.

  View details for DOI 10.1016/j.jaut.2019.06.001

  View details for PubMedID 31202617

• Insulin Receptor-Mediated Stimulation Boosts T Cell Immunity during Inflammation and Infection CELL METABOLISM Tsai, S., Clemente-Casares, X., Zhou, A. C., Lei, H., Ahn, J. J., Chan, Y., Choi, O., Luck, H., Woo, M., Dunn, S. E., Engleman, E. G., Watts, T. H., Winer, S., Winer, D. A. 2018; 28 (6): 922-+

  Abstract

  T cells represent a critical effector of cell-mediated immunity. Activated T cells engage in metabolic reprogramming during effector differentiation to accommodate dynamic changes in energy demands. Here, we show that the hormone, insulin, and downstream signaling through its insulin receptor shape adaptive immune function through modulating T cell metabolism. T cells lacking insulin receptor expression (LckCre+ Insrfl/fl) show reduced antigen-specific proliferation and compromised production of pro-inflammatory cytokines. In vivo, T cell-specific insulin receptor deficiency reduces T cell-driven colonic inflammation. In a model of severe influenza infection with A/PR8 (H1N1), lack of insulin receptor on T cells curtails antigen-specific immunity to influenza viral antigens. Mechanistically, insulin receptor signaling reinforces a metabolic program that supports T cell nutrient uptake and associated glycolytic and respiratory capacities. These data highlight insulin receptor signaling as an important node integrating immunometabolic pathways to drive optimal T cell effector function in health and disease.

  View details for PubMedID 30174303

• Leveraging heterogeneity across multiple datasets increases cell-mixture deconvolution accuracy and reduces biological and technical biases. Nature communications Vallania, F., Tam, A., Lofgren, S., Schaffert, S., Azad, T. D., Bongen, E., Haynes, W., Alsup, M., Alonso, M., Davis, M., Engleman, E., Khatri, P. 2018; 9 (1): 4735

  Abstract

  In silico quantification of cell proportions from mixed-cell transcriptomics data (deconvolution) requires a reference expression matrix, called basis matrix. We hypothesize that matrices created using only healthy samples from a single microarray platform would introduce biological and technical biases in deconvolution. We show presence of such biases in two existing matrices, IRIS and LM22, irrespective of deconvolution method. Here, we present immunoStates, a basis matrix built using 6160 samples with different disease states across 42 microarray platforms. We find that immunoStates significantly reduces biological and technical biases. Importantly, we find that different methods have virtually no or minimal effect once the basis matrix is chosen. We further show that cellular proportion estimates using immunoStates are consistently more correlated with measured proportions than IRIS and LM22, across all methods. Our results demonstrate the need and importance of incorporating biological and technical heterogeneity in a basis matrix for achieving consistently high accuracy.

  View details for PubMedID 30413720

• Use of Flow Cytometry for Diagnosis of Epilepsy Associated With Homozygous PIGW Variants. Pediatric neurology Foskett, G. K., Engleman, E., Klotz, J., Choi, O., Tolentino, L., Kochhar, A., Yang, Q. Z., Stevenson, D. A. 2018

  Abstract

  BACKGROUND: Biallelic variants in PIGW have been suggested to cause infantile spasms and hyperphosphatasia. PIGW encodes for a protein involved in the third step of glycosylphosphatidylinositol (GPI) synthesis. GPI anchored proteins are increasingly recognized as important structures for cellular interactions and neuronal development.METHODS: Molecular testing of PIGW was performed followed by fluorescence activating cell sorting analysis of granulocytes, lymphocytes, and monocytes, and compared to controls.FINDINGS: An infant was homozygous for variants in PIGW (c.199C>G; p.Pro67Ala) with an associated phenotype of infantile spasms, myoclonic seizures, cortical visual impairment, developmental delay, and minor dysmorphic features. Alkaline phosphatase levels ranged from normal to mildly elevated. Flow cytometric studies showed significantly decreased expression of important GPIs, providing functional evidence of pathogenicity.CONCLUSION: Our data provide further evidence of a novel autosomal recessive PIGW-related epilepsy disorder. Flow cytometry provided functional evidence of the pathogenicity of homozygous variants of uncertain significance in PIGW, and supports the use of flow cytometry as a functional tool to demonstrate decreased surface expression of GPI anchored proteins in cases where there are variants of unknown significance.

  View details for PubMedID 30078644

• The Interface of Pancreatic Cancer With Diabetes, Obesity, and Inflammation: Research Gaps and Opportunities: Summary of a National Institute of Diabetes and Digestive and Kidney Diseases Workshop PANCREAS Abbruzzese, J. L., Andersen, D. K., Borrebaeck, C. K., Chari, S. T., Costello, E., Cruz-Monserrate, Z., Eibl, G., Engleman, E. G., Fisher, W. E., Habtezion, A., Kim, S. K., Korc, M., Logsdon, C., Lyssiotis, C. A., Pandol, S. J., Rustgi, A., Wolfe, B. M., Zheng, L., Powers, A. C. 2018; 47 (5): 516–25

  Abstract

  A workshop on "The Interface of Pancreatic Cancer with Diabetes, Obesity, and Inflammation: Research Gaps and Opportunities" was held by the National Institute of Diabetes and Digestive and Kidney Diseases on October 12, 2017. The purpose of the workshop was to explore the relationship and possible mechanisms of the increased risk of pancreatic ductal adenocarcinoma (PDAC) related to diabetes, the role of altered intracellular energy metabolism in PDAC, the mechanisms and biomarkers of diabetes caused by PDAC, the mechanisms of the increased risk of PDAC associated with obesity, and the role of inflammatory events and mediators as contributing causes of the development of PDAC. Workshop faculty reviewed the state of the current knowledge in these areas and made recommendations for future research efforts. Further knowledge is needed to elucidate the basic mechanisms contributing to the role of hyperinsulinemia, hyperglycemia, adipokines, and acute and chronic inflammatory events on the development of PDAC.

  View details for DOI 10.1097/MPA.0000000000001037

  View details for Web of Science ID 000431180300005

  View details for PubMedID 29702529

• Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Santana-Magal, N., Rasoulouniriana, D., Saperia, C., Gutwillig, A., Rider, P., Engleman, E. G., Carmi, Y. 2018

  Abstract

  Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

  View details for PubMedID 29912184

• Macrochimerism and clinical transplant tolerance. Human immunology Scandling, J. D., Busque, S., Lowsky, R., Shizuru, J., Shori, A., Engleman, E., Jensen, K., Strober, S. 2018

  Abstract

  Current theory holds that macrochimerism is essential to the development of transplant tolerance. Hematopoietic cell transplantation from the solid organ donor is necessary to achieve macrochimerism. Over the last 10-20 years, trials of tolerance induction with combined kidney and hematopoietic cell transplantation have moved from the preclinical to the clinical arena. The achievement of macrochimerism in the clinical setting is challenging, and potentially toxic due to the conditioning regimen necessary to hematopoietic cell transplantation and due to the risk of graft-versus-host disease. There are differences in chimerism goals and methods of the three major clinical stage tolerance induction strategies in both HLA-matched and HLA-mismatched living donor kidney transplantation, with consequent differences in efficacy and safety. The Stanford protocol has proven efficacious in the induction of tolerance in HLA-matched kidney transplantation, allowing cessation of immunosuppressive drug therapy in 80% of study participants, with the safety profile of conventional transplantation. In HLA-mismatched transplantation, multi-lineage macrochimerism of over a year's duration can now be consistently achieved with the Stanford protocol, with complete withdrawal of immunosuppressive drug therapy during the second post-transplant year as the next experimental step and test of tolerance.

  View details for PubMedID 29330112

• In vitro and in vivo metabolite identification of a novel benzimidazole compound ZLN005 by LC-MS/MS. Rapid communications in mass spectrometry : RCM Sun, W. n., Nguyen, K. D., Fitch, W. L., Banister, S. D., Tang, H. n., Zhang, X. n., Yu, L. n., Engleman, E. G., Rajadas, J. n. 2018

  Abstract

  A novel benzimidazole compound ZLN005 was previously identified as a transcriptional activator of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in certain metabolic tissues. Upregulation of PGC-1α by ZLN005 has been shown to have beneficial effect in a diabetic mouse model and in a coronary artery disease model in vitro. ZLN005 could also have therapeutic potential in neurodegenerative diseases involving down-regulation of PGC-1α. Given the phenotypic efficacy of ZLN005 in several animal models of human disease, its metabolic profile was investigated to guide the development of novel therapeutics using ZLN005 as the lead compound.ZLN005 was incubated with both rat and human liver microsomes and S9 fractions to identify in vitro metabolites. Urine from rats dosed with ZLN005 was used to identify in vivo metabolites. Extracted metabolites were analyzed by LC-MS/MS using a hybrid linear ion trap triple quadrupole mass spectrometer under full scan, enhanced product ion scan, neutral loss scan and precursor scan modes. Metabolites in plasma and brain of ZLN005-treated rats were also profiled using multiple reaction monitoring.Identified in vitro transformations of ZLN005 include mono- and dihydroxylation, further oxidation to carboxylic acids, and mono-O-glucuronide and sulfate conjugation to hydroxy ZLN005 as well as glutathione conjugation. Identified in vivo metabolites are mainly glucuronide and sulfate conjugates of dihydroxyl, carboxyl, and hydroxy acid of the parent compound. The parent compound as well as several major phase I metabolites were found in rat plasma and brain.Using both in vitro and in vivo methods, we elucidated the metabolic pathway of ZLN005. Phase I metabolites with hydroxylation and carboxylation, as well as phase II metabolites with glucuronide, sulfate and glutathione conjugation, were identified.

  View details for PubMedID 29334584

• Accelerated, but not conventional, radiotherapy of murine B-cell lymphoma induces potent T cell-mediated remissions. Blood advances Dutt, S. n., Atallah, M. B., Minamida, Y. n., Filatenkov, A. n., Jensen, K. P., Iliopoulou, B. P., Tamosiuniene, R. n., Waters, J. n., Engleman, E. G., Strober, S. n. 2018; 2 (19): 2568–80

  Abstract

  Conventional local tumor irradiation (LTI), delivered in small daily doses over several weeks, is used clinically as a palliative, rather than curative, treatment for chemotherapy-resistant diffuse large B-cell lymphoma (DLBCL) for patients who are ineligible for hematopoietic cell transplantation. Our goal was to test the hypothesis that accelerated, but not conventional, LTI would be more curative by inducing T cell-mediated durable remissions. We irradiated subcutaneous A20 and BL3750 lymphoma tumors in mice with a clinically relevant total radiation dose of 30 Gy LTI, delivered in 10 doses of 3 Gy over 4 days (accelerated irradiation) or as 10 doses of 3 Gy over 12 days (conventional irradiation). Compared with conventional LTI, accelerated LTI resulted in more complete and durable tumor remissions. The majority of these mice were resistant to rechallenge with lymphoma cells, demonstrating the induction of memory antitumor immunity. The increased efficacy of accelerated LTI correlated with higher levels of tumor cell necrosis vs apoptosis and expression of "immunogenic cell death" markers, including calreticulin, heat shock protein 70 (Hsp70), and Hsp90. Accelerated LTI-induced remissions were not seen in immunodeficient Rag-2-/- mice, CD8+ T-cell-depleted mice, or Batf-3-/- mice lacking CD8α+ and CD103+ dendritic cells. Accelerated, but not conventional, LTI in immunocompetent hosts induced marked increases in tumor-infiltrating CD4+ and CD8+ T cells and MHCII+CD103+CD11c+ dendritic cells and corresponding reductions in exhausted PD-1+Eomes+CD8+ T cells and CD4+CD25+FOXP3+ regulatory T cells. These findings raise the possibility that accelerated LTI can provide effective immune control of human DLBCL.

  View details for PubMedID 30301812

• N-Carboxyanhydride Polymerization of Glycopolypeptides That Activate Antigen-Presenting Cells through Dectin-1 and Dectin-2. Angewandte Chemie (International ed. in English) Zhou, M. N., Delaveris, C. S., Kramer, J. R., Kenkel, J. A., Engleman, E. G., Bertozzi, C. R. 2018; 57 (12): 3137–42

  Abstract

  The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.

  View details for PubMedID 29370452

  View details for PubMedCentralID PMC5842139

• An Immunosuppressive Dendritic Cell Subset Accumulates at Secondary Sites and Promotes Metastasis in Pancreatic Cancer. Cancer research Kenkel, J. A., Tseng, W. W., Davidson, M. G., Tolentino, L. L., Choi, O., Bhattacharya, N., Seeley, E. S., Winer, D. A., Reticker-Flynn, N. E., Engleman, E. G. 2017; 77 (15): 4158-4170

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) after complete surgical resection is often followed by distant metastatic relapse for reasons that remain unclear. In this study, we investigated how the immune response at secondary sites affects tumor spread in murine models of metastatic PDAC. Early metastases were associated with dense networks of CD11b+CD11c+MHC-II+CD24+CD64lowF4/80lowdendritic cells (DC), which developed from monocytes in response to tumor-released GM-CSF. These cells uniquely expressed MGL2 and PD-L2 in the metastatic microenvironment and preferentially induced the expansion of T regulatory cells (Treg)in vitroandin vivoTargeted depletion of this DC population inMgl2DTRhosts activated cytotoxic lymphocytes, reduced Tregs, and inhibited metastasis development. Moreover, blocking PD-L2 selectively activated CD8 T cells at secondary sites and suppressed metastasis, suggesting that the DCs use this particular pathway to inhibit CD8 T-cell-mediated tumor immunity. Phenotypically similar DCs accumulated at primary and secondary sites in other models and in human PDAC. These studies suggest that a discrete DC subset both expands Tregs and suppresses CD8 T cells to establish an immunosuppressive microenvironment conducive to metastasis formation. Therapeutic strategies to block the accumulation and immunosuppressive activity of such cells may help prevent PDAC progression and metastatic relapse after surgical resection.Cancer Res; 77(15); 4158-70. ©2017 AACR.

  View details for DOI 10.1158/0008-5472.CAN-16-2212

  View details for PubMedID 28611041

  View details for PubMedCentralID PMC5550516

• Type I Interferon Responses Drive Intrahepatic T cells to Promote Metabolic Syndrome. Science immunology Ghazarian, M., Revelo, X. S., Nøhr, M. K., Luck, H., Zeng, K., Lei, H., Tsai, S., Schroer, S. A., Park, Y. J., Chng, M. H., Shen, L., D'Angelo, J. A., Horton, P., Chapman, W. C., Brockmeier, D., Woo, M., Engleman, E. G., Adeyi, O., Hirano, N., Jin, T., Gehring, A. J., Winer, S., Winer, D. A. 2017; 2 (10)

  Abstract

  Obesity-related insulin resistance is driven by low-grade chronic inflammation of metabolic tissues. In the liver, non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance and systemic glucose dysregulation. However, the immunological factors supporting these processes are poorly understood. We found that the liver accumulates pathogenic CD8+ T cell subsets which control hepatic insulin sensitivity and gluconeogenesis during diet-induced obesity in mice. In a cohort of human patients, CD8+ T cells represent a dominant intrahepatic immune cell population which links to glucose dysregulation. Accumulation and activation of these cells are largely supported by type I interferon (IFN-I) responses in the liver. Livers from obese mice upregulate critical interferon regulatory factors (IRFs), interferon stimulatory genes (ISGs), and IFNα protein, while IFNαR1(-/-) mice, or CD8-specific IFNαR1(-/-) chimeric mice are protected from disease. IFNαR1 inhibitors improve metabolic parameters in mice, while CD8+ T cells and IFN-I responses correlate with NAFLD activity in human patients. Thus, IFN-I responses represent a central immunological axis that governs intrahepatic T cell pathogenicity during metabolic disease.

  View details for DOI 10.1126/sciimmunol.aai7616

  View details for PubMedID 28567448

• Tolerogenic interactions between CD8(+) dendritic cells and NKT cells prevent rejection of bone marrow and organ grafts BLOOD Hongo, D., Tang, X., Zhang, X., Engleman, E. G., Strober, S. 2017; 129 (12): 1718-1728

  Abstract

  The combination of total lymphoid irradiation and anti-T-cell antibodies safely induces immune tolerance to combined hematopoietic cell and organ allografts in humans. Our mouse model required host natural killer T (NKT) cells to induce tolerance. Because NKT cells normally depend on signals from CD8(+) dendritic cells (DCs) for their activation, we used the mouse model to test the hypothesis that, after lymphoid irradiation, host CD8(+) DCs play a requisite role in tolerance induction through interactions with NKT cells. Selective deficiency of either CD8(+) DCs or NKT cells abrogated chimerism and organ graft acceptance. After radiation, the CD8(+) DCs increased expression of surface molecules required for NKT and apoptotic cell interactions and developed suppressive immune functions, including production of indoleamine 2,3-deoxygenase. Injection of naive mice with apoptotic spleen cells generated by irradiation led to DC changes similar to those induced by lymphoid radiation, suggesting that apoptotic body ingestion by CD8(+) DCs initiates tolerance induction. Tolerogenic CD8(+) DCs induced the development of tolerogenic NKT cells with a marked T helper 2 cell bias that, in turn, regulated the differentiation of the DCs and suppressed rejection of the transplants. Thus, reciprocal interactions between CD8(+) DCs and invariant NKT cells are required for tolerance induction in this system that was translated into a successful clinical protocol.

  View details for DOI 10.1182/blood-2016-07-723015

  View details for Web of Science ID 000397297300023

  View details for PubMedCentralID PMC5364338

• A distinct subset of plasmacytoid dendritic cells induces activation and differentiation of B and T lymphocytes. Proceedings of the National Academy of Sciences of the United States of America Zhang, H., Gregorio, J. D., Iwahori, T., Zhang, X., Choi, O., Tolentino, L. L., Prestwood, T., Carmi, Y., Engleman, E. G. 2017; 114 (8): 1988-1993

  Abstract

  Plasmacytoid dendritic cells (pDCs) are known mainly for their secretion of type I IFN upon viral encounter. We describe a CD2(hi)CD5(+)CD81(+) pDC subset, distinguished by prominent dendrites and a mature phenotype, in human blood, bone marrow, and tonsil, which can be generated from CD34(+) progenitors. These CD2(hi)CD5(+)CD81(+) cells express classical pDC markers, as well as the toll-like receptors that enable conventional pDCs to respond to viral infection. However, their gene expression profile is distinct, and they produce little or no type I IFN upon stimulation with CpG oligonucleotides, likely due to their diminished expression of IFN regulatory factor 7. A similar population of CD5(+)CD81(+) pDCs is present in mice and also does not produce type I IFN after CpG stimulation. In contrast to conventional CD5(-)CD81(-) pDCs, human CD5(+)CD81(+) pDCs are potent stimulators of B-cell activation and antibody production and strong inducers of T-cell proliferation and Treg formation. These findings reveal the presence of a discrete pDC population that does not produce type I IFN and yet mediates important immune functions previously attributed to all pDCs.

  View details for DOI 10.1073/pnas.1610630114

  View details for PubMedID 28167780

  View details for PubMedCentralID PMC5338447

• Progression of EGFR-Mutant Lung Adenocarcinoma is Driven By Alveolar Macrophages. Clinical cancer research : an official journal of the American Association for Cancer Research Wang, D., Lee, H., Yoon, D., Berry, G., Wheeler, T. M., Sugarbaker, D. J., Kheradmand, F., Engleman, E., Burt, B. M. 2017; 23 (3): 778-788

  Abstract

  Lung adenocarcinomas with mutations in the EGFR have unprecedented initial responses to targeted therapy against the EGFR. Over time, however, these tumors invariably develop resistance to these drugs. We set out to investigate alternative treatment approaches for these tumors.To investigate the immunologic underpinnings of EGFR-mutant lung adenocarcinoma, we utilized a bitransgenic mouse model in which a mutant human EGFR gene is selectively expressed in the lungs.EGFR oncogene-dependent progression and remission of lung adenocarcinoma was respectively dependent upon the expansion and contraction of alveolar macrophages, and the mechanism underlying macrophage expansion was local proliferation. In tumor-bearing mice, alveolar macrophages downregulated surface expression of MHC-II and costimulatory molecules; increased production of CXCL1, CXCL2, IL1 receptor antagonist; and increased phagocytosis. Depletion of alveolar macrophages in tumor-bearing mice resulted in reduction of tumor burden, indicating a critical role for these cells in the development of EGFR-mutant adenocarcinoma. Treatment of mice with EGFR-targeting clinical drugs (erlotinib and cetuximab) resulted in a significant decrease in alveolar macrophages in these mice. An activated alveolar macrophage mRNA signature was dominant in human EGFR-mutant lung adenocarcinomas, and the presence of this alveolar macrophage activation signature was associated with unfavorable survival among patients undergoing resection for EGFR-mutant lung adenocarcinoma.Because of the inevitability of failure of targeted therapy in EGFR-mutant non-small cell lung cancer (NSCLC), these data suggest that therapeutic strategies targeting alveolar macrophages in EGFR-mutant NSCLC have the potential to mitigate progression and survival in this disease. Clin Cancer Res; 23(3); 778-88. ©2016 AACR.

  View details for DOI 10.1158/1078-0432.CCR-15-2597

  View details for PubMedID 27496865

• Systemic Immunity Is Required for Effective Cancer Immunotherapy. Cell Spitzer, M. H., Carmi, Y., Reticker-Flynn, N. E., Kwek, S. S., Madhireddy, D., Martins, M. M., Gherardini, P. F., Prestwood, T. R., Chabon, J., Bendall, S. C., Fong, L., Nolan, G. P., Engleman, E. G. 2017; 168 (3): 487-502 e15

  Abstract

  Immune responses involve coordination across cell types and tissues. However, studies in cancer immunotherapy have focused heavily on local immune responses in the tumor microenvironment. To investigate immune activity more broadly, we performed an organism-wide study in genetically engineered cancer models using mass cytometry. We analyzed immune responses in several tissues after immunotherapy by developing intuitive models for visualizing single-cell data with statistical inference. Immune activation was evident in the tumor and systemically shortly after effective therapy was administered. However, during tumor rejection, only peripheral immune cells sustained their proliferation. This systemic response was coordinated across tissues and required for tumor eradication in several immunotherapy models. An emergent population of peripheral CD4 T cells conferred protection against new tumors and was significantly expanded in patients responding to immunotherapy. These studies demonstrate the critical impact of systemic immune responses that drive tumor rejection.

  View details for DOI 10.1016/j.cell.2016.12.022

  View details for PubMedID 28111070

• Tolerogenic interactions between CD8+ dendritic cells and NKT cells prevent rejection of bone marrow and organ grafts. Blood Hongo, D., Tang, X., Zhang, X., Engleman, E. G., Strober, S. 2017

  Abstract

  The combination of total lymphoid irradiation and anti-T-cell antibodies safely induces immune tolerance to combined hematopoietic cell and organ allografts in humans. Our mouse model required host natural killer T (NKT) cells to induce tolerance. Because NKT cells normally depend on signals from CD8(+) dendritic cells (DCs) for their activation, we used the mouse model to test the hypothesis that, after lymphoid irradiation, host CD8(+) DCs play a requisite role in tolerance induction through interactions with NKT cells. Selective deficiency of either CD8(+) DCs or NKT cells abrogated chimerism and organ graft acceptance. After radiation, the CD8(+) DCs increased expression of surface molecules required for NKT and apoptotic cell interactions and developed suppressive immune functions, including production of indoleamine 2,3-deoxygenase. Injection of naive mice with apoptotic spleen cells generated by irradiation led to DC changes similar to those induced by lymphoid radiation, suggesting that apoptotic body ingestion by CD8(+) DCs initiates tolerance induction. Tolerogenic CD8(+) DCs induced the development of tolerogenic NKT cells with a marked T helper 2 cell bias that, in turn, regulated the differentiation of the DCs and suppressed rejection of the transplants. Thus, reciprocal interactions between CD8(+) DCs and invariant NKT cells are required for tolerance induction in this system that was translated into a successful clinical protocol.

  View details for DOI 10.1182/blood-2016-07-723015

  View details for PubMedID 28096089

• Role of innate and adaptive immunity in obesity-associated metabolic disease JOURNAL OF CLINICAL INVESTIGATION McLaughlin, T., Ackerman, S. E., Shen, L., Engleman, E. 2017; 127 (1): 5-13

  Abstract

  Chronic inflammation in adipose tissue, possibly related to adipose cell hypertrophy, hypoxia, and/or intestinal leakage of bacteria and their metabolic products, likely plays a critical role in the development of obesity-associated insulin resistance (IR). Cells of both the innate and adaptive immune system residing in adipose tissues, as well as in the intestine, participate in this process. Thus, M1 macrophages, IFN-γ-secreting Th1 cells, CD8+ T cells, and B cells promote IR, in part through secretion of proinflammatory cytokines. Conversely, eosinophils, Th2 T cells, type 2 innate lymphoid cells, and possibly Foxp3+ Tregs protect against IR through local control of inflammation.

  View details for DOI 10.1172/JCI88876

  View details for Web of Science ID 000392271300002

  View details for PubMedID 28045397

  View details for PubMedCentralID PMC5199693

• Adipose tissue macrophages impair preadipocyte differentiation in humans. PloS one Liu, L. F., Craig, C. M., Tolentino, L. L., Choi, O., Morton, J., Rivas, H., Cushman, S. W., Engleman, E. G., McLaughlin, T. 2017; 12 (2)

  Abstract

  The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation.Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified.Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001). With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance.The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots.

  View details for DOI 10.1371/journal.pone.0170728

  View details for PubMedID 28151993

  View details for PubMedCentralID PMC5289462

• High-Dimensional Phenotypic Mapping of Human Dendritic Cells Reveals Interindividual Variation and Tissue Specialization. Immunity Alcántara-Hernández, M. n., Leylek, R. n., Wagar, L. E., Engleman, E. G., Keler, T. n., Marinkovich, M. P., Davis, M. M., Nolan, G. P., Idoyaga, J. n. 2017

  Abstract

  Given the limited efficacy of clinical approaches that rely on ex vivo generated dendritic cells (DCs), it is imperative to design strategies that harness specialized DC subsets in situ. This requires delineating the expression of surface markers by DC subsets among individuals and tissues. Here, we performed a multiparametric phenotypic characterization and unbiased analysis of human DC subsets in blood, tonsil, spleen, and skin. We uncovered previously unreported phenotypic heterogeneity of human cDC2s among individuals, including variable expression of functional receptors such as CD172a. We found marked differences in DC subsets localized in blood and lymphoid tissues versus skin, and a striking absence of the newly discovered Axl+ DCs in the skin. Finally, we evaluated the capacity of anti-receptor monoclonal antibodies to deliver vaccine components to skin DC subsets. These results offer a promising path for developing DC subset-specific immunotherapies that cannot be provided by transcriptomic analysis alone.

  View details for PubMedID 29221729

• Restoring Retinoic Acid Attenuates Intestinal Inflammation and Tumorigenesis in APCMin/+ Mice. Cancer immunology research Penny, H. L., Prestwood, T. R., Bhattacharya, N., Sun, F., Kenkel, J. A., Davidson, M. G., Shen, L., Zuniga, L. A., Seeley, E. S., Pai, R., Choi, O., Tolentino, L., Wang, J., Napoli, J. L., Engleman, E. G. 2016; 4 (11): 917-926

  Abstract

  Chronic intestinal inflammation accompanies familial adenomatous polyposis (FAP) and is a major risk factor for colorectal cancer in patients with this disease, but the cause of such inflammation is unknown. Because retinoic acid (RA) plays a critical role in maintaining immune homeostasis in the intestine, we hypothesized that altered RA metabolism contributes to inflammation and tumorigenesis in FAP. To assess this hypothesis, we analyzed RA metabolism in the intestines of patients with FAP as well as APC(Min/+) mice, a model that recapitulates FAP in most respects. We also investigated the impact of intestinal RA repletion and depletion on tumorigenesis and inflammation in APC(Min/+) mice. Tumors from both FAP patients and APC(Min/+) mice displayed striking alterations in RA metabolism that resulted in reduced intestinal RA. APC(Min/+) mice placed on a vitamin A-deficient diet exhibited further reductions in intestinal RA with concomitant increases in inflammation and tumor burden. Conversely, restoration of RA by pharmacologic blockade of the RA-catabolizing enzyme CYP26A1 attenuated inflammation and diminished tumor burden. To investigate the effect of RA deficiency on the gut immune system, we studied lamina propria dendritic cells (LPDC) because these cells play a central role in promoting tolerance. APC(Min/+) LPDCs preferentially induced Th17 cells, but reverted to inducing Tregs following restoration of intestinal RA in vivo or direct treatment of LPDCs with RA in vitro These findings demonstrate the importance of intestinal RA deficiency in tumorigenesis and suggest that pharmacologic repletion of RA could reduce tumorigenesis in FAP patients. Cancer Immunol Res; 4(11); 917-26. ©2016 AACR.

  View details for PubMedID 27638841

• Normalizing Microbiota-Induced Retinoic Acid Deficiency Stimulates Protective CD8(+) T Cell-Mediated Immunity in Colorectal Cancer. Immunity Bhattacharya, N., Yuan, R., Prestwood, T. R., Penny, H. L., DiMaio, M. A., Reticker-Flynn, N. E., Krois, C. R., Kenkel, J. A., Pham, T. D., Carmi, Y., Tolentino, L., Choi, O., Hulett, R., Wang, J., Winer, D. A., Napoli, J. L., Engleman, E. G. 2016; 45 (3): 641-655

  Abstract

  Although all-trans-retinoic acid (atRA) is a key regulator of intestinal immunity, its role in colorectal cancer (CRC) is unknown. We found that mice with colitis-associated CRC had a marked deficiency in colonic atRA due to alterations in atRA metabolism mediated by microbiota-induced intestinal inflammation. Human ulcerative colitis (UC), UC-associated CRC, and sporadic CRC specimens have similar alterations in atRA metabolic enzymes, consistent with reduced colonic atRA. Inhibition of atRA signaling promoted tumorigenesis, whereas atRA supplementation reduced tumor burden. The benefit of atRA treatment was mediated by cytotoxic CD8(+) T cells, which were activated due to MHCI upregulation on tumor cells. Consistent with these findings, increased colonic expression of the atRA-catabolizing enzyme, CYP26A1, correlated with reduced frequencies of tumoral cytotoxic CD8(+) T cells and with worse disease prognosis in human CRC. These results reveal a mechanism by which microbiota drive colon carcinogenesis and highlight atRA metabolism as a therapeutic target for CRC.

  View details for DOI 10.1016/j.immuni.2016.08.008

  View details for PubMedID 27590114

  View details for PubMedCentralID PMC5132405

• Nucleic Acid-Targeting Pathways Promote Inflammation in Obesity-Related Insulin Resistance. Cell reports Revelo, X. S., Ghazarian, M., Chng, M. H., Luck, H., Kim, J. H., Zeng, K., Shi, S. Y., Tsai, S., Lei, H., Kenkel, J., Liu, C. L., Tangsombatvisit, S., Tsui, H., Sima, C., Xiao, C., Shen, L., Li, X., Jin, T., Lewis, G. F., Woo, M., Utz, P. J., Glogauer, M., Engleman, E., Winer, S., Winer, D. A. 2016; 16 (3): 717-730

  Abstract

  Obesity-related inflammation of metabolic tissues, including visceral adipose tissue (VAT) and liver, are key factors in the development of insulin resistance (IR), though many of the contributing mechanisms remain unclear. We show that nucleic-acid-targeting pathways downstream of extracellular trap (ET) formation, unmethylated CpG DNA, or ribonucleic acids drive inflammation in IR. High-fat diet (HFD)-fed mice show increased release of ETs in VAT, decreased systemic clearance of ETs, and increased autoantibodies against conserved nuclear antigens. In HFD-fed mice, this excess of nucleic acids and related protein antigens worsens metabolic parameters through a number of mechanisms, including activation of VAT macrophages and expansion of plasmacytoid dendritic cells (pDCs) in the liver. Consistently, HFD-fed mice lacking critical responders of nucleic acid pathways, Toll-like receptors (TLR)7 and TLR9, show reduced metabolic inflammation and improved glucose homeostasis. Treatment of HFD-fed mice with inhibitors of ET formation or a TLR7/9 antagonist improves metabolic disease. These findings reveal a pathogenic role for nucleic acid targeting as a driver of metabolic inflammation in IR.

  View details for DOI 10.1016/j.celrep.2016.06.024

  View details for PubMedID 27373163

• Normalizing microbiota-induced retinoic acid deficiency stimulates protective CD8+ T-cell-mediated immunity in colorectal cancer Immunity Bhattacharya, N., Yuan, R., Prestwood, T., Penny, H., DiMaio, M., Reticker-Flynn, N., Krois, C., Kenkel, J., Pham, T., Carmi, Y., Tolentino, L., Choi, O., Hulett, R., Wang, J., Winer, D., Napoli, J., Engleman, E. 2016; 45: 641–55

  Abstract

  Although all-trans-retinoic acid (atRA) is a key regulator of intestinal immunity, its role in colorectal cancer (CRC) is unknown. We found that mice with colitis-associated CRC had a marked deficiency in colonic atRA due to alterations in atRA metabolism mediated by microbiota-induced intestinal inflammation. Human ulcerative colitis (UC), UC-associated CRC, and sporadic CRC specimens have similar alterations in atRA metabolic enzymes, consistent with reduced colonic atRA. Inhibition of atRA signaling promoted tumorigenesis, whereas atRA supplementation reduced tumor burden. The benefit of atRA treatment was mediated by cytotoxic CD8(+) T cells, which were activated due to MHCI upregulation on tumor cells. Consistent with these findings, increased colonic expression of the atRA-catabolizing enzyme, CYP26A1, correlated with reduced frequencies of tumoral cytotoxic CD8(+) T cells and with worse disease prognosis in human CRC. These results reveal a mechanism by which microbiota drive colon carcinogenesis and highlight atRA metabolism as a therapeutic target for CRC.

  View details for DOI 10.1016/j.immuni.2016.08.008

  View details for PubMedCentralID PMC5132405

• Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity. JCI insight Carmi, Y. n., Prestwood, T. R., Spitzer, M. H., Linde, I. L., Chabon, J. n., Reticker-Flynn, N. E., Bhattacharya, N. n., Zhang, H. n., Zhang, X. n., Basto, P. A., Burt, B. M., Alonso, M. N., Engleman, E. G. 2016; 1 (18): e89020

  Abstract

  BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.

  View details for PubMedID 27812544

• Hypothesis: The Intratumoral Immune Response against a Cancer Progenitor Cell Impacts the Development of Well-Differentiated versus Dedifferentiated Disease in Liposarcoma. Frontiers in oncology Tseng, W. W., Chopra, S., Engleman, E. G., Pollock, R. E. 2016; 6: 134-?

  Abstract

  Well-differentiated/dedifferentiated (WD/DD) liposarcoma is a rare malignancy of adipocyte origin ("fat cancer"). Tumors may be entirely WD, WD with a DD component, or rarely DD without a clear WD component. WD tumors are low grade and generally indolent, while tumors with a DD component are high grade and behave much more aggressively, with a modest potential for distant metastasis. The presence of cancer progenitor cells in WD/DD liposarcoma is suggested by clinical evidence and reported research findings. In addition, there are emerging data to support the existence of a naturally occurring, antigen-driven, and adaptive immune response within the tumor microenvironment. We hypothesize that the intratumoral immune response is directed against a cancer progenitor cell and that the outcome of this response impacts the development of WD versus DD disease. Further study will likely provide interesting insights into the disease biology of WD/DD liposarcoma that may be readily translated to other more common cancers.

  View details for DOI 10.3389/fonc.2016.00134

  View details for PubMedID 27376027

• Instructive roles for cytokine-receptor binding parameters in determining signaling and functional potency SCIENCE SIGNALING Moraga, I., Richter, D., Wilmes, S., Winkelmann, H., Jude, K., Thomas, C., Suhoski, M. M., Engleman, E. G., Piehler, J., Garcia, K. C. 2015; 8 (402)

  Abstract

  Cytokines dimerize cell surface receptors to activate signaling and regulate many facets of the immune response. Many cytokines have pleiotropic effects, inducing a spectrum of redundant and distinct effects on different cell types. This pleiotropy has hampered cytokine-based therapies, and the high doses required for treatment often lead to off-target effects, highlighting the need for a more detailed understanding of the parameters controlling cytokine-induced signaling and bioactivities. Using the prototypical cytokine interleukin-13 (IL-13), we explored the interrelationships between receptor binding and a wide range of downstream cellular responses. We applied structure-based engineering to generate IL-13 variants that covered a spectrum of binding strengths for the receptor subunit IL-13Rα1. Engineered IL-13 variants representing a broad range of affinities for the receptor exhibited similar potencies in stimulating the phosphorylation of STAT6 (signal transducer and activator of transcription 6). Delays in the phosphorylation and nuclear translocation of STAT6 were only apparent for those IL-13 variants with markedly reduced affinities for the receptor. From these data, we developed a mechanistic model that quantitatively reproduced the kinetics of STAT6 phosphorylation for the entire spectrum of binding affinities. Receptor endocytosis played a key role in modulating STAT6 activation, whereas the lifetime of receptor-ligand complexes at the plasma membrane determined the potency of the variant for inducing more distal responses. This complex interrelationship between extracellular ligand binding and receptor function provides the foundation for new mechanism-based strategies that determine the optimal cytokine dose to enhance therapeutic efficacy.

  View details for DOI 10.1126/scisignal.aab2677

  View details for PubMedID 26554818

  View details for PubMedCentralID PMC5568809

• Ablative Tumor Radiation Can Change the Tumor Immune Cell Microenvironment to Induce Durable Complete Remissions. Clinical cancer research Filatenkov, A., Baker, J., Mueller, A. M., Kenkel, J., Ahn, G., Dutt, S., Zhang, N., Kohrt, H., Jensen, K., Dejbakhsh-Jones, S., Shizuru, J. A., Negrin, R. N., Engleman, E. G., Strober, S. 2015; 21 (16): 3727-3739

  Abstract

  The goals of the study were to elucidate the immune mechanisms that contribute to desirable complete remissions of murine colon tumors treated with single radiation dose of 30 Gy. This dose is at the upper end of the ablative range used clinically to treat advanced or metastatic colorectal, liver, and non-small cell lung tumors.Changes in the tumor immune microenvironment of single tumor nodules exposed to radiation were studied using 21-day (>1 cm in diameter) CT26 and MC38 colon tumors. These are well-characterized weakly immunogenic tumors.We found that the high-dose radiation transformed the immunosuppressive tumor microenvironment resulting in an intense CD8(+) T-cell tumor infiltrate, and a loss of myeloid-derived suppressor cells (MDSC). The change was dependent on antigen cross-presenting CD8(+) dendritic cells, secretion of IFNγ, and CD4(+)T cells expressing CD40L. Antitumor CD8(+) T cells entered tumors shortly after radiotherapy, reversed MDSC infiltration, and mediated durable remissions in an IFNγ-dependent manner. Interestingly, extended fractionated radiation regimen did not result in robust CD8(+) T-cell infiltration.For immunologically sensitive tumors, these results indicate that remissions induced by a short course of high-dose radiotherapy depend on the development of antitumor immunity that is reflected by the nature and kinetics of changes induced in the tumor cell microenvironment. These results suggest that systematic examination of the tumor immune microenvironment may help in optimizing the radiation regimen used to treat tumors by adding a robust immune response. Clin Cancer Res; 21(16); 3727-39. ©2015 AACR.

  View details for DOI 10.1158/1078-0432.CCR-14-2824

  View details for PubMedID 25869387

• A Three-Gene Assay for Monitoring Immune Quiescence in Kidney Transplantation JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Roedder, S., Li, L., Alonso, M. N., Hsieh, S., Minh Thien Vu, M. T., Dai, H., Sigdel, T. K., Bostock, I., Macedo, C., Metes, D., Zeevi, A., Shapiro, R., Salvatierra, O., Scandling, J., Alberu, J., Engleman, E., Sarwal, M. M. 2015; 26 (8): 2042-2053

  Abstract

  Organ transplant recipients face life-long immunosuppression and consequently are at high risk of comorbidities. Occasionally, kidney transplant recipients develop a state of targeted immune quiescence (operational tolerance) against an HLA-mismatched graft, allowing them to withdraw all immunosuppression and retain stable graft function while resuming immune responses to third-party antigens. Methods to better understand and monitor this state of alloimmune quiescence by transcriptional profiling may reveal a gene signature that identifies patients for whom immunosuppression could be titrated to reduce patient and graft morbidities. Therefore, we investigated 571 unique peripheral blood samples from 348 HLA-mismatched renal transplant recipients and 101 nontransplant controls in a four-stage study including microarray, quantitative PCR, and flow cytometry analyses. We report a refined and highly validated (area under the curve, 0.95; 95% confidence interval, 0.92 to 0.97) peripheral blood three-gene assay (KLF6, BNC2, CYP1B1) to detect the state of operational tolerance by quantitative PCR. The frequency of predicted alloimmune quiescence in stable renal transplant patients receiving long-term immunosuppression (n=150) was 7.3% by the three-gene assay. Targeted cell sorting of peripheral blood from operationally tolerant patients showed a significant shift in the ratio of circulating monocyte-derived dendritic cells with significantly different expression of the genes constituting the three-gene assay. Our results suggest that incorporation of patient screening by specific cellular and gene expression assays may support the safety of drug minimization trials and protocols.

  View details for DOI 10.1681/ASN.2013111239

  View details for Web of Science ID 000358895100028

  View details for PubMedID 25429124

  View details for PubMedCentralID PMC4520154

• An interactive reference framework for modeling a dynamic immune system SCIENCE Spitzer, M. H., Gherardini, P. F., Fragiadakis, G. K., Bhattacharya, N., Yuan, R. T., Hotson, A. N., Finck, R., Carmi, Y., Zunder, E. R., Fantl, W. J., Bendall, S. C., Engleman, E. G., Nolan, G. P. 2015; 349 (6244): 155-?

  Abstract

  Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

  View details for DOI 10.1126/science.1259425

  View details for Web of Science ID 000357664300034

  View details for PubMedCentralID PMC4537647

• IMMUNOLOGY. An interactive reference framework for modeling a dynamic immune system. Science Spitzer, M. H., Gherardini, P. F., Fragiadakis, G. K., Bhattacharya, N., Yuan, R. T., Hotson, A. N., Finck, R., Carmi, Y., Zunder, E. R., Fantl, W. J., Bendall, S. C., Engleman, E. G., Nolan, G. P. 2015; 349 (6244)

  Abstract

  Immune cells function in an interacting hierarchy that coordinates the activities of various cell types according to genetic and environmental contexts. We developed graphical approaches to construct an extensible immune reference map from mass cytometry data of cells from different organs, incorporating landmark cell populations as flags on the map to compare cells from distinct samples. The maps recapitulated canonical cellular phenotypes and revealed reproducible, tissue-specific deviations. The approach revealed influences of genetic variation and circadian rhythms on immune system structure, enabled direct comparisons of murine and human blood cell phenotypes, and even enabled archival fluorescence-based flow cytometry data to be mapped onto the reference framework. This foundational reference map provides a working definition of systemic immune organization to which new data can be integrated to reveal deviations driven by genetics, environment, or pathology.

  View details for DOI 10.1126/science.1259425

  View details for PubMedID 26160952

• The receptor CD44 is associated with systemic insulin resistance and proinflammatory macrophages in human adipose tissue DIABETOLOGIA Liu, L. F., Kodama, K., Wei, K., Tolentino, L. L., Choi, O., Engleman, E. G., Butte, A. J., McLaughlin, T. 2015; 58 (7): 1579-1586

  Abstract

  Proinflammatory immune cell infiltration in human adipose tissue is associated with the development of insulin resistance. We previously identified, via a gene expression-based genome-wide association study, the cell-surface immune cell receptor CD44 as a functionally important gene associated with type 2 diabetes. We then showed that, compared with controls, Cd44 knockout mice were protected from insulin resistance and adipose tissue inflammation during diet-induced obesity. We thus sought to test whether CD44 is associated with adipose tissue inflammation and insulin resistance in humans.Participants included 58 healthy, overweight/moderately obese white adults who met predetermined criteria for insulin resistance or insulin sensitivity based on the modified insulin-suppression test. Serum was collected from 43 participants to measure circulating concentrations of CD44. Subcutaneous adipose tissue was obtained from 17 participants to compare CD44, its ligand osteopontin (OPN, also known as SPP1) and pro-inflammatory gene expression. CD44 expression on adipose tissue macrophage (ATM) surfaces was determined by flow cytometry.Serum CD44 concentrations were significantly increased in insulin-resistant (IR) participants. CD44 gene expression in subcutaneous adipose tissue was threefold higher in the IR subgroup. The expression of OPN, CD68 and IL6 was also significantly elevated in IR individuals. CD44 gene expression correlated significantly with CD68 and IL6 expression. CD44 density on ATMs was associated with proinflammatory M1 polarisation.CD44 and OPN in human adipose tissue are associated with localised inflammation and systemic insulin resistance. This receptor-ligand pair is worthy of further research as a potentially modifiable contributor to human insulin resistance and type 2 diabetes.

  View details for DOI 10.1007/s00125-015-3603-y

  View details for Web of Science ID 000356528900023

  View details for PubMedID 25952479

• Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity. Nature Carmi, Y., Spitzer, M. H., Linde, I. L., Burt, B. M., Prestwood, T. R., Perlman, N., Davidson, M. G., Kenkel, J. A., Segal, E., Pusapati, G. V., Bhattacharya, N., Engleman, E. G. 2015; 521 (7550): 99-104

  Abstract

  Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.

  View details for DOI 10.1038/nature14424

  View details for PubMedID 25924063

  View details for PubMedCentralID PMC4877172

• Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity NATURE Carmi, Y., Spitzer, M. H., Linde, I. L., Burt, B. M., Prestwood, T. R., Perlman, N., Davidson, M. G., Kenkel, J. A., Segal, E., Pusapati, G. V., Bhattacharya, N., Engleman, E. G. 2015; 521 (7550): 99-U254

  Abstract

  Whereas cancers grow within host tissues and evade host immunity through immune-editing and immunosuppression, tumours are rarely transmissible between individuals. Much like transplanted allogeneic organs, allogeneic tumours are reliably rejected by host T cells, even when the tumour and host share the same major histocompatibility complex alleles, the most potent determinants of transplant rejection. How such tumour-eradicating immunity is initiated remains unknown, although elucidating this process could provide the basis for inducing similar responses against naturally arising tumours. Here we find that allogeneic tumour rejection is initiated in mice by naturally occurring tumour-binding IgG antibodies, which enable dendritic cells (DCs) to internalize tumour antigens and subsequently activate tumour-reactive T cells. We exploited this mechanism to treat autologous and autochthonous tumours successfully. Either systemic administration of DCs loaded with allogeneic-IgG-coated tumour cells or intratumoral injection of allogeneic IgG in combination with DC stimuli induced potent T-cell-mediated antitumour immune responses, resulting in tumour eradication in mouse models of melanoma, pancreas, lung and breast cancer. Moreover, this strategy led to eradication of distant tumours and metastases, as well as the injected primary tumours. To assess the clinical relevance of these findings, we studied antibodies and cells from patients with lung cancer. T cells from these patients responded vigorously to autologous tumour antigens after culture with allogeneic-IgG-loaded DCs, recapitulating our findings in mice. These results reveal that tumour-binding allogeneic IgG can induce powerful antitumour immunity that can be exploited for cancer immunotherapy.

  View details for DOI 10.1038/nature14424

  View details for Web of Science ID 000354040900041

  View details for PubMedID 25924063

  View details for PubMedCentralID PMC4877172

• Regulation of Obesity-Related Insulin Resistance with Gut Anti-inflammatory Agents CELL METABOLISM Luck, H., Tsai, S., Chung, J., Clemente-Casares, X., Ghazarian, M., Revelo, X. S., Lei, H., Luk, C. T., Shi, S. Y., Surendra, A., Copeland, J. K., Ahn, J., Prescott, D., Rasmussen, B. A., Chng, M. H., Engleman, E. G., Girardin, S. E., Lam, T. K., Croitoru, K., Dunn, S., Philpott, D. J., Guttman, D. S., Woo, M., Winer, S., Winer, D. A. 2015; 21 (4): 527-542

  Abstract

  Obesity has reached epidemic proportions, but little is known about its influence on the intestinal immune system. Here we show that the gut immune system is altered during high-fat diet (HFD) feeding and is a functional regulator of obesity-related insulin resistance (IR) that can be exploited therapeutically. Obesity induces a chronic phenotypic pro-inflammatory shift in bowel lamina propria immune cell populations. Reduction of the gut immune system, using beta7 integrin-deficient mice (Beta7(null)), decreases HFD-induced IR. Treatment of wild-type HFD C57BL/6 mice with the local gut anti-inflammatory, 5-aminosalicyclic acid (5-ASA), reverses bowel inflammation and improves metabolic parameters. These beneficial effects are dependent on adaptive and gut immunity and are associated with reduced gut permeability and endotoxemia, decreased visceral adipose tissue inflammation, and improved antigen-specific tolerance to luminal antigens. Thus, the mucosal immune system affects multiple pathways associated with systemic IR and represents a novel therapeutic target in this disease.

  View details for DOI 10.1016/j.cmet.2015.03.001

  View details for Web of Science ID 000352500800009

  View details for PubMedID 25863246

• Chimerism, Graft Survival, and Withdrawal of Immunosuppressive Drugs in HLA Matched and Mismatched Patients After Living Donor Kidney and Hematopoietic Cell Transplantation. American journal of transplantation Scandling, J. D., Busque, S., Shizuru, J. A., Lowsky, R., Hoppe, R., Dejbakhsh-Jones, S., Jensen, K., Shori, A., Strober, J. A., Lavori, P., Turnbull, B. B., Engleman, E. G., Strober, S. 2015; 15 (3): 695-704

  Abstract

  Thirty-eight HLA matched and mismatched patients given combined living donor kidney and enriched CD34(+) hematopoietic cell transplants were enrolled in tolerance protocols using posttransplant conditioning with total lymphoid irradiation and anti-thymocyte globulin. Persistent chimerism for at least 6 months was associated with successful complete withdrawal of immunosuppressive drugs in 16 of 22 matched patients without rejection episodes or kidney disease recurrence with up to 5 years follow up thereafter. One patient is in the midst of withdrawal and five are on maintenance drugs. Persistent mixed chimerism was achieved in some haplotype matched patients for at least 12 months by increasing the dose of T cells and CD34(+) cells infused as compared to matched recipients in a dose escalation study. Success of drug withdrawal in chimeric mismatched patients remains to be determined. None of the 38 patients had kidney graft loss or graft versus host disease with up to 14 years of observation. In conclusion, complete immunosuppressive drug withdrawal could be achieved thus far with the tolerance induction regimen in HLA matched patients with uniform long-term graft survival in all patients.

  View details for DOI 10.1111/ajt.13091

  View details for PubMedID 25693475

• B-1a lymphocytes attenuate insulin resistance. Diabetes Shen, L., Chng, M. H., Alonso, M. N., Yuan, R., Winer, D. A., Engleman, E. G. 2015; 64 (2): 593-603

  Abstract

  Obesity-associated insulin resistance, a common precursor of type 2 diabetes, is characterized by chronic inflammation of tissues, including visceral adipose tissue (VAT). Here we show that B-1a cells, a subpopulation of B lymphocytes, are novel and important regulators of this process. B-1a cells are reduced in frequency in obese high-fat diet (HFD)-fed mice, and EGFP interleukin-10 (IL-10) reporter mice show marked reductions in anti-inflammatory IL-10 production by B cells in vivo during obesity. In VAT, B-1a cells are the dominant producers of B cell-derived IL-10, contributing nearly half of the expressed IL-10 in vivo. Adoptive transfer of B-1a cells into HFD-fed B cell-deficient mice rapidly improves insulin resistance and glucose tolerance through IL-10 and polyclonal IgM-dependent mechanisms, whereas transfer of B-2 cells worsens metabolic disease. Genetic knockdown of B cell-activating factor (BAFF) in HFD-fed mice or treatment with a B-2 cell-depleting, B-1a cell-sparing anti-BAFF antibody attenuates insulin resistance. These findings establish B-1a cells as a new class of immune regulators that maintain metabolic homeostasis and suggest manipulation of these cells as a potential therapy for insulin resistance.

  View details for DOI 10.2337/db14-0554

  View details for PubMedID 25249575

  View details for PubMedCentralID PMC4303967

• Invariant natural killer T cells in lupus patients promote IgG and IgG autoantibody production EUROPEAN JOURNAL OF IMMUNOLOGY Shen, L., Zhang, H., Caimol, M., Benike, C. J., Chakravarty, E. F., Strober, S., Engleman, E. G. 2015; 45 (2): 612-623

  Abstract

  IgG autoantibodies, including antibodies to double-stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus (SLE), but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG in vitro. Despite the rarity of iNKT cells in lupus blood (0.002-0.05% of CD3-positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti-dsDNA IgG by lupus PBMCs. Addition of anti-iNKT or anti-CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B-cell maturation. Like fresh iNKT cells, expanded iNKT-cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti-dsDNA. This activity was inhibited by anti-CD40L antibody, as well as anti-CD1d antibody, confirming a role for CD40L-CD40 and TCR-CD1d interactions in lupus iNKT-cell-mediated help. These results reveal a critical role for iNKT cells in B-cell maturation and autoantibody production in patients with lupus.

  View details for DOI 10.1002/eji.201444760

  View details for Web of Science ID 000349625400030

  View details for PubMedID 25352488

  View details for PubMedCentralID PMC4324163

• Detection of intestinal cancer by local, topical application of a quenched fluorescence probe for cysteine cathepsins. Chemistry & biology Segal, E., Prestwood, T. R., van der Linden, W. A., Carmi, Y., Bhattacharya, N., Withana, N., Verdoes, M., Habtezion, A., Engleman, E. G., Bogyo, M. 2015; 22 (1): 148-158

  Abstract

  Early detection of colonic polyps can prevent up to 90% of colorectal cancer deaths. Conventional colonoscopy readily detects the majority of premalignant lesions, which exhibit raised morphology. However, lesions that are flat and depressed are often undetected using this method. Therefore, there is a need for molecular-based contrast agents to improve detection rates over conventional colonoscopy. We evaluated a quenched fluorescent activity-based probe (qABP; BMV109) that targets multiple cysteine cathepsins that are overexpressed in intestinal dysplasia in a genetic model of spontaneous intestinal polyp formation and in a chemically induced model of colorectal carcinoma. We found that the qABP selectively targets cysteine cathepsins, resulting in high sensitivity and specificity for intestinal tumors in mice and humans. Additionally, the qABP can be administered by either intravenous injection or by local delivery to the colon, making it a highly valuable tool for improved detection of colorectal lesions using fluorescence-guided colonoscopy.

  View details for DOI 10.1016/j.chembiol.2014.11.008

  View details for PubMedID 25579207

  View details for PubMedCentralID PMC4353655

• Adaptive Immunity and Antigen-Specific Activation in Obesity-Associated Insulin Resistance. Mediators of inflammation Chng, M. H., Alonso, M. N., Barnes, S. E., Nguyen, K. D., Engleman, E. G. 2015; 2015: 593075-?

  Abstract

  Type 2 diabetes mellitus (T2D) is a metabolic disease that is strongly tied to obesity and often preceded by insulin resistance (IR). It has been established that chronic inflammation of hypertrophic adipose tissue depots in obese individuals leads to obesity-associated IR and is mediated by cells of the innate immune system, particularly macrophages. More recently, cells of the adaptive immune system, B and T lymphocytes, have also emerged as important regulators of glucose homeostasis, raising the intriguing possibility that antigen-driven immune responses play a role in disease. In this review, we critically evaluate the roles that various B and T cell subsets play in IR, and then we examine the data suggesting that antigen-driven mechanisms, such as antigen presentation and costimulation, may drive the activity of these lymphocytes.

  View details for DOI 10.1155/2015/593075

  View details for PubMedID 26146464

  View details for PubMedCentralID PMC4471324

• Adaptive Immunity and Antigen-Specific Activation in Obesity-Associated Insulin Resistance MEDIATORS OF INFLAMMATION Chng, M. H., Alonso, M. N., Barnes, S. E., Nguyen, K. D., Engleman, E. G. 2015

  Abstract

  Type 2 diabetes mellitus (T2D) is a metabolic disease that is strongly tied to obesity and often preceded by insulin resistance (IR). It has been established that chronic inflammation of hypertrophic adipose tissue depots in obese individuals leads to obesity-associated IR and is mediated by cells of the innate immune system, particularly macrophages. More recently, cells of the adaptive immune system, B and T lymphocytes, have also emerged as important regulators of glucose homeostasis, raising the intriguing possibility that antigen-driven immune responses play a role in disease. In this review, we critically evaluate the roles that various B and T cell subsets play in IR, and then we examine the data suggesting that antigen-driven mechanisms, such as antigen presentation and costimulation, may drive the activity of these lymphocytes.

  View details for DOI 10.1155/2015/593075

  View details for Web of Science ID 000356285300001

  View details for PubMedID 26146464

  View details for PubMedCentralID PMC4471324

• T-Cell Profile in Adipose Tissue Is Associated With Insulin Resistance and Systemic Inflammation in Humans ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY McLaughlin, T., Liu, L., Lamendola, C., Shen, L., Morton, J., Rivas, H., Winer, D., Tolentino, L., Choi, O., Zhang, H., Chng, M. H., Engleman, E. 2014; 34 (12): 2637-2643

  Abstract

  The biological mechanisms linking obesity to insulin resistance have not been fully elucidated. We have shown that insulin resistance or glucose intolerance in diet-induced obese mice is related to a shift in the ratio of pro- and anti-inflammatory T cells in adipose tissue. We sought to test the hypothesis that the balance of T-cell phenotypes would be similarly related to insulin resistance in human obesity.Healthy overweight or obese human subjects underwent adipose-tissue biopsies and quantification of insulin-mediated glucose disposal by the modified insulin suppression test. T-cell subsets were quantified by flow cytometry in visceral (VAT) and subcutaneous adipose tissue (SAT). Results showed that CD4 and CD8 T cells infiltrate both depots, with proinflammatory T-helper (Th)-1, Th17, and CD8 T cells, significantly more frequent in VAT as compared with SAT. T-cell profiles in SAT and VAT correlated significantly with one another and with peripheral blood. Th1 frequency in SAT and VAT correlated directly, whereas Th2 frequency in VAT correlated inversely, with plasma high-sensitivity C-reactive protein concentrations. Th2 in both depots and peripheral blood was inversely associated with systemic insulin resistance. Furthermore, Th1 in SAT correlated with plasma interleukin-6. Relative expression of associated cytokines, measured by real-time polymerase chain reaction, reflected flow cytometry results. Most notably, adipose tissue expression of anti-inflammatory interleukin-10 was inversely associated with insulin resistance.CD4 and CD8 T cells populate human adipose tissue and the relative frequency of Th1 and Th2 are highly associated with systemic inflammation and insulin resistance. These findings point to the adaptive immune system as a potential mediator between obesity and insulin resistance or inflammation. Identification of antigenic stimuli in adipose tissue may yield novel targets for treatment of obesity-associated metabolic disease.

  View details for DOI 10.1161/ATVBAHA.114.304636

  View details for Web of Science ID 000345443400019

  View details for PubMedCentralID PMC4445971

• T-cell profile in adipose tissue is associated with insulin resistance and systemic inflammation in humans. Arteriosclerosis, thrombosis, and vascular biology McLaughlin, T., Liu, L., Lamendola, C., Shen, L., Morton, J., Rivas, H., Winer, D., Tolentino, L., Choi, O., Zhang, H., Hui Yen Chng, M., Engleman, E. 2014; 34 (12): 2637-2643

  Abstract

  The biological mechanisms linking obesity to insulin resistance have not been fully elucidated. We have shown that insulin resistance or glucose intolerance in diet-induced obese mice is related to a shift in the ratio of pro- and anti-inflammatory T cells in adipose tissue. We sought to test the hypothesis that the balance of T-cell phenotypes would be similarly related to insulin resistance in human obesity.Healthy overweight or obese human subjects underwent adipose-tissue biopsies and quantification of insulin-mediated glucose disposal by the modified insulin suppression test. T-cell subsets were quantified by flow cytometry in visceral (VAT) and subcutaneous adipose tissue (SAT). Results showed that CD4 and CD8 T cells infiltrate both depots, with proinflammatory T-helper (Th)-1, Th17, and CD8 T cells, significantly more frequent in VAT as compared with SAT. T-cell profiles in SAT and VAT correlated significantly with one another and with peripheral blood. Th1 frequency in SAT and VAT correlated directly, whereas Th2 frequency in VAT correlated inversely, with plasma high-sensitivity C-reactive protein concentrations. Th2 in both depots and peripheral blood was inversely associated with systemic insulin resistance. Furthermore, Th1 in SAT correlated with plasma interleukin-6. Relative expression of associated cytokines, measured by real-time polymerase chain reaction, reflected flow cytometry results. Most notably, adipose tissue expression of anti-inflammatory interleukin-10 was inversely associated with insulin resistance.CD4 and CD8 T cells populate human adipose tissue and the relative frequency of Th1 and Th2 are highly associated with systemic inflammation and insulin resistance. These findings point to the adaptive immune system as a potential mediator between obesity and insulin resistance or inflammation. Identification of antigenic stimuli in adipose tissue may yield novel targets for treatment of obesity-associated metabolic disease.

  View details for DOI 10.1161/ATVBAHA.114.304636

  View details for PubMedID 25341798

• Requirement for Interactions of Natural Killer T Cells and Myeloid-Derived Suppressor Cells for Transplantation Tolerance AMERICAN JOURNAL OF TRANSPLANTATION Hongo, D., Tang, X., Baker, J., Engleman, E. G., Strober, S. 2014; 14 (11): 2467-2477

  Abstract

  The goal of the study was to elucidate the cellular and molecular mechanisms by which a clinically applicable immune tolerance regimen of combined bone marrow and heart transplants in mice results in mixed chimerism and graft acceptance. The conditioning regimen of lymphoid irradiation and anti-T cell antibodies changed the balance of cells in the lymphoid tissues to create a tolerogenic microenvironment favoring the increase of natural killer T (NKT) cells, CD4+ CD25+ regulatory T cells and Gr-1+ CD11b+ myeloid-derived suppressor cells (MDSCs), over conventional T cells (Tcons). The depletion of MDSCs abrogated chimerism and tolerance, and add back of these purified cells was restorative. The conditioning regimen activated the MDSCs as judged by the increased expression of arginase-1, IL-4Rα and programmed death ligand 1, and the activated cells gained the capacity to suppress the proliferation of Tcons to alloantigens in the mixed leukocyte reaction. MDSC activation was dependent on the presence of host invariant NKT cells. The conditioning regimen polarized the host invariant NKT cells toward IL-4 secretion, and MDSC activation was dependent on IL-4. In conclusion, there was a requirement for MDSCs for chimerism and tolerance, and their suppressive function was dependent on their interactions with NKT cells and IL-4.

  View details for DOI 10.1111/ajt.12914

  View details for Web of Science ID 000344178800008

  View details for PubMedID 25311657

  View details for PubMedCentralID PMC4205183

• Potential for immunotherapy in soft tissue sarcoma HUMAN VACCINES & IMMUNOTHERAPEUTICS Tseng, W. W., Somaiah, N., Engleman, E. G. 2014; 10 (11): 3117-3124

  Abstract

  Soft tissue sarcomas (STS) are rare, heterogeneous tumors of mesenchymal origin. Despite optimal treatment, a large proportion of patients will develop recurrent and metastatic disease. For these patients, current treatment options are quite limited. Significant progress has been made recently in the use of immunotherapy for the treatment of other solid tumors (e.g. prostate cancer, melanoma). There is a strong rationale for immunotherapy in STS, based on an understanding of disease biology. For example, STS frequently have chromosomal translocations which result in unique fusion proteins and specific subtypes have been shown to express cancer testis antigens. In this review, we discuss the current status of immunotherapy in STS, including data from human studies with cancer vaccines, adoptive cell therapy, and immune checkpoint blockade. Further research into STS immunology is needed to help design logical, subtype-specific immunotherapeutic strategies.

  View details for DOI 10.4161/21645515.2014.983003

  View details for Web of Science ID 000349364300011

  View details for PubMedID 25625925

• Treatment of 4T1 Metastatic Breast Cancer with Combined Hypofractionated Irradiation and Autologous T-Cell Infusion. Radiation research Filatenkov, A., Baker, J., Müller, A. M., Ahn, G., Kohrt, H., Dutt, S., Jensen, K., Dejbakhsh-Jones, S., Negrin, R. S., Shizuru, J. A., Engleman, E. G., Strober, S. 2014; 182 (2): 163-169

  Abstract

  The goal of this study was to determine whether a combination of local tumor irradiation and autologous T-cell transplantation can effectively treat metastatic 4T1 breast cancer in mice. BALB/c mice were injected subcutaneously with luciferase-labeled 4T1 breast tumor cells and allowed to grow for 21 days, at which time metastases appeared in the lungs. Primary tumors were treated at that time with 3 daily fractions of 20 Gy of radiation each. Although this approach could eradicate primary tumors, tumors in the lungs grew progressively. We attempted to improve efficacy of the radiation by adding autologous T-cell infusions. Accordingly, T cells were purified from the spleens of tumor-bearing mice after completion of irradiation and cryopreserved. Cyclophosphamide was administered thereafter to induce lymphodepletion, followed by T-cell infusion. Although the addition of cyclophosphamide to irradiation did not improve survival or reduce tumor progression, the combination of radiation, cyclophosphamide and autologous T-cell infusion induced durable remissions and markedly improved survival. We conclude that the combination of radiation and autologous T-cell infusion is an effective treatment for metastatic 4T1 breast cancer.

  View details for DOI 10.1667/RR13471.1

  View details for PubMedID 24992165

• Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin. Immunologic research Alonso, M. N., Gregorio, J. G., Davidson, M. G., Gonzalez, J. C., Engleman, E. G. 2014; 58 (2-3): 374-377

  Abstract

  Monocytes rapidly infiltrate inflamed tissues and differentiate into CD209(+) inflammatory dendritic cells (DCs) that promote robust immunity or, if unregulated, inflammatory disease. Previous studies in experimental animal models indicate that inflammatory DC depletion through systemic elimination of their monocyte precursors with clodronate-loaded liposomes ameliorates the development of psoriasis and other diseases. However, translation of systemic monocyte depletion strategies is difficult due to the importance of monocytes during homeostasis and infection clearance. Here, we describe a strategy that avoids the monocyte intermediates to deplete inflammatory DCs through antibody-loaded toxin. Mice with an abundance of inflammatory DCs as a consequence of lipopolysaccharide exposure were treated with anti-CD209 antibody conjugated to saporin, a potent ribosome inactivator. The results demonstrate depletion of CD209(+) DCs. This strategy could prove useful for the targeted reduction of inflammatory DCs in disease.

  View details for DOI 10.1007/s12026-014-8511-6

  View details for PubMedID 24781193

• Immune correlates of talactoferrin alfa in biopsied tumor of relapsed/refractory metastatic non-small cell lung cancer patients. Immunopharmacology and immunotoxicology Riess, J. W., Bhattacharya, N., Blenman, K. R., Neal, J. W., Hwang, G., Pultar, P., San-Pedro Salcedo, M., Engleman, E., Lee, P. P., Malik, R., Wakelee, H. A. 2014; 36 (2): 182-186

  Abstract

  Talactoferrin alfa (TLF) is a unique recombinant form of human lactoferrin. The hypothesized mechanism of action involves TLF binding to the intestinal endothelium inducing dendritic cell maturation and cytokine release leading to infiltration of tumor with monocytes and T-lymphocytes and inhibition of tumor growth.Based on promising phase II trial results, this correlative study was undertaken to examine immune mechanism of action of TLF in metastatic non-small cell lung cancer (NSCLC) patients.Talactoferrin was administered orally at 1.5 g bid weeks 1-12 with 2 weeks off on a 14-week cycle. Enrolled patients had a pathologic diagnosis of NSCLC previously treated with at least two lines of systemic treatment. Patients had core biopsy of tumor before initiation of talactoferrin and at week 7 on TLF. Flow cytometry and quantitative immunohistochemistry for immune correlates were performed on the biopsied specimens.Four patients with metastatic NSCLC were enrolled. The trial was halted pre-maturely in light of negative phase III trial results. For the two patients who had repeat on-treatment tumor biopsies, a consistent increase in monocytes as a percentage of total immune cells was observed. Otherwise, no clear trend of increase or decrease was observed in any other immune cell parameters compared to matched patient pre-treatment biopsies.Repeat biopsies for immune correlates by flow cytometry and quantitative immunohistochemistry in NSCLC patients are feasible. In the few patients sampled before trial closure, increased monocytes as a total percentage of the immune cell population within tumor was observed in response to TLF.

  View details for DOI 10.3109/08923973.2013.864671

  View details for PubMedID 24494587

• B Lymphocytes in obesity-related adipose tissue inflammation and insulin resistance CELLULAR AND MOLECULAR LIFE SCIENCES Winer, D. A., Winer, S., Chng, M. H., Shen, L., Engleman, E. G. 2014; 71 (6): 1033-1043

  Abstract

  Obesity-related insulin resistance is a chronic inflammatory condition that often gives rise to type 2 diabetes (T2D). Much evidence supports a role for pro-inflammatory T cells and macrophages in promoting local inflammation in tissues such as visceral adipose tissue (VAT) leading to insulin resistance. More recently, B cells have emerged as an additional critical player in orchestrating these processes. B cells infiltrate VAT and display functional and phenotypic changes in response to diet-induced obesity. B cells contribute to insulin resistance by presenting antigens to T cells, secreting inflammatory cytokines, and producing pathogenic antibodies. B cell manipulation represents a novel approach to the treatment of obesity-related insulin resistance and potentially to the prevention of T2D. This review summarizes the roles of B cells in governing VAT inflammation and the mechanisms by which these cells contribute to altered glucose homeostasis in insulin resistance.

  View details for DOI 10.1007/s00018-013-1486-y

  View details for Web of Science ID 000331653900006

  View details for PubMedID 24127133

  View details for PubMedCentralID PMC3954849

• In Vivo T Cell Activation Induces the Formation of CD209(+) PDL-2(+) Dendritic Cells PLOS ONE Davidson, M. G., Alonso, M. N., Kenkel, J. A., Suhoski, M. M., Gonzalez, J. C., Yuan, R., Engleman, E. G. 2013; 8 (10)

  Abstract

  Two critical functions of dendritic cells (DC) are to activate and functionally polarize T cells. Activated T cells can, in turn, influence DC maturation, although their effect on de novo DC development is poorly understood. Here we report that activation of T cells in mice, with either an anti-CD3 antibody or super antigen, drives the rapid formation of CD209(+)CD11b(+)CD11c(+) MHC II(+) DC from monocytic precursors (Mo-DC). GM-CSF is produced by T cells following activation, but surprisingly, it is not required for the formation of CD209(+) Mo-DC. CD40L, however, is critical for the full induction of Mo-DC following T cell activation. T cell induced CD209(+) Mo-DC are comparable to conventional CD209(-) DC in their ability to stimulate T cell proliferation. However, in contrast to conventional CD209(-) DC, CD209(+) Mo-DC fail to effectively polarize T cells, as indicated by a paucity of T cell cytokine production. The inability of CD209(+) Mo-DC to polarize T cells is partly explained by increased expression of PDL-2, since blockade of this molecule restores some polarizing capacity to the Mo-DC. These findings expand the range of signals capable of driving Mo-DC differentiation in vivo beyond exogenous microbial factors to include endogenous factors produced following T cell activation.

  View details for DOI 10.1371/journal.pone.0076258

  View details for Web of Science ID 000325434500056

  View details for PubMedID 24098455

  View details for PubMedCentralID PMC3788745

• Th17 cells induce Th1-polarizing monocyte-derived dendritic cells. Journal of immunology Davidson, M. G., Alonso, M. N., Yuan, R., Axtell, R. C., Kenkel, J. A., Suhoski, M. M., González, J. C., Steinman, L., Engleman, E. G. 2013; 191 (3): 1175-1187

  Abstract

  In chronically inflamed tissues, such as those affected by autoimmune disease, activated Th cells often colocalize with monocytes. We investigate in this study how murine Th cells influence the phenotype and function of monocytes. The data demonstrate that Th1, Th2, and Th17 subsets promote the differentiation of autologous monocytes into MHC class II(+), CD11b(+), CD11c(+) DC that we call DCTh. Although all Th subsets induce the formation of DCTh, activated Th17 cells uniquely promote the formation of IL-12/IL-23-producing DCTh (DCTh17) that can polarize both naive and Th17 cells to a Th1 phenotype. In the inflamed CNS of mice with Th17-mediated experimental autoimmune encephalomyelitis, Th cells colocalize with DC, as well as monocytes, and the Th cells obtained from these lesions drive the formation of DCTh that are phenotypically indistinguishable from DCTh17 and polarize naive T cells toward a Th1 phenotype. These results suggest that DCTh17 are critical in the interplay of Th17- and Th1-mediated responses and may explain the previous finding that IL-17-secreting Th cells become IFN-γ-secreting Th1 cells in experimental autoimmune encephalomyelitis and other autoimmune disorders.

  View details for DOI 10.4049/jimmunol.1203201

  View details for PubMedID 23794631

  View details for PubMedCentralID PMC3954848

• Uniform Long-Term Graft Survival in a Clincial Trial of the Induction of Tolerance to Kidney Transplants. 13th American Transplant Congress (ATC) Scandling, J., Busque, S., Shori, A., Dejbakhsh-Jones, S., Shizuru, J., Lowsky, R., Benike, C., Engleman, E., Strober, S. WILEY-BLACKWELL. 2013: 200–200

  View details for Web of Science ID 000318240300549

• In vivo T cell activation induces the formation of CD209(+) PDL-2(+) dendritic cells. PloS one Davidson, M. G., Alonso, M. N., Kenkel, J. A., Suhoski, M. M., González, J. C., Yuan, R., Engleman, E. G. 2013; 8 (10)

  Abstract

  Two critical functions of dendritic cells (DC) are to activate and functionally polarize T cells. Activated T cells can, in turn, influence DC maturation, although their effect on de novo DC development is poorly understood. Here we report that activation of T cells in mice, with either an anti-CD3 antibody or super antigen, drives the rapid formation of CD209(+)CD11b(+)CD11c(+) MHC II(+) DC from monocytic precursors (Mo-DC). GM-CSF is produced by T cells following activation, but surprisingly, it is not required for the formation of CD209(+) Mo-DC. CD40L, however, is critical for the full induction of Mo-DC following T cell activation. T cell induced CD209(+) Mo-DC are comparable to conventional CD209(-) DC in their ability to stimulate T cell proliferation. However, in contrast to conventional CD209(-) DC, CD209(+) Mo-DC fail to effectively polarize T cells, as indicated by a paucity of T cell cytokine production. The inability of CD209(+) Mo-DC to polarize T cells is partly explained by increased expression of PDL-2, since blockade of this molecule restores some polarizing capacity to the Mo-DC. These findings expand the range of signals capable of driving Mo-DC differentiation in vivo beyond exogenous microbial factors to include endogenous factors produced following T cell activation.

  View details for DOI 10.1371/journal.pone.0076258

  View details for PubMedID 24098455

• Redirecting cell-type specific cytokine responses with engineered interleukin-4 superkines NATURE CHEMICAL BIOLOGY Junttila, I. S., Creusot, R. J., Moraga, I., Bates, D. L., Wong, M. T., Alonso, M. N., Suhoski, M. M., Lupardus, P., Meier-Schellersheim, M., Engleman, E. G., Utz, P. J., Fathman, C. G., Paul, W. E., Garcia, K. C. 2012; 8 (12): 990-998

  Abstract

  Cytokines dimerize their receptors, with the binding of the 'second chain' triggering signaling. In the interleukin (IL)-4 and IL-13 system, different cell types express varying numbers of alternative second receptor chains (γc or IL-13Rα1), forming functionally distinct type I or type II complexes. We manipulated the affinity and specificity of second chain recruitment by human IL-4. A type I receptor-selective IL-4 'superkine' with 3,700-fold higher affinity for γc was three- to ten-fold more potent than wild-type IL-4. Conversely, a variant with high affinity for IL-13Rα1 more potently activated cells expressing the type II receptor and induced differentiation of dendritic cells from monocytes, implicating the type II receptor in this process. Superkines showed signaling advantages on cells with lower second chain numbers. Comparative transcriptional analysis reveals that the superkines induce largely redundant gene expression profiles. Variable second chain numbers can be exploited to redirect cytokines toward distinct cell subsets and elicit new actions, potentially improving the selectivity of cytokine therapy.

  View details for DOI 10.1038/NCHEMBIO.1096

  View details for Web of Science ID 000311491200014

  View details for PubMedID 23103943

  View details for PubMedCentralID PMC3508151

• Cd14 SNPs regulate the innate immune response MOLECULAR IMMUNOLOGY Liu, H., Hu, Y., Zheng, M., Suhoski, M. M., Engleman, E. G., Dill, D. L., Hudnall, M., Wang, J., Spolski, R., Leonard, W. J., Peltz, G. 2012; 51 (2): 112-127

  Abstract

  CD14 is a monocytic differentiation antigen that regulates innate immune responses to pathogens. Here, we show that murine Cd14 SNPs regulate the length of Cd14 mRNA and CD14 protein translation efficiency, and consequently the basal level of soluble CD14 (sCD14) and type I IFN production by murine macrophages. This has substantial downstream consequences for the innate immune response; the level of expression of at least 40 IFN-responsive murine genes was altered by this mechanism. We also observed that there was substantial variation in the length of human CD14 mRNAs and in their translation efficiency. sCD14 increased cytokine production by human dendritic cells (DCs), and sCD14-primed DCs augmented human CD4T cell proliferation. These findings may provide a mechanism for exploring the complex relationship between CD14 SNPs, serum sCD14 levels, and susceptibility to human infectious and allergic diseases.

  View details for DOI 10.1016/j.molimm.2012.02.112

  View details for Web of Science ID 000304687500002

  View details for PubMedID 22445606

  View details for PubMedCentralID PMC3341513

• Tolerance and Withdrawal of Immunosuppressive Drugs in Patients Given Kidney and Hematopoietic Cell Transplants AMERICAN JOURNAL OF TRANSPLANTATION Scandling, J. D., Busque, S., Dejbakhsh-Jones, S., Benike, C., Sarwal, M., Millan, M. T., Shizuru, J. A., Lowsky, R., Engleman, E. G., Strober, S. 2012; 12 (5): 1133-1145

  Abstract

  Sixteen patients conditioned with total lymphoid irradiation (TLI) and antithymocyte globulin (ATG) were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA-matched donors in a tolerance induction protocol. Blood cell monitoring included changes in chimerism, balance of T-cell subsets and responses to donor alloantigens. Fifteen patients developed multilineage chimerism without graft-versus-host disease (GVHD), and eight with chimerism for at least 6 months were withdrawn from antirejection medications for 1-3 years (mean, 28 months) without subsequent rejection episodes. Four chimeric patients have just completed or are in the midst of drug withdrawal, and four patients were not withdrawn due to return of underlying disease or rejection episodes. Blood cells from all patients showed early high ratios of CD4+CD25+ regulatory T cells and NKT cells versus conventional naive CD4+ T cells, and those off drugs showed specific unresponsiveness to donor alloantigens. In conclusion, TLI and ATG promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and hematopoietic donor cell infusions. All 16 patients had excellent graft function at the last observation point with or without maintenance drugs.

  View details for DOI 10.1111/j.1600-6143.2012.03992.x

  View details for Web of Science ID 000303235100012

  View details for PubMedID 22405058

  View details for PubMedCentralID PMC3338901

• T(H)1, T(H)2, and T(H)17 cells instruct monocytes to differentiate into specialized dendritic cell subsets BLOOD Alonso, M. N., Wong, M. T., Zhang, A. L., Winer, D., Suhoski, M. M., Tolentino, L. L., Gaitan, J., Davidson, M. G., Kung, T. H., Galel, D. M., Nadeau, K. C., Kim, J., Utz, P. J., Soederstroem, K., Engleman, E. G. 2011; 118 (12): 3311-3320

  Abstract

  Monocytes and T helper (T(H)) cells rapidly infiltrate inflamed tissues where monocytes differentiate into inflammatory dendritic cells (DCs) through undefined mechanisms. Our studies indicate that T(H) cells frequently interact with monocytes in inflamed skin and elicit the differentiation of specialized DC subsets characteristic of these lesions. In psoriasis lesions, T(H)1 and T(H)17 cells interact with monocytes and instruct these cells to differentiate into T(H)1- and T(H)17-promoting DCs, respectively. Correspondingly, in acute atopic dermatitis, T(H)2 cells interact with monocytes and elicit the formation of T(H)2-promoting DCs. DC formation requires GM-CSF and cell contact, whereas T(H) subset specific cytokines dictate DC function and the expression of DC subset specific surface molecules. Moreover, the phenotypes of T cell-induced DC subsets are maintained after subsequent stimulation with a panel of TLR agonists, suggesting that T(H)-derived signals outweigh downstream TLR signals in their influence on DC function. These findings indicate that T(H) cells govern the formation and function of specialized DC subsets.

  View details for DOI 10.1182/blood-2011-03-341065

  View details for Web of Science ID 000295120900018

  View details for PubMedID 21813450

  View details for PubMedCentralID PMC3179399

• B cells promote insulin resistance through modulation of T cells and production of pathogenic IgG antibodies NATURE MEDICINE Winer, D. A., Winer, S., Shen, L., Wadia, P. P., Yantha, J., Paltser, G., Tsui, H., Wu, P., Davidson, M. G., Alonso, M. N., Leong, H. X., Glassford, A., Caimol, M., Kenkel, J. A., Tedder, T. F., McLaughlin, T., Miklos, D. B., Dosch, H., Engleman, E. G. 2011; 17 (5): 610-U134

  Abstract

  Chronic inflammation characterized by T cell and macrophage infiltration of visceral adipose tissue (VAT) is a hallmark of obesity-associated insulin resistance and glucose intolerance. Here we show a fundamental pathogenic role for B cells in the development of these metabolic abnormalities. B cells accumulate in VAT in diet-induced obese (DIO) mice, and DIO mice lacking B cells are protected from disease despite weight gain. B cell effects on glucose metabolism are mechanistically linked to the activation of proinflammatory macrophages and T cells and to the production of pathogenic IgG antibodies. Treatment with a B cell-depleting CD20 antibody attenuates disease, whereas transfer of IgG from DIO mice rapidly induces insulin resistance and glucose intolerance. Moreover, insulin resistance in obese humans is associated with a unique profile of IgG autoantibodies. These results establish the importance of B cells and adaptive immunity in insulin resistance and suggest new diagnostic and therapeutic modalities for managing the disease.

  View details for DOI 10.1038/nm.2353

  View details for Web of Science ID 000290250400038

  View details for PubMedID 21499269

  View details for PubMedCentralID PMC3270885

• The role of vanin-1 and oxidative stress-related pathways in distinguishing acute and chronic pediatric ITP BLOOD Zhang, B., Lo, C., Shen, L., Sood, R., Jones, C., Cusmano-Ozog, K., Park-Snyder, S., Wong, W., Jeng, M., Cowan, T., Engleman, E. G., Zehnder, J. L. 2011; 117 (17): 4569-4579

  Abstract

  Pediatric immune thrombocytopenia (ITP) is usually self-limited. However, approximately 20% of children develop chronic ITP, which can be associated with significant morbidity because of long-term immunosuppression and splenectomy in refractory cases. To explore the molecular mechanism of chronic ITP compared with acute ITP, we studied 63 pediatric patients with ITP. Gene expression analysis of whole blood revealed distinct signatures for acute and chronic ITP. Oxidative stress-related pathways were among the most significant chronic ITP-associated pathways. Overexpression of VNN1, an oxidative stress sensor in epithelial cells, was most strongly associated with progression to chronic ITP. Studies of normal persons demonstrated VNN1 expression in a variety of blood cells. Exposure of blood mononuclear cells to oxidative stress inducers elicited dramatic up-regulation of VNN1 and down-regulation of PPARγ, indicating a role for VNN1 as a peripheral blood oxidative stress sensor. Assessment of redox state by tandem mass spectrometry demonstrated statistically significant lower glutathione ratios in patients with ITP versus healthy controls; lower glutathione ratios were also seen in untreated patients with ITP compared with recently treated patients. Our work demonstrates distinct patterns of gene expression in acute and chronic ITP and implicates oxidative stress pathways in the pathogenesis of chronic pediatric ITP.

  View details for DOI 10.1182/blood-2010-09-304931

  View details for PubMedID 21325602

• Successful Tolerance Induction with a Post KidneyTransplantation (Tx) Regimen of Total Lymphoid Irradiation (TLI), Antithymocyte Globulin (ATG) and Donor Purified CD34 Progenitor Cells in HLA-Matched Recipients American Transplant Congress Busque, S., Scandling, J. D., Dejbakhsh-Jones, S., Shizuru, J. A., Lowsky, R., Benike, C., Engleman, E. G., Sarwal, M., Millan, M. T., Shori, A., Hoppe, R. T., Srober, S. WILEY-BLACKWELL. 2011: 79–79

  View details for Web of Science ID 000289318400163

• Changes in T Cell Subsets in 12 Patients Enrolled in a Tolerance Induction Protocol with Combined Kidney and Hematopoietic Cell Transplantation. American Transplant Congress Dejbakhsh-Jones, S., Takahashi, K., Jensen, K. P., Busque, S., Scandling, J. D., Shizuru, J., Lowsky, R., Engleman, E., Strober, S. WILEY-BLACKWELL. 2011: 177–177

  View details for Web of Science ID 000289318400491

• Plasmacytoid dendritic cell dichotomy: identification of IFN-a producing cells as a phenotypically and functionally distinct subset. Journal of immunology Björck, P., Leong, H. X., Engleman, E. G. 2011; 186 (3): 1477-1485

  Abstract

  Plasmacytoid dendritic cells (pDC) produce large amounts of type I IFN in response to invading pathogens, but can also suppress immune responses and promote tolerance. In this study, we show that in mice, these functions are attributable to two distinct pDC subsets, one of which gives rise to the other. CD9(pos)Siglec-H(low) pDC secrete IFN-α when stimulated with TLR agonists, induce CTLs, and promote protective antitumor immunity. By contrast, CD9(neg)Siglec-H(high) pDC secrete negligible amounts of IFN-α, induce Foxp3(+) CD4(+) T cells, and fail to promote antitumor immunity. Although newly formed pDC in the bone marrow are CD9(pos) and are capable of producing IFN-α, after these cells traffic to peripheral tissues, they lose CD9 expression and the ability to produce IFN-α. We propose that newly generated pDC mobilized from the bone marrow, rather than tissue-resident pDC, are the major source of IFN-α in infected hosts.

  View details for DOI 10.4049/jimmunol.1000454

  View details for PubMedID 21172865

  View details for PubMedCentralID PMC3138736

• Plasmacytoid Dendritic Cell Dichotomy: Identification of IFN-alpha Producing Cells as a Phenotypically and Functionally Distinct Subset JOURNAL OF IMMUNOLOGY Bjoerck, P., Leong, H. X., Engleman, E. G. 2011; 186 (3): 1477-1485

  View details for DOI 10.4049/jimmunol.1000454

  View details for Web of Science ID 000286381200026

• Aldehyde Dehydrogenase (ALDH) Activity Segregates Murine Pancreatic Cancer Stem Cells into Distinct Phenotypic Subtypes Which Dictate Histopathologic Tumor Grade. 100th Annual Meeting United States-and-Canadian-Academy-of-Pathology WINER, D. A., Seeley, S. E., Tseng, W. W., Zahn, J. M., ALONSO, M. N., Winer, S., Leong, H., Kvezereli, M., Ji, H., Lowy, A. M., Engleman, E. G. NATURE PUBLISHING GROUP. 2011: 447A–447A

  View details for Web of Science ID 000287282302350

• Regulation of human Th9 differentiation by type I interferons and IL-21 IMMUNOLOGY AND CELL BIOLOGY Wong, M. T., Ye, J. J., Alonso, M. N., Landrigan, A., Cheung, R. K., Engleman, E., Utz, P. J. 2010; 88 (6): 624-631

  Abstract

  Interleukin (IL)-9-producing CD4(+) T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-beta and IL-4 directed human naive CD4(+) T cells to produce IL-9. Whereas IL-4 suppressed TGF-beta-induced Foxp3 expression, TGF-beta failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-3. Addition of IL-1 beta, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-gamma and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4(+) T cells.

  View details for DOI 10.1038/icb.2010.53

  View details for Web of Science ID 000280830700007

  View details for PubMedID 20421880

  View details for PubMedCentralID PMC3090036

• Natural killer cells trigger osteoclastogenesis and bone destruction in arthritis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Soederstroem, K., Stein, E., Colmenero, P., Purath, U., Mueller-Ladner, U., de Matos, C. T., Tarner, I. H., Robinson, W. H., Engleman, E. G. 2010; 107 (29): 13028-13033

  Abstract

  Osteoclasts are bone-eroding cells that develop from monocytic precursor cells in the presence of receptor activator of NF-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Osteoclasts are essential for physiological bone remodeling, but localized excessive osteoclast activity is responsible for the periarticular bone destruction that characteristically occurs in patients with rheumatoid arthritis (RA). The origin of osteoclasts at sites of bone erosion in RA is unknown. Natural killer (NK) cells, as well as monocytes, are abundant in the inflamed joints of patients with RA. We show here that such NK cells express both RANKL and M-CSF and are frequently associated with CD14(+) monocytes in the RA synovium. Moreover, when synovial NK cells are cocultured with monocytes in vitro, they trigger their differentiation into osteoclasts, a process dependent on RANKL and M-CSF. As in RA, NK cells in the joints of mice with collagen-induced arthritis (CIA) express RANKL. Depletion of NK cells from mice before the induction of CIA reduces the severity of subsequent arthritis and almost completely prevents bone erosion. These results suggest that NK cells may play an important role in the destruction of bone associated with inflammatory arthritis.

  View details for DOI 10.1073/pnas.1000546107

  View details for PubMedID 20615964

• Development of an Orthotopic Model of Invasive Pancreatic Cancer in an Immunocompetent Murine Host CLINICAL CANCER RESEARCH Tseng, W. W., Winer, D., Kenkel, J. A., Choi, O., Shain, A. H., Pollack, J. R., French, R., Lowy, A. M., Engleman, E. G. 2010; 16 (14): 3684-3695

  Abstract

  The most common preclinical models of pancreatic adenocarcinoma utilize human cells or tissues that are xenografted into immunodeficient hosts. Several immunocompetent, genetically engineered mouse models of pancreatic cancer exist; however, tumor latency and disease progression in these models are highly variable. We sought to develop an immunocompetent, orthotopic mouse model of pancreatic cancer with rapid and predictable growth kinetics.Cell lines with epithelial morphology were derived from liver metastases obtained from Kras(G12D/+);LSL-Trp53(R172H/+);Pdx-1-Cre mice. Tumor cells were implanted in the pancreas of immunocompetent, histocompatible B6/129 mice, and the mice were monitored for disease progression. Relevant tissues were harvested for histologic, genomic, and immunophenotypic analysis.All mice developed pancreatic tumors by two weeks. Invasive disease and liver metastases were noted by six to eight weeks. Histologic examination of tumors showed cytokeratin-19-positive adenocarcinoma with regions of desmoplasia. Genomic analysis revealed broad chromosomal changes along with focal gains and losses. Pancreatic tumors were infiltrated with dendritic cells, myeloid-derived suppressor cells, macrophages, and T lymphocytes. Survival was decreased in RAG(-/-) mice, which are deficient in T cells, suggesting that an adaptive immune response alters the course of disease in wild-type mice.We have developed a rapid, predictable orthotopic model of pancreatic adenocarcinoma in immunocompetent mice that mimics human pancreatic cancer with regard to genetic mutations, histologic appearance, and pattern of disease progression. This model highlights both the complexity and relevance of the immune response to invasive pancreatic cancer and may be useful for the preclinical evaluation of new therapeutic agents.

  View details for DOI 10.1158/1078-0432.CCR-09-2384

  View details for Web of Science ID 000279903100017

  View details for PubMedID 20534740

  View details for PubMedCentralID PMC3085509

• Ineffective Vaccination against Solid Tumors Can Be Enhanced by Hematopoietic Cell Transplantation JOURNAL OF IMMUNOLOGY Filatenkov, A., Mueller, A. M., Tseng, W. W., Dejbakhsh-Jones, S., Winer, D., Luong, R., Shizuru, J. A., Engleman, E. G., Strober, S. 2009; 183 (11): 7196-7203

  Abstract

  Vaccination with tumor Ags has not been an effective treatment for solid tumors. The goal of the current study was to determine whether a combination of vaccination and hematopoietic cell transplantation (HCT) can effectively treat primary, disseminated, or metastatic CT26 and MC38 murine colon tumors. Vaccination of tumor-bearing mice with irradiated tumor cells and CpG adjuvant failed to alter progressive tumor growth. However, mice bearing primary, disseminated lung, or metastatic liver tumors were uniformly cured after administration of total body irradiation, followed by the transplantation of hematopoietic progenitor cells and T cells from syngeneic, but not allogeneic vaccinated donors. Requirements for effective treatment of tumors included irradiation of hosts, vaccination of donors with both tumor cells and CpG, transfer of both CD4(+) and CD8(+) T cells along with progenitor cells, and ability of donor cells to produce IFN-gamma. Irradiation markedly increased the infiltration of donor T cells into the tumors, and the combined irradiation and HCT altered the balance of tumor-infiltrating cells to favor CD8(+) effector memory T cells as compared with CD4(+)CD25(+)FoxP3(+) T regulatory cells. The combination of vaccination and autologous hematopoietic cell transplantation was also effective in treating tumors. In conclusion, these findings show that otherwise ineffective vaccination to solid nonhematologic tumors can be dramatically enhanced by HCT.

  View details for DOI 10.4049/jimmunol.0900159

  View details for Web of Science ID 000272478800039

  View details for PubMedID 19890041

  View details for PubMedCentralID PMC2783632

• Increased VNN1/PPARG Gene Expression Ratio Is Correlated with Developing Chronic ITP and Oxidative Stress Exposure to PBMC in Vitro 51st Annual Meeting and Exposition of the American-Society-of-Hematology Zhang, B., Shen, L., Jeng, M., Jones, C., Wong, W., Engleman, E. E., Zehnder, J. L. AMER SOC HEMATOLOGY. 2009: 368–68

  View details for Web of Science ID 000272725801073

• Obesity predisposes to Th17 bias EUROPEAN JOURNAL OF IMMUNOLOGY Winer, S., Paltser, G., Chan, Y., Tsui, H., Engleman, E., Winer, D., Dosch, H. 2009; 39 (9): 2629-2635

  Abstract

  Obesity is associated with numerous inflammatory conditions including atherosclerosis, autoimmune disease and cancer. Although the precise mechanisms are unknown, obesity-associated rises in TNF-alpha, IL-6 and TGF-beta are believed to contribute. Here we demonstrate that obesity selectively promotes an expansion of the Th17 T-cell sublineage, a subset with prominent pro-inflammatory roles. T-cells from diet-induced obese mice expand Th17 cell pools and produce progressively more IL-17 than lean littermates in an IL-6-dependent process. The increased Th17 bias was associated with more pronounced autoimmune disease as confirmed in two disease models, EAE and trinitrobenzene sulfonic acid colitis. In both, diet-induced obese mice developed more severe early disease and histopathology with increased IL-17(+) T-cell pools in target tissues. The well-described association of obesity with inflammatory and autoimmune disease is mechanistically linked to a Th17 bias.

  View details for DOI 10.1002/eji.200838893

  View details for Web of Science ID 000269786000034

  View details for PubMedID 19662632

• Chemoselective Ligation in the Functionalization of Polysaccharide-Based Particles JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Beaudette, T. T., Cohen, J. A., Bachelder, E. M., Broaders, K. E., Cohen, J. L., Engleman, E. G., Frechet, J. M. 2009; 131 (30): 10360-?

  Abstract

  Despite the promise of precisely targeted or otherwise functionalized polymeric particulate drug delivery vehicles, typical biocompatible particles are generally not amenable to facile and selective surface modification. Herein, we report the development of a simple, mild, and chemoselective strategy for the conjugation of biologically active molecules to the surface of dextran-based microparticles. Alkoxyamine-bearing reagents were used to form stable oxime conjugates with latent aldehyde functionality present in reducing carbohydrate chain ends. We demonstrate the functionalization of dextran-based microparticles with a fluorophore as well as a cell-penetrating peptide sequence, which facilitated the delivery of cargo to nonphagocytic cells leading to a 60-fold increase in the expression of a reporter gene when plasmid DNA-loaded particles were used.

  View details for DOI 10.1021/ja903984s

  View details for Web of Science ID 000268644400013

  View details for PubMedID 19591467

• Normalization of obesity-associated insulin resistance through immunotherapy NATURE MEDICINE Winer, S., Chan, Y., Paltser, G., Truong, D., Tsui, H., Bahrami, J., Dorfman, R., Wang, Y., Zielenski, J., Mastronardi, F., Maezawa, Y., Drucker, D. J., Engleman, E., Winer, D., Dosch, H. 2009; 15 (8): 921-U126

  Abstract

  Obesity and its associated metabolic syndromes represent a growing global challenge, yet mechanistic understanding of this pathology and current therapeutics are unsatisfactory. We discovered that CD4(+) T lymphocytes, resident in visceral adipose tissue (VAT), control insulin resistance in mice with diet-induced obesity (DIO). Analyses of human tissue suggest that a similar process may also occur in humans. DIO VAT-associated T cells show severely biased T cell receptor V(alpha) repertoires, suggesting antigen-specific expansion. CD4(+) T lymphocyte control of glucose homeostasis is compromised in DIO progression, when VAT accumulates pathogenic interferon-gamma (IFN-gamma)-secreting T helper type 1 (T(H)1) cells, overwhelming static numbers of T(H)2 (CD4(+)GATA-binding protein-3 (GATA-3)(+)) and regulatory forkhead box P3 (Foxp3)(+) T cells. CD4(+) (but not CD8(+)) T cell transfer into lymphocyte-free Rag1-null DIO mice reversed weight gain and insulin resistance, predominantly through T(H)2 cells. In obese WT and ob/ob (leptin-deficient) mice, brief treatment with CD3-specific antibody or its F(ab')(2) fragment, reduces the predominance of T(H)1 cells over Foxp3(+) cells, reversing insulin resistance for months, despite continuation of a high-fat diet. Our data suggest that the progression of obesity-associated metabolic abnormalities is under the pathophysiological control of CD4(+) T cells. The eventual failure of this control, with expanding adiposity and pathogenic VAT T cells, can successfully be reversed by immunotherapy.

  View details for DOI 10.1038/nm.2001

  View details for Web of Science ID 000268770400037

  View details for PubMedID 19633657

  View details for PubMedCentralID PMC3063199

• In Vivo Studies on the Effect of Co-Encapsulation of CpG DNA and Antigen in Acid-Degradable Microparticle Vaccines MOLECULAR PHARMACEUTICS Beaudette, T. T., Bachelder, E. M., Cohen, J. A., Obermeyer, A. C., Broaders, K. E., Frechet, J. M., Kang, E., Mende, I., Tseng, W. W., Davidson, M. G., Engleman, E. G. 2009; 6 (4): 1160-1169

  Abstract

  Protein-based vaccines have been explored as a safer alternative to traditional weakened or killed whole organism based vaccination strategies and have been investigated for their ability to activate the immune system against certain cancers. For optimal stimulation of T lymphocytes, protein-based vaccines should deliver protein antigens to antigen presenting cells in the context of appropriate immunostimulatory signals, thus mimicking actual pathogens. In this report, we describe the synthesis, characterization, and biological evaluation of immunostimulatory acid-degradable microparticles, which are suitable delivery vehicles for use in protein-based vaccines and cancer immunotherapy. Using a 3' conjugation strategy, we optimized the attachment of immunostimulatory CpG DNA to our vaccine carriers and demonstrated that under acidic conditions similar to those found in endosomal compartments, these new particles were capable of simultaneously releasing a model protein antigen and a CpG DNA adjuvant. We found in an in vivo cytotoxicity assay that the co-encapsulation of ovalbumin, a model antigen, and immunostimulatory agent in the same particle led to superior cytotoxic T lymphocyte activity compared to particles coadministered with adjuvant in an unbound form. In addition, we investigated the ability of these acid-degradable particles to induce protective immunity in the MO5 murine melanoma model and found that they were effective until tumor escape, which appeared to result from a loss of antigen expression by the cancer cells due to in vivo selection pressure.

  View details for DOI 10.1021/mp900038e

  View details for Web of Science ID 000268456300014

  View details for PubMedID 19415922

• T-Cell Activation by Antigen-Loaded pH-Sensitive Hydrogel Particles in Vivo: The Effect of Particle Size BIOCONJUGATE CHEMISTRY Cohen, J. A., Beaudette, T. T., Tseng, W. W., Bachelder, E. M., Mende, I., Engleman, E. G., Frechet, J. M. 2009; 20 (1): 111-119

  Abstract

  Polymeric carriers designed to encapsulate protein antigens have great potential for improving the efficacy of vaccines and immunotherapeutics for diseases such as cancer. We recently developed a carrier system based on polyacrylamide hydrogel microparticles cross-linked with acid-labile moieties. After being phagocytosed by antigen-presenting cells, the protein encapsulated within the carrier is released and processed for subsequent presentation of antigenic epitopes. To understand the impact of particle size on the activation of T-cells following uptake by antigen-presenting cells, particles with mean diameters of 3.5 microm and 35 nm encapsulating a model protein antigen were synthesized by emulsion and microemulsion based polymerization techniques, respectively. In vivo tests demonstrated that both sizes of particles were effective at stimulating the proliferation of T-cells and were capable of generating an antigen-specific cytotoxic T-cell response when coadministered with immunostimulatory DNA. Contrary to previous reports in the literature, our results suggest that there is no significant difference in the magnitude of T-cell activation for the two sizes of particles used in these experiments. This disparity in findings may be related to fundamental differences in material properties of the carriers used in these studies, such as the hydrophilicity of the polyacrylamide particles described here versus the hydrophobic nature of carriers investigated by other groups.

  View details for DOI 10.1021/bc800338n

  View details for Web of Science ID 000262659700015

  View details for PubMedID 19102625

• Chimerism and Tolerance after Combined Human Kidney and Hematopoietic Cell Transplantation Using Conditioning with Total Lymphoid Irradiation and Anti-Thymocyte Globulin 50th Annual Meeting of the American-Society-of-Hematology/ASH/ASCO Joint Symposium Lowsky, R., Shizuru, J., Busque, S., Scandling, J., Sarwal, M., Jones, S., Benike, C., Hoppe, R., Engleman, E., Strober, S. AMER SOC HEMATOLOGY. 2008: 758–58

  View details for Web of Science ID 000262104702507

• Plasmacytoid dendritic cells take up opsonized antigen leading to CD4(+) and CD8(+) T cell activation in vivo JOURNAL OF IMMUNOLOGY Bjorck, P., Beilhack, A., Herman, E. I., Negrin, R. S., Engleman, E. G. 2008; 181 (6): 3811-3817

  Abstract

  Plasmacytoid dendritic cells (pDC) are the body's main source of IFN-alpha, but, unlike classical myeloid DC (myDC), they lack phagocytic activity and are generally perceived as playing only a minor role in Ag processing and presentation. We show that murine pDC, as well as myDC, express Fcgamma receptors (CD16/CD32) and can use these receptors to acquire Ag from immune complexes (IC), resulting in the induction of robust Ag-specific CD4(+) and CD8(+) T cell responses. IC-loaded pDC stimulate CD4(+) T cells to proliferate and secrete a mixture of IL-4 and IFN-gamma, and they induce CD8(+) T cells to secrete IL-10 as well as IFN-gamma. In contrast, IC-loaded myDC induce both CD4(+) and CD8(+) T cells to secrete mainly IFN-gamma. These results indicate that pDC can shape an immune response by acquiring and processing opsonized Ag, leading to a predominantly Th2 response.

  View details for PubMedID 18768834

• Tolerance and chimerism after renal and hematopoietic-cell transplantation. New England journal of medicine Scandling, J. D., Busque, S., Dejbakhsh-Jones, S., Benike, C., Millan, M. T., Shizuru, J. A., Hoppe, R. T., Lowsky, R., Engleman, E. G., Strober, S. 2008; 358 (4): 362-368

  Abstract

  We describe a recipient of combined kidney and hematopoietic-cell transplants from an HLA-matched donor. A post-transplantation conditioning regimen of total lymphoid irradiation and antithymocyte globulin allowed engraftment of the donor's hematopoietic cells. The patient had persistent mixed chimerism, and the function of the kidney allograft has been normal for more than 28 months since discontinuation of all immunosuppressive drugs. Adverse events requiring hospitalization were limited to a 2-day episode of fever with neutropenia. The patient has had neither rejection episodes nor clinical manifestations of graft-versus-host disease.

  View details for DOI 10.1056/NEJMoa074191

  View details for PubMedID 18216356

• Brief report: Tolerance and chimerism after renal and hematopoietic-cell transplantation NEW ENGLAND JOURNAL OF MEDICINE Scandling, J. D., Busque, S., Dejbakhsh-Jones, S., Benike, C., Millan, M. T., Shizuru, J. A., Hoppe, R. T., Lowsky, R., Engleman, E. G., Strober, S. 2008; 358 (4): 362-368

  Abstract

  We describe a recipient of combined kidney and hematopoietic-cell transplants from an HLA-matched donor. A post-transplantation conditioning regimen of total lymphoid irradiation and antithymocyte globulin allowed engraftment of the donor's hematopoietic cells. The patient had persistent mixed chimerism, and the function of the kidney allograft has been normal for more than 28 months since discontinuation of all immunosuppressive drugs. Adverse events requiring hospitalization were limited to a 2-day episode of fever with neutropenia. The patient has had neither rejection episodes nor clinical manifestations of graft-versus-host disease.

  View details for Web of Science ID 000252507900006

• Qa-1(b)-dependent modulation of dendritic cell and NK cell cross-talk in vivo. Journal of immunology Colmenero, P., Zhang, A. L., Qian, T., Lu, L., Cantor, H., Söderström, K., Engleman, E. G. 2007; 179 (7): 4608-4615

  Abstract

  Dendritic cells (DC) trigger activation and IFN-gamma release by NK cells in lymphoid tissues, a process important for the polarization of Th1 responses. Little is known about the molecular signals that regulate DC-induced NK cell IFN-gamma synthesis. In this study, we analyzed whether the interaction between Qa-1(b) expressed on DC and its CD94/NKG2A receptor on NK cells affects this process. Activation of DC using CpG-oligodeoxynucleotides in Qa-1(b)-deficient mice, or transfer of CpG-oligodeoxynucleotide-activated Qa-1(b)-deficient DC into wild-type mice, resulted in dramatically increased IFN-gamma production by NK cells, as compared with that induced by Qa-1(b)-expressing DC. Masking the CD94/NKG2A inhibitory receptor on NK cells in wild-type mice similarly enhanced the IFN-gamma response of these cells to Qa-1(b)-expressing DC. Furthermore, NK cells from CD94/NKG2A-deficient mice displayed higher IFN-gamma production upon DC stimulation. These results demonstrate that Qa-1(b) is critically involved in regulating IFN-gamma synthesis by NK cells in vivo through its interaction with CD94/NKG2A inhibitory receptors. This receptor-ligand interaction may be essential to prevent unabated cytokine production by NK cells during an inflammatory response.

  View details for PubMedID 17878358

• Natural killer cells trigger differentiation of monocytes into dendritic cells BLOOD Zhang, A. L., Colmenero, P., Purath, U., de Matos, C. T., Hueber, W., Klareskog, L., Tarner, I. H., Engleman, E. G., Soderstrom, K. 2007; 110 (7): 2484-2493

  Abstract

  Circulating monocytes can differentiate into dendritic cells (moDCs), which are potent inducers of adaptive immune responses. Previous reports show that granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 induce monocyte differentiation into moDCs in vitro, but little is known about the physiological requirements that initiate moDC differentiation in vivo. Here we show that a unique natural killer (NK) cell subset (CD3(-)CD56(bright)) that accumulates in lymph nodes and chronically inflamed tissues triggers CD14(+) monocytes to differentiate into potent T-helper-1 (T(H)1) promoting DC. This process requires direct contact of monocytes with NK cells and is mediated by GM-CSF and CD154 derived from NK cells. It is noteworthy that synovial fluid (SF) from patients with rheumatoid arthritis (RA) and psoriatic arthritis (PsA), but not osteoarthritis (OA), induces monocytes to differentiate into DC. However, this process occurs only in the presence of NK cells. We propose that NK cells play a role in the maintenance of T(H)1-mediated inflammatory diseases such as RA by providing a local milieu for monocytes to differentiate into DC.

  View details for DOI 10.1182/blood-2007-02-076364

  View details for Web of Science ID 000249800900046

  View details for PubMedID 17626840

  View details for PubMedCentralID PMC1988958

• CD101 surface expression discriminates potency among murine FoxP3(+) regulatory T cells JOURNAL OF IMMUNOLOGY Fernandez, I., Zeiser, R., Karsunky, H., Kambham, N., Beilhack, A., Soderstrom, K., Negrin, R. S., Engleman, E. 2007; 179 (5): 2808-2814

  Abstract

  CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25-30% of CD4+CD25+ Treg and approximately 20% of conventional memory T cells. CD101(high) Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101(low) Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101(high) Treg, compared with CD101(low) Treg. Moreover, treatment with CD101(high) Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101(high) Treg all of the in vivo suppressor activity was contained within the CD62L(high) subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.

  View details for Web of Science ID 000248991800020

  View details for PubMedID 17709494

• Breaking self-tolerance to tumor-associated antigens by in vivo manipulation of dendritic cells. Methods in molecular biology (Clifton, N.J.) Mende, I., Engleman, E. G. 2007; 380: 457-468

  Abstract

  Dendritic cells (DC) are extremely potent antigen-presenting cells, which can prime both naive CD4+ and CD8+ T lymphocytes. In their immature state, DC continuously sample and process antigens from the surrounding environment, but only mature DC express sufficient levels of costimulatory molecules to activate naive T cells. DC present in tumors are functionally immature owing to the immunosuppressive actions of tumor-derived factors and regulatory T cells, and such immature DC promote immune tolerance to the tumor. Recent studies from animal models suggest that Toll-like receptor (TLR) agonists such as CpG can reverse the tolerogenic state of tumoral DC. Strategies that allow DC to gain access to both tumor antigens and TLR agonists, in situ, can overcome tumor tolerance leading to the induction of potent systemic antitumor immunity.

  View details for PubMedID 17876112

• Orthotopic mouse model of colorectal cancer. Journal of visualized experiments : JoVE Tseng, W., Leong, X., Engleman, E. 2007: 484-?

  Abstract

  The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

  View details for DOI 10.3791/484

  View details for PubMedID 18989400

• Incorporation of CpG oligonucleotide ligand into protein-loaded particle vaccines promotes antigen-specific CD8 T-cell immunity BIOCONJUGATE CHEMISTRY Standley, S. M., Mende, I., Goh, S. L., Kwon, Y. J., Beaudette, T. T., Engleman, E. G., Frechet, J. M. 2007; 18 (1): 77-83

  Abstract

  The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as important in order to generate more potent immune responses. This article presents the synthesis and biological evaluation of carrier particles that both deliver a protein payload to APCs and display receptor ligands for the enhancement of APC immunostimulation. Particles displaying CpG oligonucleotide ligands for Toll-like receptor 9 were synthesized. The addition of CpG DNA to the particles led to a 45-fold increase in the secretion of interleukin-12, a cytokine that aids in T-cell activation, and a significant increase in the expression of costimulatory molecules by APCs. Moreover, vaccination with particles containing both ovalbumin (OVA) and CpG DNA induced a superior OVA-specific CD8 T-cell response in vivo, as measured by increased OVA-specific CD8 T-cell proliferation, secretion of the proinflammatory cytokine IFN-gamma, and the induction of OVA-specific cytotoxicity.

  View details for DOI 10.1021/bc060165i

  View details for Web of Science ID 000243503400011

  View details for PubMedID 17226959

• Systemic antitumor effect of intratumoral injection of dendritic cells in combination with local photodynamic therapy CLINICAL CANCER RESEARCH Saji, H., Song, W. R., Furumoto, K., Kato, H., Engleman, E. G. 2006; 12 (8): 2568-2574

  Abstract

  Photodynamic therapy (PDT), which is used clinically for the palliative treatment of cancer, induces local tumor cell death but has no effect on tumors in untreated sites. The purpose of this study was to determine if local PDT followed by intratumoral injection of naïve dendritic cells (IT-DC) induces systemic antitumor immunity that can inhibit the growth of untreated as well as PDT + IT-DC-treated tumors.BALB/c or C57Bl/6 mice were injected s.c. with CT26 colorectal carcinoma cells and B16 melanoma cells, respectively, and following 10 to 12 days of tumor growth, the tumors were treated with PDT alone or PDT followed by IT-DC or IT-PBS. In other studies, tumors were established simultaneously in both lower flanks or in one flank and in the lungs, but only one flank was treated.Whereas neither PDT nor IT-DC alone was effective, PDT + IT-DC eradicated both CT26 and B16 tumors in a significant proportion of animals and prolonged the survival of mice of which the tumors were not cured. The spleens of mice treated with PDT + IT-DC contained tumor-specific cytotoxic and IFN-gamma-secreting T cells whereas the spleens of control groups did not. Moreover, adoptive transfer of splenocytes from successfully treated CT26 tumor-free mice protected naïve animals from a subsequent challenge with CT26, and this was mediated mainly by CD8 T cells. Most importantly, PDT plus IT-DC administered to one tumor site led to tumor regression at distant sites, including multiple lung metastases.PDT + IT-DC induces potent systemic antitumor immunity in mice and should be evaluated in the treatment of human cancer.

  View details for DOI 10.1158/1078-0432.CCR-05-1986

  View details for Web of Science ID 000237173300026

  View details for PubMedID 16638867

• Flk2(+) myeloid progenitors are the main source of Langerhans cells BLOOD Mende, I., Karsunky, H., Weissman, I. L., Engleman, E. G., Merad, M. 2006; 107 (4): 1383-1390

  Abstract

  Langerhans cells (LCs) are antigen-presenting cells (APCs) residing in the epidermis that play a major role in skin immunity. Our earlier studies showed that when skin is inflamed LCs are replaced by bone marrow-derived progenitor cells, while during steady-state conditions LCs are able to self-renew in the skin. Identification of the LC progenitors in bone marrow would represent a critical step toward identifying the factors that regulate LC generation as well as their trafficking to the skin. To determine LC lineage origin, we reconstituted lethally irradiated CD45.2 mice with rigorously purified lymphoid and myeloid progenitors from CD45.1 congenic mice. Twenty-four hours later, we exposed the mice to UV light to deplete resident LCs and induce their replacement by progenitors. Reconstitution with common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs), or early thymic progenitors led to LC generation within 2 to 3 weeks. CMPs were at least 20 times more efficient at generating LCs than CLPs. LCs from both lineages were derived almost entirely from fetal liver kinase-2+ (Flk-2+) progenitors, displayed typical dendritic-cell (DC) morphology, and showed long-term persistence in the skin. These results indicate that LCs are derived mainly from myeloid progenitors and are dependent on Flt3-ligand for their development.

  View details for DOI 10.1182/blood-2005-05-1878

  View details for Web of Science ID 000235296100026

  View details for PubMedID 16263793

• In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self antigens and induces potent antitumor immunity JOURNAL OF IMMUNOLOGY Okano, F., Miriam, M., Furumoto, K., Engleman, E. G. 2005; 174 (5): 2645-2652

  Abstract

  Most tumor-associated Ags are self proteins that fail to elicit a T cell response as a consequence of immune tolerance. Dendritic cells (DCs) generated ex vivo have been used to break tolerance against such self Ags; however, in vitro manipulation of DCs is cumbersome and difficult to control, resulting in vaccines of variable potency. To address this problem we developed a method for loading and activating DCs, in situ, by first directing sufficient numbers of DCs to peripheral tissues using Flt3 ligand and then delivering a tumor-associated Ag and oligonucleotide containing unmethylated CG motifs to these tissues. In this study, we show in three different tumor models that this method can overcome tolerance and induce effective antitumor immunity. Vaccination resulted in the generation of CD8(+) T and NK cell effectors that mediated durable tumor responses without attacking normal tissues. These findings demonstrate that unmodified tumor-associated self Ags can be targeted to DCs in vivo to induce potent systemic antitumor immunity.

  View details for Web of Science ID 000227172100027

  View details for PubMedID 15728471

• Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors EXPERIMENTAL HEMATOLOGY Karsunky, H., Merad, M., Mende, I., Manz, M. G., Engleman, E. G., Weissman, I. L. 2005; 33 (2): 173-181

  Abstract

  The aim of this study was to determine the lineage origin of interferon-alpha-producing cells (IPCs), also called plasmacytoid dendritic cells, in mice by evaluating the ability of common lymphoid (CLP) and myeloid (CMP) progenitors to give rise to IPCs.Sublethally irradiated C57Bl/6 mice were intravenously transplanted with rigorously purified lymphoid and myeloid progenitors from a congenic mouse strain. At various time points posttransplantation mice were analyzed for donor-derived cells by flow cytometry. The developmental potential of all progenitor populations was also tested in in vitro cultures. In addition, in vitro and in vivo derived IPCs were functionally assessed for their interferon-alpha production after virus challenge.Transplantation of 1 x 10(4) common myeloid progenitors, 1 x 10(4) common lymphoid progenitors or 2.5 x 10(4) granulocyte/macrophage progenitors all led to the generation of IPCs within 2 to 3 weeks. In general, IPC reconstitution in spleen and liver by CMPs was more efficient than by CLP. Adding Flt3L alone to in vitro cultures was sufficient to support the development of IPCs from myeloid progenitors whereas CLPs required additional survival factors provided either by stroma cells or by introduction of transgenic Bcl-2. Both myeloid- and lymphoid-derived IPC were indistinguishable by function, gene expression, and morphology.Surprisingly, our results clearly show that murine IPCs differentiate from both lineages but are mainly of myeloid origin. These results extend to IPCs the observation made originally in classical dendritic cells that cellular expression of so called lineage markers does not correlate with lineal origin.

  View details for DOI 10.1016/j.exphem.2004.10.010

  View details for Web of Science ID 000227147000007

  View details for PubMedID 15676211

• Breaking tolerance to tumors with dendritic cell-based immunotherapy 7th NIH Symposium on Therapeutic Oligonucleotides Mende, I., Engleman, E. G. NEW YORK ACAD SCIENCES. 2005: 96–104

  Abstract

  Dendritic cells (DC) are the most potent antigen-presenting cells known, capable of priming both naïve CD4 and CD8 T lymphocytes. In their immature state DC are especially efficient at ingesting dying cells and processing their antigens while in their mature state DC express high levels of both MHC class I and II antigens as well as a variety of costimulatory molecules needed to induce a primary T cell response. In addition to stimulating adaptive T cell responses, DC can stimulate NK cells and B cells. Their dual role in innate and adaptive immunity led us to explore their potential utility in tumor immunotherapy. The results of clinical trials in which autologous DC loaded with tumor antigen are administered to tumor-bearing patients have been promising, but overall immunologic potency and clinical efficacy have been unsatisfactory. It has become clear that more potent and more efficient DC based immunotherapies are needed, preferably based on in vivo targeting of DC rather than in vitro manipulation followed by systemic administration of the cells. Recent studies in animal models suggest that DC can be loaded with antigen and activated in vivo in a manner that results in potent antitumor immunity. Two approaches to DC targeting are described in this review, both of which have been shown to overcome immune tolerance to tumors and induce dramatic tumor regression.

  View details for Web of Science ID 000236466000009

  View details for PubMedID 16394129

• Using signaling pathways to overcome immune tolerance to tumors. Science's STKE : signal transduction knowledge environment Engleman, E. G., Brody, J., Soares, L. 2004; 2004 (241): pe28-?

  Abstract

  The ability of tumors to evade the immune system is thought to result from the inability of T lymphocytes to recognize and respond to tumor antigens. This lack of T cell response may depend on a failure of dendritic cells to present antigen in the proper context so that T cells become tolerant to tumor antigens rather than primed to undergo an immune response. The inability of tumor-associated dendritic cells to effectively present antigen may in turn depend on inhibitory factors in the tumor milieu. Recent experiments suggest that the administration of toll-like receptor ligands stimulate dendritic cell activation and maturation and may thus help overcome T cell tolerance to tumor antigens. Whether or not such an approach is clinically feasible remains to be seen.

  View details for PubMedID 15252217

• Depletion of host Langerhans cells before transplantation of donor alloreactive T cells prevents skin graft-versus-host disease NATURE MEDICINE Merad, M., Hoffmann, P., Ranheim, E., Slaymaker, S., Manz, M. G., Lira, S. A., Charo, I., Cook, D. N., Weissman, I. L., Strober, S., Engleman, E. G. 2004; 10 (5): 510-517

  Abstract

  Skin is the most commonly affected organ in graft-versus-host disease (GVHD). To explore the role of Langerhans cells in GVHD, the principal dendritic cells of the skin, we studied the fate of these cells in mice transplanted with allogeneic bone marrow. In contrast to other dendritic cells, host Langerhans cells were replaced by donor Langerhans cells only when donor T cells were administered along with bone marrow, and the extent of Langerhans cell chimerism correlated with the dose of donor T cells injected. Donor T cells depleted host Langerhans cells through a Fas-dependent pathway and induced the production in skin of CCL20, which was required for the recruitment of donor Langerhans cells. Administration of donor T cells to bone marrow-chimeric mice with persistent host Langerhans cells, but not to mice whose Langerhans cells had been replaced, resulted in marked skin GVHD. These findings indicate a crucial role for donor T cells in host Langerhans cell replacement, and show that host dendritic cells can persist in nonlymphoid tissue for the duration of an animal's life and can trigger GVHD despite complete blood chimerism.

  View details for DOI 10.1038/nm1038

  View details for Web of Science ID 000221242400029

  View details for PubMedID 15098028

• DC-based cancer vaccines: lessons from clinical trials CYTOTHERAPY Brody, J. D., Engleman, E. G. 2004; 6 (2): 122-127

  View details for DOI 10.1080/14653240410005267

  View details for Web of Science ID 000221162400006

  View details for PubMedID 15203988

• Induction of potent antitumor immunity by in situ targeting of intraturnoral DCs JOURNAL OF CLINICAL INVESTIGATION Furumoto, K., Soares, L., Engleman, E. G., Merad, M. 2004; 113 (5): 774-783

  Abstract

  Recent reports of tumor regression following delivery of autologous tumor antigen-pulsed DCs suggest that defective antigen presentation may play a key role in tumor escape. Here we show in two different murine tumor models, CT26 (colon adenocarcinoma) and B16 (melanoma), that the number and activation state of intratumoral DCs are critical factors in the host response to tumors. We used CCL20/macrophage inflammatory protein-3alpha (MIP-3alpha) chemokine to increase the number of tumoral DCs and intratumoral injections of CG-rich motifs (CpGs) to activate such cells. Expression of CCL20 in the tumor site attracted large numbers of circulating DCs into the tumor mass and, in the case of CT26 tumors, led to complete tumor regression. Intratumoral CpG injections, in addition to CCL20, were required to induce therapeutic immunity against B16 tumors. In this model CpG overcame tumor-mediated inhibition of DC activation and enabled tumoral DCs to cross-present tumor antigens to naive CD8 T cells. CpG activation of tumoral DCs alone was not sufficient to induce tumor regression in either tumor model, nor was systemic delivery of the DC growth factor, Flt3 ligand, which dramatically increased the number of circulating DCs but not the number of tumoral DCs. These results indicate that the number of tumoral DCs as well as the tumor milieu determines the ability of tumor-bearing hosts to mount an effective antitumor immune response. Our results also suggest that DCs can be manipulated in vivo without delivery of defined tumor antigens to induce a specific T cell-mediated antitumor response and provide the basis for the use of chemokines in DC-targeted clinical strategies.

  View details for DOI 10.1172/JCI200419762

  View details for Web of Science ID 000189379500020

  View details for PubMedID 14991076

  View details for PubMedCentralID PMC351319

• Two isoforms of otubain 1 regulate T cell anergy via GRAIL NATURE IMMUNOLOGY Soares, L., Seroogy, C., Skrenta, H., Anandasabapathy, N., Lovelace, P., Chung, C. D., Engleman, E., Fathman, C. G. 2004; 5 (1): 45-54

  Abstract

  The active ubiquitin E3 ligase GRAIL is crucial in the induction of CD4 T cell anergy. Here we show that GRAIL is associated with and regulated by two isoforms of the ubiquitin-specific protease otubain 1. In lethally irradiated mice reconstituted with bone marrow cells from T cell receptor-transgenic mice retrovirally transduced to express the genes encoding these proteases, otubain 1-expressing cells contained negligible amounts of endogenous GRAIL, proliferated well and produced large amounts of interleukin 2 after antigenic stimulation. In contrast, cells expressing the alternatively spliced isoform, otubain 1 alternative reading frame 1, contained large amounts of endogenous GRAIL and were functionally anergic, and they proliferated poorly and produced undetectable interleukin 2 when stimulated in a similar way. Thus, these two proteins have opposing epistatic functions in controlling the stability of GRAIL expression and the resultant anergy phenotype in T cells.

  View details for DOI 10.1038/ni1017

  View details for Web of Science ID 000187635800013

  View details for PubMedID 14661020

• Dendritic cell-based cancer immunotherapy SEMINARS IN ONCOLOGY Engleman, E. G. 2003; 30 (3): 23-29

  Abstract

  The development of effective cancer vaccines depends heavily on the ability to deliver target antigens to generate an immune response. Dendritic cells are the most potent antigen-processing cells, capable of sensitizing T cells to new and recall antigens. Dendritic cells express high levels of major histocompatibility complex class I and II antigens, which are crucial to cancer immunotherapy, as well as a variety of important immunomodulatory proteins, adhesins, and a potent cytokine. Dendritic cells must undergo activation to induce an immune response, and this can be achieved through the use of certain carrier proteins, adjuvants, cytokines, or genetically engineered viruses. Dendritic cells are scattered throughout many tissues of the body, as well as bone marrow and peripheral blood. Most studies have used dendritic cells from peripheral blood; however, these cells are not prevalent in peripheral blood mononuclear cells. The cytokine, granulocyte-macrophage colony-stimulating factor, has been found to induce the maturation and enhance the viability of dendritic cells isolated from peripheral blood. Numerous clinical trials of antigen-pulsed dendritic cells have been conducted in various types of cancer, including non-Hodgkin lymphoma, multiple myeloma, prostate cancer, malignant melanoma, colorectal cancer, and non-small cell lung cancer. These studies show that antigen-loaded dendritic cell vaccinations are safe and promising in the treatment of cancer. This review discusses the use of dendritic cells in immunotherapy and some of the clinical trials that have been conducted.

  View details for DOI 10.1016/S0093-7754(03)00229-X

  View details for Web of Science ID 000184407200004

  View details for PubMedID 12881809

• Induction of immunity to tumor-associated antigens following dendritic cell vaccination of cancer patients CLINICAL IMMUNOLOGY Engleman, E. G., Fong, L. 2003; 106 (1): 10-15

  View details for DOI 10.1016/S1521-6616(02)00016-5

  View details for Web of Science ID 000181308900003

  View details for PubMedID 12584045

• Langerhans cells renew in the skin throughout life under steady-state conditions NATURE IMMUNOLOGY Merad, M., Manz, M. G., Karsunky, H., Wagers, A., Peters, W., Charo, I., Weissman, I. L., Cyster, J. G., Engleman, E. G. 2002; 3 (12): 1135-1141

  Abstract

  Langerhans cells (LCs) are bone marrow (BM)-derived epidermal dendritic cells (DCs) that represent a critical immunologic barrier to the external environment, but little is known about their life cycle. Here, we show that in lethally irradiated mice that had received BM transplants, LCs of host origin remained for at least 18 months, whereas DCs in other organs were almost completely replaced by donor cells within 2 months. In parabiotic mice with separate organs, but a shared blood circulation, there was no mixing of LCs. However, in skin exposed to ultraviolet light, LCs rapidly disappeared and were replaced by circulating LC precursors within 2 weeks. The recruitment of new LCs was dependent on their expression of the CCR2 chemokine receptor and on the secretion of CCR2-binding chemokines by inflamed skin. These data indicate that under steady-state conditions, LCs are maintained locally, but inflammatory changes in the skin result in their replacement by blood-borne LC progenitors.

  View details for DOI 10.1038/ni852

  View details for Web of Science ID 000179467800010

  View details for PubMedID 12415265

• Productive infection of plasmacytoid dendritic cells with human immunodeficiency virus type 1 is triggered by CD40 ligation JOURNAL OF VIROLOGY Fong, L., Mengozzi, M., Abbey, N. W., Herndier, B. G., Engleman, E. G. 2002; 76 (21): 11033-11041

  Abstract

  Immature plasmacytoid dendritic cells are the principal alpha interferon-producing cells (IPC), responsible for primary antiviral immunity. IPC express surface molecules CD4, CCR5, and CXCR4, which are known coreceptors required for human immunodeficiency virus (HIV) infection. Here we show that IPC are susceptible to and replicate HIV type 1 (HIV-1). Importantly, viral replication is triggered upon activation of IPC with CD40 ligand, a signal physiologically delivered by CD4 T cells. Immunohistochemical staining of tonsil from HIV-infected individuals reveals HIV p24(+) IPC, consistent with in vivo infection of these cells. IPC exposed in vitro to HIV produce alpha interferon, which partially inhibits viral replication. Nevertheless, IPC efficiently transmit HIV-1 to CD4 T-cells, and such transmission is also augmented by CD40 ligand activation. IPC produce RANTES/CCL5 and MIP-1alpha/CCL3 when exposed to HIV in vitro. IPC also induce naïve CD4 T cells to proliferate and would therefore preferentially infect these cells. These results indicate that IPC may play an important role in the dissemination of HIV.

  View details for DOI 10.1128/JVI.76.21.11033-11041.2002

  View details for Web of Science ID 000178512300047

  View details for PubMedID 12368346

  View details for PubMedCentralID PMC136607

• In vivo manipulation of dendritic cells to induce therapeutic immunity BLOOD Merad, M., Sugie, T., Engleman, E. G., Fong, L. 2002; 99 (5): 1676-1682

  Abstract

  Efficient antigen presentation and T-cell priming are essential components of effective antitumor immunity. Dendritic cells are critical to both of these functions but to date no method has been devised that both targets antigen to these cells and activates them, in situ, in a manner that induces systemic immunity. In this study we combined a dendritic cell growth factor, Flt3 ligand, with a dendritic cell activator, immunostimulatory DNA, and a tumor antigen to activate and load dendritic cells in vivo. Initial studies showed that immunostimulatory DNA not only activates dendritic cells but also prolongs their survival in vivo and in vitro. Following treatment of mice with Flt3 ligand, coadministration of immunostimulatory DNA and antigen induced potent antitumor immunity, resulting in both tumor prevention and regression of existing tumors. CD8 cytotoxic T lymphocytes but not CD4 T cells were required for tumor protection. Natural killer cells also contributed to tumor protection. These results show that dendritic cells can be loaded with antigen and activated, in situ, and provide the basis for dendritic cell- targeted clinical strategies.

  View details for Web of Science ID 000174042700025

  View details for PubMedID 11861283

• Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: clinical and immune responses in 35 patients BLOOD Timmerman, J. M., Czerwinski, D. K., Davis, T. A., Hsu, F. J., Benike, C., Hao, Z. M., Taidi, B., Rajapaksa, R., Caspar, C. B., Okada, C. Y., van Beckhoven, A., Liles, T. M., Engleman, E. G., Levy, R. 2002; 99 (5): 1517-1526

  Abstract

  Tumor-specific clonal immunoglobulin expressed by B-cell lymphomas (idiotype [Id]) can serve as a target for active immunotherapy. We have previously described the vaccination of 4 patients with follicular lymphoma using dendritic cells (DCs) pulsed with tumor-derived Id protein and now report on 35 patients treated using this approach. Among 10 initial patients with measurable lymphoma, 8 mounted T-cell proliferative anti-Id responses, and 4 had clinical responses--2 complete responses (CRs) (progression-free [PF] for 44 and 57 months after vaccination), 1 partial response (PR) (PF for 12 months), and 1 molecular response (PF for 75+ months). Subsequently, 25 additional patients were vaccinated after first chemotherapy, and 15 of 23 (65%) who completed the vaccination schedule mounted T-cell or humoral anti-Id responses. Induction of high-titer immunoglobulin G anti-Id antibodies required coupling of Id to the immunogenic carrier protein keyhole limpet hemocyanin (Id-KLH). These antibodies could bind to and induce tyrosine phosphorylation in autologous tumor cells. Among 18 patients with residual tumor at the time of vaccination, 4 (22%) had tumor regression, and 16 of 23 patients (70%) remain without tumor progression at a median of 43 months after chemotherapy. Six patients with disease progression after primary DC vaccination received booster injections of Id-KLH protein, and tumor regression was observed in 3 of them (2 CRs and 1 PR). We conclude that Id-pulsed DC vaccination can induce T-cell and humoral anti-Id immune responses and durable tumor regression. Subsequent boosting with Id-KLH can lead to tumor regression despite apparent resistance to the primary DC vaccine.

  View details for Web of Science ID 000174042700004

  View details for PubMedID 11861263

• Dendritic cell-based xenoantigen vaccination for prostate cancer immunotherapy JOURNAL OF IMMUNOLOGY Fong, L., Brockstedt, D., Benike, C., Breen, J. K., Strang, G., Ruegg, C. L., Engleman, E. G. 2001; 167 (12): 7150-7156

  Abstract

  Many tumor-associated Ags represent tissue differentiation Ags that are poorly immunogenic. Their weak immunogenicity may be due to immune tolerance to self-Ags. Prostatic acid phosphatase (PAP) is just such an Ag that is expressed by both normal and malignant prostate tissue. We have previously demonstrated that PAP can be immunogenic in a rodent model. However, generation of prostate-specific autoimmunity was seen only when a xenogeneic homolog of PAP was used as the immunogen. To explore the potential role of xenoantigen immunization in cancer patients, we performed a phase I clinical trial using dendritic cells pulsed with recombinant mouse PAP as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly vaccinations of xenoantigen-loaded dendritic cells with minimal treatment-associated side effects. All patients developed T cell immunity to mouse PAP following immunization. Eleven of the 21 patients also developed T cell proliferative responses to the homologous self-Ag. These responses were associated with Ag-specific IFN-gamma and/or TNF-alpha secretion, but not IL-4, consistent with induction of Th1 immunity. Finally, 6 of 21 patients had clinical stabilization of their previously progressing prostate cancer. All six of these patients developed T cell immunity to human PAP following vaccination. These results demonstrate that xenoantigen immunization can break tolerance to a self-Ag in humans, resulting in a clinically significant antitumor effect.

  View details for Web of Science ID 000172613400057

  View details for PubMedID 11739538

• Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fong, L., Hou, Y. F., Rivas, A., Benike, C., Yuen, A., Fisher, G. A., DAVIS, M. M., Engleman, E. G. 2001; 98 (15): 8809-8814

  Abstract

  Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA(+)CD27(-)CCR7(-)). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer(+) T cells, confirming the role of CD8 T cells in this treatment strategy.

  View details for Web of Science ID 000169967000107

  View details for PubMedID 11427731

• Dendritic cells injected via different routes induce immunity in cancer patients JOURNAL OF IMMUNOLOGY Fong, L., Brockstedt, D., Benike, C., Wu, L., Engleman, E. G. 2001; 166 (6): 4254-4259

  Abstract

  Dendritic cells (DC) represent potent APCs that are capable of generating tumor-specific immunity. We performed a pilot clinical trial using Ag-pulsed DC as a tumor vaccine. Twenty-one patients with metastatic prostate cancer received two monthly injections of DC enriched and activated from their PBMC. DC were cocultured ex vivo with recombinant mouse prostatic acid phosphatase as the target neoantigen. Following enrichment, DC developed an activated phenotype with up-regulation of CD80, CD86, and CD83 expression. During culture, the DC maintained their levels of various adhesion molecules, including CD44, LFA-1, cutaneous lymphocyte-associated Ag, and CD49d, up-regulated CCR7, but lost CD62 ligand and CCR5. In the absence of CD62 ligand, such cells would not be expected to prime T cells efficiently if administered i.v. due to their inability to access lymphoid tissue via high endothelial venules. To assess this possibility, three patient cohorts were immunized with Ag-pulsed DC by i.v., intradermal (i.d.), or intralymphatic (i.l.) injection. All patients developed Ag-specific T cell immune responses following immunization, regardless of route. Induction of IFN-gamma production, however, was seen only with i.d. and i.l. routes of administration, and no IL-4 responses were seen regardless of route, consistent with the induction of Th1-type immunity. Five of nine patients who were immunized by the i.v. route developed Ag-specific Abs compared with one of six for i.d. and two of six for i.l. routes. These results suggest that while activated DC can prime T cell immunity regardless of route, the quality of this response and induction of Ag-specific Abs may be affected by the route of administration.

  View details for Web of Science ID 000167437700082

  View details for PubMedID 11238679

• Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice MOLECULAR THERAPY Scappaticci, F. A., Smith, R., Pathak, A., Schloss, D., Lum, B., Cao, Y. H., Johnson, F., Engleman, E. G., Nolan, G. P. 2001; 3 (2): 186-196

  Abstract

  Angiostatin and endostatin are potent endothelial cell growth inhibitors that have been shown to inhibit angiogenesis in vivo and tumor growth in mice. However, tumor shrinkage requires chronic delivery of large doses of these proteins. Here we report synergistic antitumor activity and survival of animals when these factors are delivered in combination to tumors by retroviral gene transfer. We have demonstrated this efficacy in both murine leukemia and melanoma models. Complete loss of tumorigenicity was seen in 40% of the animals receiving tumors transduced by the combination of angiostatin and endostatin in the leukemia model. The synergy was also demonstrated in vitro on human umbilical vein endothelial cell differentiation and this antiangiogenic activity may suggest a mechanism for the antitumor activity in vivo. These findings imply separate pathways by which angiostatin and endostatin mediate their antiangiogenic effects. Together, these data suggest that a combination of antiangiogenic factors delivered by retroviral gene transfer may produce synergistic antitumor effects in both leukemia and solid tumors, thus avoiding long-term administration of recombinant proteins. The data also suggest that novel combinations of antiangiogenic factors delivered into tumors require further investigation as therapeutic modalities.

  View details for Web of Science ID 000167343800008

  View details for PubMedID 11237675

• Dendritic cell development from common myeloid progenitors 3rd International Conference on Hematopoietic Stem Cells: Genetics and Medicine Manz, M. G., Traver, D., Akashi, K., Merad, M., Miyamoto, T., Engleman, E. G., Weissman, I. L. NEW YORK ACAD SCIENCES. 2001: 167–174

  Abstract

  Dendritic cells (DCs) are professional antigen-presenting cells which both initiate adaptive immune responses and control tolerance to self-antigens. It has been suggested that these different effects on responder cells depend on subsets of DCs arising from either myeloid or lymphoid hematopoietic origins. In this model, CD8 alpha+ Mac-1- DCs are supposed to be of lymphoid while CD8 alpha- Mac-1+ DCs are supposed to be of myeloid origin. Here we summarize our findings that both CD8 alpha+ and CD8 alpha- DCs can arise from clonogenic common myeloid progenitors (CMPs) in both thymus and spleen. Therefore CD8 alpha expression DCs does not indicate a lymphoid origin and differences among CD8 alpha+ and CD8 alpha- DCs might rather reflect maturation status than ontogeny. On the basis of transplantation studies, it seems likely that most of the DCs in secondary lymphoid organs and a substantial fraction of thymic DCs are myeloid-derived.

  View details for Web of Science ID 000172028500019

  View details for PubMedID 11458504

• Idiotype vaccination following ABMT can stimulate specific anti-idiotype immune responses in patients with B-cell lymphoma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Davis, T. A., Hsu, F. J., Caspar, C. B., van Beckhoven, A., Czerwinski, D. K., Liles, T. M., Taidi, B., Benike, C. J., Engleman, E. G., Levy, R. 2001; 7 (9): 517-522

  Abstract

  Vaccination with the idiotype (Id) protein derived from B-cell malignancies can produce Id-specific immune responses that correlate with improved remission duration and survival rates in patients with follicular non-Hodgkin's lymphoma (NHL). A state of minimal or no residual disease correlates strongly with the laboratory detection of a cellular or humoral immune response. High-dose cytotoxic therapy (HDCT) with autologous stem cell support (autologous bone marrow transplantation [ABMT]) can provide profound cytoreduction of B-cell NHL, but the potential immune suppression associated with myeloablative therapy may compromise a patient's ability to mount a specific immune response. To determine whether patients with NHL could mount detectable immuneresponses following ABMT, Id vaccines were administered at 2 to 12 months following myeloablative therapy to a series of patients with relapsed or resistant B-cell NHL. Two different vaccination strategies produced robust immune responses against KLH in all patients, supporting the capacity of the reconstituted immune system following HDCT to react against a strong antigen. Combining the results from both vaccination strategies, 10 of 12 patients mounted Id-specific humoral or cellular responses. Vaccinations were consistently well tolerated. Of the 12 patients, 7 have experienced prolonged remissions with a follow-up from HDCT ranging from 3 to more than 11 years. Our experience serves to document the ability of the recovering immune system to react against both self and xenotypic antigens and supports the feasibility and safety of antigen-specific vaccination following myeloablative therapy in patients with B-cell NHL.

  View details for Web of Science ID 000171449200006

  View details for PubMedID 11669219

• Development of CD8 alpha-positive dendritic cells from a common myeloid progenitor SCIENCE Traver, D., Akashi, K., Manz, M., Merad, M., Miyamoto, T., Engleman, E. G., Weissman, I. L. 2000; 290 (5499): 2152-2154

  Abstract

  Dendritic cells (DCs) are critical in both initiating adaptive immune responses and maintaining tolerance to self antigens. These apparently contradictory roles have been suggested to depend on different subsets of DCs that arise from either myeloid or lymphoid hematopoietic origins, respectively. Although DC expression of CD8alpha is attributed to a lymphoid origin, here we show that both CD8alpha+ and CD8alpha- DCs can arise from clonogenic common myeloid progenitors in both thymus and spleen. Thus, expression of CD8alpha is not indicative of a lymphoid origin, and phenotypic and functional differences among DC subsets are likely to reflect maturation status rather than ontogeny.

  View details for Web of Science ID 000165870600058

  View details for PubMedID 11118150

• Dendritic cell development from lymphoid and myeloid committed hematopoietic progenitors. Manz, M. G., Traver, D., Miyamoto, T., Merad, M., Engleman, E. G., Weissman, I. L., Akashi, K. AMER SOC HEMATOLOGY. 2000: 821A–821A

  View details for Web of Science ID 000165256103546

• Differentiation of myeloid dendritic cells into CD8 alpha-positive dendritic cells in vivo BLOOD Merad, M., Fong, L., Bogenberger, J., Engleman, E. G. 2000; 96 (5): 1865-1872

  Abstract

  Bone marrow-derived dendritic cells (DC) represent a family of antigen-presenting cells (APC) with varying phenotypes. For example, in mice, CD8alpha(+) and CD8alpha(-) DC are thought to represent cells of lymphoid and myeloid origin, respectively. Langerhans cells (LC) of the epidermis are typical myeloid DC; they do not express CD8alpha, but they do express high levels of myeloid antigens such as CD11b and FcgammaR. By contrast, thymic DC, which derive from a lymphoid-related progenitor, express CD8alpha but only low levels of myeloid antigens. CD8alpha(+) DC are also found in the spleen and lymph nodes (LN), but the origin of these cells has not been determined. By activating and labeling CD8alpha(-) epidermal LC in vivo, it was found that these cells expressed CD8alpha on migration to the draining LN. Similarly, CD8alpha(-) LC generated in vitro from a CD8 wild-type mouse and injected into the skin of a CD8alphaKO mouse expressed CD8alpha when they reached the draining LN. The results also show that CD8alpha(+) LC are potent APC. After migration from skin, they localized in the T-cell areas of LN, secreted high levels of interleukin-12, interferon-gamma, and chemokine-attracting T cells, and they induced antigen-specific T-cell activation. These results demonstrate that myeloid DC in the periphery can express CD8alpha when they migrate to the draining LN. CD8alpha expression on these DC appears to reflect a state of activation, mobilization, or both, rather than lineage. (Blood. 2000;96:1865-1872)

  View details for Web of Science ID 000089578300037

  View details for PubMedID 10961888

• In vivo persistence of donor cells following adoptive transfer of allogeneic dendritic cells in HIV-infected patients CELL TRANSPLANTATION Shapero, M. H., Kundu, S. K., Engleman, E., Laus, R., van Schooten, W. C., Merigan, T. C. 2000; 9 (3): 307-317

  Abstract

  Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

  View details for Web of Science ID 000088921000002

  View details for PubMedID 10972330

• In Vivo Persistence of Donor Cells following Adoptive Transfer of Allogeneic Dendritic Cells in HIV-Infected Patients. Cell transplantation Shapero, M. H., Kundu, S. K., Engleman, E., Laus, R., Van Schooten, W. C., Merigan, T. C. 2000; 9 (3): 307-317

  Abstract

  Peripheral blood samples from HIV-seropositive individuals enrolled in a pilot clinical trial investigating the use of allogeneic dendritic cell therapy were evaluated for mixed chimerism. In this study, dendritic cells from HLA-identical, HIV-seronegative siblings were used. Patients received an infusion of dendritic cells pulsed with HIV MN gp160 protein or with peptides from HLA-A2 restricted epitopes of env, gag, and pol proteins every month for 6-9 months. Of the five allogeneic dendritic cell recipients, two showed increases in HIV antigen-specific immune responses. Allele-specific polymorphisms were identified in three sib-pairs that allowed infused donor cells to be detected using sensitive PCR-based molecular methods. Analysis of blood samples from patients showed similar patterns of donor cell persistence after the first infusion, in that cells were detectable for at least 1 week. Also, differences were observed in the kinetics of cell survival between the first and subsequent infusion cycles in all three patients. This suggests variation in HIV-specific immune responses detected among these three patients was not due to differences in persistence of infused donor cells.

  View details for DOI 10.1177/096368970000900302

  View details for PubMedID 28880658

• Clinical transplantation tolerance twelve years after prospective withdrawal of immunosuppressive drugs: Studies of chimerism and anti-donor reactivity TRANSPLANTATION Strober, S., Benike, C., Krishnaswamy, S., Engleman, E. G., GRUMET, F. C. 2000; 69 (8): 1549-1554

  Abstract

  Previous studies showed the feasibility of inducing transplantation tolerance to cadaveric renal allografts in patients given pretransplant total lymphoid irradiation (TLI). Microchimerism has been theorized to be an important or necessary factor in long-term graft acceptance and tolerance in humans.A cadaveric renal transplant recipient given pretransplant total lymphoid irradiation and withdrawn from immunosuppressive drugs more than 12 years ago was tested for microchimerism using a sensitive nested polymerase chain reaction technique, and for anti-donor reactivity using the mixed leukocyte reaction and an ELISA screen for anti-HLA antibodies. Donor and recipient were mismatched for all HLA-A, B, and DR antigens.The "tolerant" recipient had good graft function, no detectable donor-type cells in the blood by polymerase chain reaction analysis, vigorous reactivity to donor stimulator cells in the mixed leukocyte reaction, and no detectable serum anti-HLA antibodies.Operational tolerance to HLA-A, B, and DR mismatched organ allografts can be induced prospectively in humans for at least 12 years after withdrawal of immunosuppressive drugs. The allograft can be maintained in the absence of detectable donor microchimerism and in the presence of anti-donor reactivity in the mixed leukocyte reaction, suggesting that neither chimerism nor clonal deletion or anergy of recipient T cells to alloantigens presented by donor Class II HLA molecules is required for persistence of the tolerant state using this total lymphoid irradiation protocol.

  View details for Web of Science ID 000086910700005

  View details for PubMedID 10836360

• Idiotype vaccination using dendritic cells after autologous peripheral blood progenitor cell transplantation for multiple myeloma BIOLOGY OF BLOOD AND MARROW TRANSPLANTATION Liso, A., Stockerl-Goldstein, K. E., Auffermann-Gretzinger, S., Benike, C. J., Reichardt, V., van Beckhoven, A., Rajapaksa, R., Engleman, E. G., Blume, K. G., Levy, R. 2000; 6 (6): 621-627

  Abstract

  The idiotype (Id) determinants on the multiple myeloma immunoglobulin can serve as tumor-specific antigens. An anti-Id immune response may stem the growth of the malignant clone. We report on 26 patients treated at our institution with high-dose chemotherapy and peripheral blood progenitor cell transplantation (PBPCT) and vaccinated with the Id protein. The patients received chemotherapy and PBPCT to establish a minimal residual disease state. After high-dose therapy, the patients received a series of monthly immunizations consisting of 2 intravenous infusions of dendritic cells (DCs) pulsed with either Id protein or Id coupled with keyhole limpet hemocyanin (KLH) as an immunogenic carrier protein, followed by subcutaneous boosts of Id-KLH conjugates. DCs were obtained in all patients from a leukapheresis product 3 to 9 months after PBPCT. Patients were observed for toxicity, immune responses, and tumor status. The DC infusions and the administration of Id-KLH boosts were well tolerated, with patients experiencing only minor and transient side effects. Of the patients, 24 of 26 generated a KLH-specific cellular proliferative immune response. Only 4 patients developed an Id-specific proliferative immune response. Three of these immune responders were in complete remission at the time of vaccination. A total of 17 patients are alive at a median follow-up of 30 months after transplantation. Id vaccination with autologous DCs is feasible for myeloma patients after transplantation. Id-specific cellular responses can be induced in patients who are in complete remission. Further studies are needed to increase the rate of anti-Id immune responses in patients who do not achieve complete remission.

  View details for Web of Science ID 000165614400004

  View details for PubMedID 11128812

• Dendritic cells in cancer immunotherapy ANNUAL REVIEW OF IMMUNOLOGY Fong, L., Engleman, E. G. 2000; 18: 245-273

  Abstract

  The potential to harness the potency and specificity of the immune system underlies the growing interest in cancer immunotherapy. One such approach uses bone marrow-derived dendritic cells, phenotypically distinct and extremely potent antigen-presenting cells, to present tumor-associated antigens and thereby generate tumor-specific immunity. Support for this strategy comes from animal studies that have demonstrated that dendritic cells, when loaded ex vivo with tumor antigens and administered to tumor-bearing hosts, can elicit T cell-mediated tumor destruction. These observations have led to clinical trials designed to investigate the immunologic and clinical effects of antigen-loaded dendritic cells administered as a therapeutic vaccine to patients with cancer. In the design and conduct of such trials, important considerations include antigen selection, methods for introducing the antigen into MHC class I and II processing pathways, methods for isolating and activating dendritic cells, and route of administration. Although current dendritic cell-based vaccination methods are cumbersome, promising results from clinical trials in patients with malignant lymphoma, melanoma, and prostate cancer suggest that immunotherapeutic strategies that take advantage of the antigen presenting properties of dendritic cells may ultimately prove both efficacious and widely applicable to human tumors.

  View details for Web of Science ID 000087236500010

  View details for PubMedID 10837059

• Idiotype-pulsed dendritic cell vaccination for B-cell lymphoma: Clinical and immunological responses in 26 patients. Timmerman, J. M., Davis, T. A., Hsu, F. J., Caspar, C. B., Benike, C., Liles, T. M., Czerwinski, D. K., Taidi, B., van Beckhoven, A., Engleman, E. G., Levy, R. AMER SOC HEMATOLOGY. 1999: 385A–385A

  View details for Web of Science ID 000083790301761

• Antiangiogenic gene therapy of murine B16F10 melanoma and L1210 leukemia tumors with angiostatin and endostatin containing MMuLV retroviral vectors. Scappaticci, F. A., Smith, R., Schloss, D., Cao, Y., Engleman, E. G., Nolan, G. P., Leung, L. AMER SOC HEMATOLOGY. 1999: 420B–420B

  View details for Web of Science ID 000083790701952

• Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration CLINICAL IMMUNOLOGY Brockstedt, D. G., Podsakoff, G. M., Fong, L., Kurtzman, G., Mueller-Ruchholtz, W., Engleman, E. G. 1999; 92 (1): 67-75

  Abstract

  Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations.

  View details for Web of Science ID 000081452100009

  View details for PubMedID 10413654

• Idiotype vaccination using dendritic cells after autologous peripheral blood stem cell transplantation for multiple myeloma - A feasibility study BLOOD Reichardt, V. L., Okada, C. Y., Liso, A., Benike, C. J., Stockerl-Goldstein, K. E., Engleman, E. G., Blume, K. G., Levy, R. 1999; 93 (7): 2411-2419

  Abstract

  The idiotype (Id) determinant on the multiple myeloma (MM) protein can be regarded as a tumor-specific marker. Immunotherapy directed at the MM Id may stem the progression of this disease. We report here on the first 12 MM patients treated at our institution with high-dose therapy and peripheral blood stem cell transplantation (PBSCT) followed by Id immunizations. MM patients received PBSCT to eradicate the majority of the disease. PBSCT produced a complete response in 2 patients, a partial response in 9 patients and stable disease in 1 patient. Three to 7 months after high-dose therapy, patients received a series of monthly immunizations that consisted of two intravenous infusions of Id-pulsed autologous dendritic cells (DC) followed by five subcutaneous boosts of Id/keyhole limpet hemocyanin (KLH) administered with adjuvant. Between 1 and 11 x 10(6) DC were obtained by leukapheresis in all patients even after PBSCT. The administration of Id-pulsed DC and Id/KLH vaccines were well tolerated with patients experiencing only minor and transient side effects. Two of 12 patients developed an Id-specific, cellular proliferative immune response and one of three patients studied developed a transient but Id-specific cytotoxic T-cell (CTL) response. Eleven of the 12 patients generated strong KLH-specific cellular proliferative immune responses showing the patients' immunocompetence at the time of vaccination. The two patients who developed a cellular Id-specific immune response remain in complete remission. Of the 12 treated patients, 9 are currently alive after autologous transplantation with a minimum follow-up of 16 months, 2 patients died because of recurrent MM and 1 patient succumbed to acute leukemia. These studies show that patients make strong anti-KLH responses despite recent high-dose therapy and that DC-based Id vaccination is feasible after PBSCT and can induce Id-specific T-cell responses. Further vaccine development is necessary to increase the proportion of patients that make Id-specific immune responses. The clinical benefits of Id vaccination in MM remain to be determined.

  View details for Web of Science ID 000079377400034

  View details for PubMedID 10090953

• Comparison of primary sensitization of naive human T cells to varicella-zoster virus peptides by dendritic cells in vitro with responses elicited in vivo by varicella vaccination 3rd International Conference of the Varicella-Zoster Virus Jenkins, D. E., Yasukawa, L. L., Bergen, R., Benike, C., Engleman, E. G., Arvin, A. M. AMER ASSOC IMMUNOLOGISTS. 1999: 560–67

  Abstract

  Dendritic cells (DC) are potent APC during primary and secondary immune responses. The first objective of this study was to determine whether human DC mediate in vitro sensitization of naive CD4+ T cells to epitopes of the immediate early 62 (IE62) protein of varicella zoster virus (VZV). The induction of CD4+ T cell proliferative responses to eight synthetic peptides representing amino acid sequences of the VZV IE62 protein was assessed using T cells and DC from VZV-susceptible donors. The second objective was to compare in vitro responses of naive T cells with responses to VZV peptides induced in vivo after immunization with varicella vaccine. T cell proliferation was induced by three peptides, P1, P4, and P7, in 71-100% of the donors tested before and after vaccination using DC as APC. Monocytes were effective APC for VZV peptides only after immunization. Two peptides, P2 and P8, induced naive T cell proliferation less effectively and were also less immunogenic for T cells from vaccinated or naturally immune donors. T cell recognition of specific peptides was concordant between naive, DC-mediated responses, and postvaccine responses using monocytes as APC in 69% of comparisons (p = 0.05; chi2); the predictive value of a positive response to an IE62 peptide before immunization for T cell sensitization in vivo was 82%. These observations indicate that primary T cell responses detected in vitro using DC as APC may be useful to characterize the potential immunogenicity of viral protein epitopes in vivo.

  View details for Web of Science ID 000077748100072

  View details for PubMedID 9886433

• Effect of WF10 (TCDO) on antigen presentation 3rd International Conference on New Trends in Clinical and Experimental Immunosuppression McGrath, M. S., Benike, C., Kuehne, F. W., Engleman, E. ELSEVIER SCIENCE INC. 1998: 4200–4204

  View details for Web of Science ID 000077593000097

  View details for PubMedID 9865348

• Isolation and utilization of human dendritic cells from peripheral blood to assay an in vitro primary immune response to varicella-zoster virus peptides 3rd International Conference of the Varicella-Zoster Virus Jenkins, D. E., Yasukawa, L. L., Benike, C. J., Engleman, E. G., Arvin, A. M. UNIV CHICAGO PRESS. 1998: S39–S42

  Abstract

  A human dendritic cell-based assay used to monitor a T cell proliferation response to viral peptides in vitro is described. Dendritic cells and autologous CD4+ T cells were isolated from peripheral blood by a series of density-gradient centrifugations or magnetic bead separations (or both). Peptides corresponding to residues of the immediate early protein, IE62, of varicella-zoster virus (VZV) were used as stimulating antigens, and persons with no history of varicella and no humoral or cellular immunity to VZV served as naive donors for the assays. Three VZV-susceptible donors were tested, and all demonstrated an in vitro response to multiple VZV peptides. This assay has potential as a screen to establish the immunogenicity of viral antigens in vitro using T cells from naive donors.

  View details for Web of Science ID 000077034100009

  View details for PubMedID 9852971

• Donor blood monocytes but not T or B cells facilitate long-term allograft survival after total lymphoid irradiation TRANSPLANTATION Hayamizu, K., Zeng, D., Huie, P., Garcia-Ojeda, M. E., Bloch, D. A., Fong, L., Engleman, E. G., Sibley, R. K., Strober, S. 1998; 66 (5): 585-593

  Abstract

  Previous studies showed that a combination of posttransplant total lymphoid irradiation (TLI), rabbit antithymocyte globulin (ATG), and a single donor blood transfusion induced tolerance to ACI heart allografts in Lewis rats. All three modalities were required to achieve tolerance. The objective of the current study was to determine the subset(s) of cells in the donor blood that facilitated long-term allograft survival.Lewis hosts received TLI, ATG, and donor cell infusion after heart transplantation. Graft survival, mixed leukocyte reaction (MLR), and intragraft cytokine mRNA were studied.The intravenous injection of 25 x 10(6) ACI peripheral blood mononuclear cells (PBMC) significantly prolonged graft survival as compared with that of Lewis hosts given TLI and ATG alone. Injection of highly enriched blood T cells or splenic B cells adjusted for the number contained in 25 x 10(6) PBMC failed to induce significant graft prolongation. Unexpectedly, depletion of monocytes (CD11b+ cells) from PBMC resulted in the loss of graft prolongation activity. Enriched populations of monocytes obtained by plastic adherence were more efficient in prolonging graft survival than PBMC on a per cell basis. Hosts with long-term grafts (>100-day survival) showed evidence of immune deviation, because the MLR to ACI stimulator cells was vigorous, but secretion of interferon-gamma in the MLR was markedly reduced. In situ hybridization studies of long-term grafts showed markedly reduced levels of interferon-gamma mRNA as compared with rejecting grafts.Infusion of donor monocytes facilitated graft prolongation via immune deviation.

  View details for Web of Science ID 000075996500006

  View details for PubMedID 9753336

• CTL response to Mycobacterium tuberculosis: Identification of an immunogenic epitope in the 19-kDa lipoprotein JOURNAL OF IMMUNOLOGY Mohagheghpour, N., Gammon, D., Kawamura, L. M., van Vollenhoven, A., Benike, C. J., Engleman, E. G. 1998; 161 (5): 2400-2406

  Abstract

  The successful resolution of infection with Mycobacterium tuberculosis (M.tb) is believed to involve the induction of CTLs that are capable of killing cells harboring this pathogen, although little information is known about the MHC restriction or fine specificity of such CTLs. In this study, we used knowledge of the HLA-A*0201-binding motif and an immunofluorescence-based peptide-binding assay to screen for potential HLA-A*0201-binding epitopes contained in the 19-kDa lipoprotein of M.tb (M.tb19). CD8+ T cells derived from HLA-A*0201+ patients with active tuberculosis (TB) as well as tuberculin skin test-positive individuals who had no history of TB were used as effector cells to determine whether these epitopes are recognized by in vivo-primed CTLs. An in vitro vaccination system using HLA-A*0201+ dendritic cells (DCs) as APCs was used to determine whether these epitopes can sensitize naive CD8+ T cells in vitro, leading to the generation of Ag-specific CTLs. The results show that an HLA-A*0201-binding peptide comprised of residues 88 to 97 of M.tb19 (P88-97) is recognized by circulating CD8+ CTLs from both healthy tuberculin skin test-positive individuals and patients with active TB but not by tuberculin skin test-negative subjects. Moreover, dendritic cells pulsed with this peptide induced class I MHC-restricted CTLs from the T cells of healthy unsensitized persons. Finally, CTL lines that were specific for P88-97 were shown to lyse autologous monocytes that had been infected acutely with the H37Ra strain of M.tb. These results demonstrate that M.tb19 elicits HLA class I-restricted CTLs in vitro and in vivo that recognize endogenously processed Ag. Epitopes of the type identified here may prove useful in the design of an M.tb vaccine.

  View details for Web of Science ID 000075511600041

  View details for PubMedID 9725236

• V7 (CD101) ligation inhibits TCR/CD3-induced IL-2 production by blocking Ca2+ flux and nuclear factor of activated T cell nuclear translocation JOURNAL OF IMMUNOLOGY Soares, L. R., Tsavaler, L., Rivas, A., Engleman, E. G. 1998; 161 (1): 209-217

  Abstract

  Ligation of the V7 (CD101) molecule on T cells with anti-V7 mAb blocks TCR/CD3-induced proliferation by inhibiting IL-2 transcription. To explore the basis for this observation, we analyzed the effects of V7 ligation on CD3/TCR-induced changes in intracellular free Ca2+ and Ca2+-dependent nuclear factor of activated T cells (NF-AT) translocation to the nucleus, which is required for IL-2 transcription. T cells exposed to anti-V7 mAb fluxed Ca2+ transiently, but did not flux Ca2+ in response to subsequent treatment with anti-CD3; however, they recovered the capacity to flux Ca2+ after treatment with pervanadate, indicating that tyrosine dephosphorylation of a critical V7-related substrate is required in the desensitization process. One such substrate, phospholipase C (PLC)-gamma1, becomes tyrosine phosphorylated on CD3/TCR activation and mediates inositol triphosphate-dependent Ca2+ flux. Co-cross-linking of T cells with anti-CD3 and anti-V7 resulted in selective inhibition of PLC-gamma1 tyrosine phosphorylation, which may explain V7-mediated blockade of anti-CD3-induced Ca2+ flux. Moreover, anti-CD3-induced binding of transcription factors to a consensus NF-AT-binding oligonucleotide, which is dependent on Ca2+, was blocked completely by treatment of the cells with anti-V7, whereas binding to a consensus-activating protein-1 oligonucleotide was unaffected. Western blot analysis of cytoplasmic and nuclear extracts confirmed that anti-V7 prevented nuclear translocation of NF-ATc induced by anti-CD3. We conclude that V7 ligation interferes with T cell activation and IL-2 secretion through a Ca2+ and tyrosine kinase-dependent pathway that inhibits PLC-gamma1 phosphorylation and prevents NF-AT translocation to the nucleus.

  View details for Web of Science ID 000074302700028

  View details for PubMedID 9647226

• A pilot clinical trial of HIV antigen-pulsed allogeneic and autologous dendritic cell therapy in HIV-infected patients AIDS RESEARCH AND HUMAN RETROVIRUSES Kundu, S. K., Engleman, E., Benike, C., Shapero, M. H., Dupuis, M., van Schooten, W. C., Eibl, M., Merigan, T. C. 1998; 14 (7): 551-560

  Abstract

  A pilot study was carried out to assess the safety and antigen-presenting properties of allogeneic or autologous dendritic cells (DCs) in six HLA-A2+, HIV-infected patients. Allogeneic DCs obtained from the peripheral blood of HLA-identical, HIV-seronegative siblings were pulsed with recombinant HIV-1 MN gp160 or synthetic peptides corresponding to HLA-A2-restricted cytotoxic epitopes of envelope, Gag, and Pol proteins. The antigen-pulsed cells were infused intravenously six to nine times at monthly intervals and HIV-specific immune responses were monitored. One allogeneic DC recipient with a CD4+ T cell count of 460/mm3 showed increases in envelope-specific CTL- and lymphocyte-proliferative responses, as well as in IFN-gamma and IL-2 production. Another allogeneic DC recipient with a CD4+ T cell count of 434/mm3 also showed an increase in HIV envelope-specific lymphocyte-proliferative responses. A recipient of autologous DCs with a CD4+ T cell count of 730/mm3 showed an increase in peptide-specific lymphocyte-proliferative responses after three infusions. Three other allogeneic DC recipients with CD4+ T cell counts <410/mm3 did not show increases in their HIV-specific immune responses. No clinically significant adverse effects were noted in this study and CD4+ T cell numbers and plasma HIV-1 RNA detected by RT-PCR of all six patients were stable during the study period. Thus, both allogeneic and autologous DC infusions were well tolerated and in patients with normal or near normal CD4+ T cell counts administration of these antigen-pulsed cells enhanced the immune response to HIV. However, since no effect on viral load was observed there was no evidence that this approach provided clinical benefit.

  View details for Web of Science ID 000073421600004

  View details for PubMedID 9591709

• Generation of primary peptide-specific CD8(+) cytotoxic T-lymphocytes in vitro using allogeneic dendritic cells CELL TRANSPLANTATION Peshwa, M. V., Benike, C., Dupuis, M., Kundu, S. K., Engleman, E. G., Merigan, T. C., van Schooten, W. C. 1998; 7 (1): 1-9

  Abstract

  Dendritic cells (DC) are potent antigen-presenting cells (APC) capable of inducing strong T-cell-mediated immunity. Infusion of lymphoma-specific antigen-loaded autologous DC has been demonstrated to result in the generation of antigen-specific immunity and reduction in tumor burden in B-cell lymphoma patients. Cellular immunotherapy employing antigen-loaded DC could have a potential therapeutic impact in tumors and viral infections, including HIV infection. However, DC in HIV-infected individuals and breast cancer patients are believed to be functionally defective. Therefore, the potential of using allogeneic DC offers significant implications for DC immunotherapy in AIDS and immunocompromised cancer patients. To explore the potential of allogeneic DC therapy in vivo, we tested the ability of allogeneic DC to generate primary peptide-specific CD8+ cytotoxic T-lymphocyte (CTL) responses in vitro. Our results indicate that DC from HLA class I-matched individuals elicit primary immune responses in vitro using viral peptides as naive antigens. A primary peptide-specific immune response could also be detected even when only one HLA allele (HLA-A*0201) was matched between the allogeneic DC and T-lymphocytes. The ability to generate primary peptide-specific responses in vitro is strongly indicative of the in vivo therapeutic potential of allogeneic DC.

  View details for Web of Science ID 000072003100001

  View details for PubMedID 9489758

• Clinical trials of dendritic cell vaccination for B-cell non-Hodgkin's lymphoma. Timmerman, J. M., Davis, T. A., Hsu, F. J., Benike, C., Liles, T. M., Czerwinski, D., Taidi, B., van Beckhoven, A., Fazio, M., Engleman, E. G., Levy, R. FEDERATION AMER SOC EXP BIOL. 1998: 15–15

  View details for Web of Science ID 000076309900019

• Induction of tissue-specific autoimmune prostatitis with prostatic acid phosphatase immunization - Implications for immunotherapy of prostate cancer JOURNAL OF IMMUNOLOGY Fong, L., Ruegg, C. L., Brockstedt, D., Engleman, E. G., Laus, R. 1997; 159 (7): 3113-3117

  Abstract

  Prostatic acid phosphatase (PAP) is uniquely expressed in prostatic tissue and prostate cancer. In this study, the immunogenicity of PAP was investigated in a male rat model. We show that immunization with recombinant rat or human PAP in CFA leads to a significant Ab response, but does not generate CTL or result in autoimmune prostatitis. In contrast, immunization with recombinant vaccinia expressing human PAP, but not rat PAP, generates a CTL response and tissue-specific prostatitis in the absence of detectable PAP-specific Abs. These findings suggest that a cellular immune response to PAP, rather than Abs, mediates destructive autoimmune prostatitis. Thus, xenogeneic forms of PAP are a new tool for the induction of prostate-specific immunity and may prove useful for the immunotherapy of prostate cancer.

  View details for Web of Science ID A1997XY46100006

  View details for PubMedID 9317107

• Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide JOURNAL OF IMMUNOLOGY Kim, D. T., Mitchell, D. J., Brockstedt, D. G., Fong, L., Nolan, G. P., Fathman, C. G., Engleman, E. G., Rothbard, J. B. 1997; 159 (4): 1666-1668

  Abstract

  Protection against most intracellular pathogens requires T cells that recognize pathogen-derived peptides in association with MHC class I molecules on the surface of infected cells. However, because exogenous proteins do not ordinarily enter the cytosol and access the MHC class I-processing pathway, protein-based vaccines that induce class I-restricted CTL responses have proved difficult to design. We have addressed this problem by conjugating proteins, such as OVA, to a short cationic peptide derived from HIV-1 tat (residues 49-57). When APC were exposed in vitro to such protein conjugates, they processed and presented the peptides in association with MHC class I molecules and stimulated CD8+ Ag-specific T cells. Moreover, Ag-specific CTLs were generated in vivo by immunizing mice with histocompatible dendritic cells that had been exposed to protein-tat conjugates.

  View details for Web of Science ID A1997XP43800012

  View details for PubMedID 9257826

• Ligation of the V7 molecule on T cells blocks anergy induction through a CD28-independent mechanism JOURNAL OF IMMUNOLOGY Soares, L. R., Rivas, A., Tsavaler, L., Engleman, E. G. 1997; 159 (3): 1115-1124

  Abstract

  Previous studies have demonstrated that a mAb that recognizes the leukocyte surface Ag V7 inhibits TCR/CD3-dependent T cell activation. In the current study, we demonstrate that in addition to inhibiting T cell proliferation and IL-2 production, anti-V7 blocks tyrosine phosphorylation of TCR/CD3-associated substrates. PMA overcomes this effect, and both PMA and exogenous IL-2 overcome anti-V7-mediated inhibition of T cell proliferation and IL-2 production. T cells stimulated with anti-CD3 in the absence of CD28 or V7 ligation become unresponsive (anergic) to restimulation with anti-CD3; T cells primed in the presence of either anti-V7 or anti-CD28 retain their ability to respond to restimulation with anti-CD3. When T cells are primed in the presence of optimal concentrations of anti-V7 and anti-CD28 Abs, they proliferate normally, indicating that the costimulatory signals generated through CD28 dominate the inhibitory signals generated through V7. However, as the anti-CD28 stimulus is diluted, the V7 effect becomes dominant and proliferation is inhibited. Thus, although both anti-V7 and anti-CD28 Abs prevent anergy, they induce distinct, competing intracellular signals. Wortmannin, which blocks phosphoinositol 3-kinase-dependent signaling, has little effect on V7-mediated inhibition, while herbimycin, an inhibitor of tyrosine kinase, synergizes with anti-V7 to inhibit T cell activation. On the basis of these findings, V7-mediated signals appear to inhibit TCR-dependent tyrosine kinases that are required for IL-2 production and cellular proliferation.

  View details for Web of Science ID A1997XL78900010

  View details for PubMedID 9233604

• Differential response of CD4(+) V7(+) and CD4(+) V7(-) cells to T cell receptor-dependent signals: CD4(+) V7(+) T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen EUROPEAN JOURNAL OF IMMUNOLOGY Soares, L. R., Rivas, A., Ruegg, C., Engleman, E. G. 1997; 27 (6): 1413-1421

  Abstract

  V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells. In the current study, CD4+ V7+ and CD4+ V7- T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4+ V7+ T cells alone produced IL-2 and proliferated vigorously. By contrast, CD4+ V7- cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28-antibody were added to the cultures. The enhancement of the V7- T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up-regulate such signals through their interaction with counter-receptors on T cells. To assess the role of V7 in a system independent of co-stimulation, CD4+ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.

  View details for Web of Science ID A1997XD60100017

  View details for PubMedID 9209493

• Dendritic cells that process and present nominal antigens to naive T lymphocytes are derived from CD2(+) precursors JOURNAL OF IMMUNOLOGY Takamizawa, M., Rivas, A., Fagnoni, F., Benike, C., Kosek, J., Hyakawa, H., Engleman, E. G. 1997; 158 (5): 2134-2142

  Abstract

  Dendritic cells (DC) are potent APC that, in mature form, can be distinguished from other mononuclear cells on the basis of their distinct morphology, absence of lineage markers, and dense expression of MHC and costimulatory molecules. While comparing different DC preparation methods, we observed that DC derived from cultured PBMC that had been depleted of CD2+ cells before culture were functionally distinct from DC derived from PBMC that had not been depleted of CD2+ cells. Thus, both types of DC stimulated allogeneic T cells to proliferate in the MLR, but only DC derived from CD2+ precursors could sensitize naive T cells to soluble Ags such as keyhole limpet hemocyanin and HIV gp160 glycoprotein. Subsequent studies confirmed the existence of CD2+ and CD2- DC precursor populations among HLA-DRbright, lineage-negative PBMC. Immediately after their isolation, these populations were morphologically similar to one another by light and electron microscopy, and neither had substantial Ag-presenting activity. After culture for 24 to 48 h with supernatant from PHA-activated PBMC, both populations developed dendrites, formed clusters with T cells, and stimulated allogeneic T cell responses in the MLR as well as autologous T cell responses to tetanus toxoid, a recall Ag. However, CD2+ DC precursors alone gave rise to APC that presented soluble Ags to naive CD4+ T cells, a property that could be inhibited by Abs to CD4, CD11a, and CD28 on T cells or CD86 on DC. The expression of CD54 and CD86 on CD2+ DC precursors was increased markedly after their culture and differentiation, while the expression of these molecules on CD2- DC precursors was not remarkably changed. These findings reveal the existence of two functionally distinct populations of DC, each derived from a phenotypically distinct precursor present in monocyte-depleted peripheral blood.

  View details for Web of Science ID A1997WJ66000019

  View details for PubMedID 9036958

• Dendritic cells: Potential role in cancer therapy 9th Meeting of the Japanese-Association-for-Animal-Cell-Technology (JAACT 96) Engleman, E. G. SPRINGER. 1997: 1–8

  Abstract

  Dendritic cells (DC) are extremely potent antigen presenting cells, uniquely capable of sensitizing naive T cells to protein antigens and eliciting antigen specific immune responses. Studies of human DC isolated from peripheral blood indicate that these cells can be used to stimulate and expand antigen specific CD4+ and CD8+ T cells, in vitro. On the basis of these findings we have initiated pilot clinical studies to investigate the ability of DC pulsed ex vivo with tumor associated proteins to stimulate host anti-tumor immunity when re-infused as a vaccine. In the first such study DC pulsed with tumor derived idiotype protein were infused into patients with low grade malignant B cell lymphoma who had failed conventional chemotherapy. The majority of treated patients developed T cell mediated anti-idiotype immune responses and some of the patients experienced tumor regression. These results suggest that DC based immunotherapy is a potentially useful approach to B cell lymphoma and raises the possibility that the approach may prove useful in the treatment of other tumors as well.

  View details for Web of Science ID 000071912900002

  View details for PubMedID 9474803

• Transport of immunogens into the MHC class I and II pathways by a peptide from HIV tat 40th Symposium of the Alfred-Benzon-Foundation on HLA and Disease - the Molecular Basis Rothbard, J., Kim, D., Mitchell, D., Bockstedt, D., Fong, L., Nolan, G., Fathman, C. G., Engleman, E. MUNKSGAARD. 1997: 161–175

  View details for Web of Science ID A1997BJ08M00012

• Allogeneic dendritic cell induction of HIV-specific cytotoxic T lymphocyte responses from T cells of HIV type 1-infected and uninfected individuals AIDS RESEARCH AND HUMAN RETROVIRUSES Dupuis, M., Peshwa, M. V., Benike, C., Kundu, S. K., Engleman, E. G., VANSCHOOTEN, W. C., Merigan, T. C. 1997; 13 (1): 33-39

  Abstract

  The potential benefit of T cell-based vaccination for HIV-1 infection remains to be determined. Cytotoxic T lymphocytes (CTLs) appear to clear substantial populations of HIV-1 virus in vivo, although CTL activity may contribute to the decline in CD4+ T cell count observed in the course of the disease. To investigate further the role of specific CTL responses in the control of HIV-1 replication, we raised primary CTL lines against a panel of conserved HIV-1 epitopes using blood-derived dendritic cells as antigen-presenting cells (APCs). Specific primary human CTL responses were induced against HLA-A*0201-restricted peptides with dendritic cells from HIV-1-seronegative donors. This method of immunization elicited cytotoxic activities capable of recognizing endogenously processed antigen. The CTL induction protocol was extended in order to explore the capacity of HLA-matched allogeneic dendritic cells to evoke novel CTL responses in T cells from an HIV-seropositive asymptomatic individual. Allogeneic peptide-pulsed dendritic cells from a healthy sibling were capable of eliciting a CTL response directed against an HIV epitope (env814: SLLNATDIAV) that was initially not detected in the CTL effector population of the HIV-1-infected patient. The possibility of manipulating CTL specificity directed against multiple conserved HIV-1 epitopes represents a significant step in the evaluation of T cell-based vaccination for treatment of disease.

  View details for Web of Science ID A1997WA16700006

  View details for PubMedID 8989425

• Role of natural killer cells in the generation of influenza virus-specific cytotoxic T cells CELLULAR IMMUNOLOGY Kos, F. J., Engleman, E. G. 1996; 173 (1): 1-6

  Abstract

  Recently we reported that natural killer (NK) cells are critical accessory cells required for the differentiation of alloantigen-stimulated CD8+ T cells into effector cytotoxic T lymphocytes (CTL) in vitro. In this study we provide evidence that NK cells are also required for the generation of influenza virus-specific CTL. Depletion of NK cells from responder human peripheral blood mononuclear cells or mouse splenocytes abolished the induction of influenza A virus-specific CTL under culture conditions. Treatment of C57BL/6 mice with the NK cell-depleting NK1.1 monoclonal antibody (mAb) before primary or secondary immunization with influenza A virus abrogated the capacity of CTL precursors to differentiate into influenza virus-specific CTL effectors in vivo. These results extend our previous findings and demonstrate that NK cells critically influence the induction of antigen-specific CTL, both in vitro and in vivo.

  View details for Web of Science ID A1996VN24100001

  View details for PubMedID 8871595

• B4B, a novel growth-arrest gene, is expressed by a subset of progenitor/pre-B lymphocytes negative for cytoplasmic mu-chain JOURNAL OF IMMUNOLOGY Ruegg, C. L., Wu, H. Y., Fagnoni, F. F., Engleman, E. G., Laus, R. 1996; 157 (1): 72-80

  Abstract

  We subjected PBMC of normal adults to density fractionation to enrich for an immunoblast fraction that would include early immune lineage precursors. Differential display PCR experiments identified one transcript that is expressed specifically in this immunoblast fraction. This cDNA, designated B4B, encodes a novel gene product containing four putative transmembrane-spanning domains. B4B+ cells, detected with anti-B4B Ig, were found at very low frequency in PBMC (0.01%) and were enriched significantly in intermediate density fractions (0.1-1.0%). B4B+ cells were shown to be CD19+CD45+HLA-DR+ and negative for CD20, cytoplasmic mu-chain, CD3, CD16, CD56, CD34, and CD68 (monocyte), consistent with a progenitor/pre-B lymphocyte subset that does not express cytoplasmic mu-chain and thus may lack productive Ig rearrangement. This phenotypic description of the B4B+ subset agrees with our finding that the frequency of B4B+ cells was greatly increased in bone marrow (3-10%) as compared with PBMC (0.01%). The B4B polypeptide sequence exhibits significant homology to only one known protein, PMP-22/gas-3, a Schwann cell-specific protein that induces cell growth arrest. Transient expression of B4B specifically inhibited cellular proliferation by more than 50%. Based on its antiproliferative effect and pattern of expression restricted to a subpopulation of immature B cells, the B4B gene product may be involved in the elimination of B cells before productive VDJC rearrangement of Ig loci or, alternatively, in the growth arrest of transformed progenitor B cells.

  View details for Web of Science ID A1996UT10900010

  View details for PubMedID 8683158

• Immune regulation: A critical link between NK cells and CTLs IMMUNOLOGY TODAY Kos, F. J., Engleman, E. G. 1996; 17 (4): 174-176

  View details for Web of Science ID A1996UC44900009

  View details for PubMedID 8871349

• Selective depletion of myelin-reactive T cells with the anti-OX-40 antibody ameliorates autoimmune encephalomyelitis NATURE MEDICINE Weinberg, A. D., Bourdette, D. N., Sullivan, T. J., Lemon, M., Wallin, J. J., Maziarz, R., Davey, M., Palida, F., Godfrey, W., Engleman, E., Fulton, R. J., Offner, H., Vandenbark, A. A. 1996; 2 (2): 183-189

  Abstract

  The OX-40 protein was selectively upregulated on encephalitogenic myelin basic protein (MBP)-specific T cells at the site of inflammation during the onset of experimental autoimmune encephalomyelitis (EAE). An OX-40 immunotoxin was used to target and eliminate MBP-specific T cells within the central nervous system without affecting peripheral T cells. When injected in vivo, the OX-40 immunotoxin bound exclusively to myelin-reactive T cells isolated from the CNS, which resulted in amelioration of EAE. Expression of the human OX-40 antigen was also found in peripheral blood of patients with acute graft-versus-host disease and the synovia of patients with rheumatoid arthritis during active disease. The unique expression of the OX-40 molecule may provide a novel therapeutic strategy for eliminating autoreactive CD4+T cells that does not require prior knowledge of the pathogenic autoantigen.

  View details for Web of Science ID A1996TU06000038

  View details for PubMedID 8574963

• Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells NATURE MEDICINE Hsu, F. J., Benike, C., Fagnoni, F., Liles, T. M., Czerwinski, D., Taidi, B., Engleman, E. G., Levy, R. 1996; 2 (1): 52-58

  Abstract

  In this pilot study, we investigated the ability of autologous dendritic cells pulsed ex vivo with tumor-specific idiotype protein to stimulate host antitumor immunity when infused as a vaccine. Four patients with follicular B-cell lymphoma received a series of three or four infusions of antigen-pulsed dendritic cells followed, in each instance, by subcutaneous injections of soluble antigen two weeks later. All patients developed measurable antitumor cellular immune responses. In addition, clinical responses have been measured with one patient experiencing complete tumor regression, a second patient having partial tumor regression, and a third patient resolving all evidence of disease as detected by a sensitive tumor-specific molecular analysis.

  View details for Web of Science ID A1996TP57900037

  View details for PubMedID 8564842

• Dehydroepiandrosterone in systemic lupus erythematosus - Results of a double-blind, placebo-controlled, randomized clinical trial ARTHRITIS AND RHEUMATISM VANVOLLENHOVEN, R. F., Engleman, E. G., McGuire, J. L. 1995; 38 (12): 1826-1831

  Abstract

  To determine if dehydroepiandrosterone (DHEA) is beneficial in the treatment of systemic lupus erythematosus (SLE).In a double-blind, placebo-controlled, randomized trial, 28 female patients with mild to moderate SLE were given DHEA 200 mg/day or placebo for 3 months. Outcomes included the SLE Disease Activity Index (SLEDAI) score, patient's and physician's overall assessments of disease activity, and concurrent corticosteroid dosages (which were adjusted as clinically indicated).In the patients who were receiving DHEA, the SLEDAI score, patient's and physician's overall assessment of disease activity, and concurrent prednisone dosage decreased, while in the patients taking placebo, small increases were seen. The difference in patient's assessment between the groups was statistically significant (P = 0.022, adjusted). Lupus flares occurred more frequently in the placebo group (P = 0.053). Mild acne was a frequent side effect of DHEA.DHEA may be useful as a therapeutic agent for the treatment of mild to moderate SLE. Further studies of DHEA in the treatment of SLE are warranted.

  View details for Web of Science ID A1995TL67900015

  View details for PubMedID 8849355

• INHIBITION OF ANTIGEN-PRESENTING CELL-FUNCTION BY ALENDRONATE IN-VITRO JOURNAL OF BONE AND MINERAL RESEARCH Sansoni, P., Passeri, G., Fagnoni, F., Mohagheghpour, N., SNELLI, G., BRIANTI, V., Engleman, E. G. 1995; 10 (11): 1719-1725

  Abstract

  Bisphosphonates are potent inhibitors of bone resorption in vivo and are emerging as important and widely used drugs for the treatment of a variety of abnormal bone resorptive processes. In the current study we investigated the in vitro effects of 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), a recently developed, extremely potent bisphosphonate, on the immune functions of human peripheral blood mononuclear cells (PBMCs). PBMC proliferation induced by lectins, alloantigens, and a nominal antigen (tetanus toxoid) was inhibited in a dose-dependent manner by alendronate. Pretreatment of monocytes, but not T cells, with the compound at concentrations ranging from 10(-4) to 10(-8) M was inhibitory, indicating that alendronate acts selectively on antigen-presenting cells (APCs). Alendronate did not affect the viability of monocytes or T cells or the expression of cell surface molecules known to play critical roles in antigen presentation. Alendronate exhibited dose-dependent inhibition of the production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) by activated monocytes. The inhibitory effect of 10(-6) M alendronate on PBMC proliferation was reversed by 10 U/ml recombinant rIL-1 beta, whereas other cytokines such as IL-6, TNF-alpha, and granulocyte-macrophage colony-stimulating factor (GM-CSF) had no effect. Thus, alendronate acts on monocytes to inhibit their antigen-presenting/accessory cell functions through a mechanism that can be overcome by exogenous IL-1. The inhibitory effect of this agent on cytokine production may contribute to its inhibitory effect on bone resorption.

  View details for Web of Science ID A1995TD33700014

  View details for PubMedID 8592949

• CHARACTERIZATION OF THE EPITOPE RECOGNIZED BY A MAB THAT REACTS DIFFERENTIALLY WITH MURINE SUPPRESSOR T-CELLS INTERNATIONAL IMMUNOLOGY Kapp, J. A., Pierce, C. W., Webb, D. R., Devens, B., Godfrey, W., FUKUSE, S., Engleman, E., Lake, J. P., MAGNANI, J. I., Maiti, P. K., Sehon, A. 1995; 7 (8): 1319-1330

  Abstract

  Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

  View details for Web of Science ID A1995RP59000017

  View details for PubMedID 7495739

• REQUIREMENT FOR NATURAL-KILLER-CELLS IN THE INDUCTION OF CYTOTOXIC T-CELLS JOURNAL OF IMMUNOLOGY Kos, F. J., Engleman, E. G. 1995; 155 (2): 578-584

  Abstract

  Cell-mediated immunity involves the participation of both regulatory and cytotoxic cells. The conversion of precursors to effector CD8+ cytotoxic T (Tc) cells requires cell-cell collaboration in which CD4+ T cells are traditionally viewed as helper cells. An in vitro system was used here to demonstrate that the generation of human alloantigen-specific CD8+ Tc cells requires the participation of CD3-CD16+CD56+ NK cells but not CD4+ T helper cells. Depletion of NK cells from responders abolished the induction of alloantigen-specific Tc cells in mixed lymphocyte cultures (MLC). Purified CD5+CD8+ T cells stimulated with alloantigen proliferated but did not differentiate into fully functional effector Tc cells. Coculture of responder CD5+CD8+ T cells with NK cells promoted the conversion of CD8+ Tc cell precursors (pTc) into effector Tc cells. Anti-CD56 mAbs blocked Tc cell induction in MLC, suggesting a role for CD56 molecules expressed on NK cells in either alloantigen recognition or delivery of accessory signals to pTc cells. These findings suggest a novel critical link between the natural and specific immune responses.

  View details for Web of Science ID A1995RG97300009

  View details for PubMedID 7541820

• ROLE OF B70/B7-2 IN CD4(+) T-CELL IMMUNE-RESPONSES INDUCED BY DENDRITIC CELLS IMMUNOLOGY Fagnoni, F. F., Takamizawa, M., Godfrey, W. R., Rivas, A., Azuma, M., Okumura, K., Engleman, E. G. 1995; 85 (3): 467-474

  Abstract

  Dendritic cells (DC) are potent antigen-presenting cells (APC). However, the molecular basis underlying this activity remains incompletely understood. To address this question, we generated murine monoclonal antibodies (mAb) against human peripheral blood-derived DC. One such antibody, designated IT209, stained differentiated DC and adherent monocytes, but failed to stain freshly isolated peripheral blood mononuclear cells (PBMC). The antigen recognized by IT209 was identified as B70 (B7-2; also recently identified as CD86). Using this mAb we studied the role of B70 in CD4+ T-cell activation by DC in vitro. IT209 partly inhibited the proliferative response of CD4+ T cells to allogeneic DC and to recall antigens, such as tetanus toxoid (TT) and purified protein derivative (PPD) of tuberculin, presented by autologous DC. More importantly, the mAb had a potent inhibitory effect on the primary response of CD4+ T cells to autologous DC pulsed with human immunodeficiency virus (HIV) gp160 or keyhole limpet haemocyanin (KLH). Adherent monocytes, despite their expression of B70, failed to induce T-cell responses to these antigens. IT209-mediated inhibition of CD4+ T-cell responses was equivalent to that produced by anti-CD25 mAb, whereas an anti-CD80 mAb was only marginally inhibitory and did not augment the effect of IT209. These findings indicate that the B70 antigen plays an important role in DC-dependent CD4+ T-cell activation, particularly in the induction of primary CD4+ T-cell responses to soluble antigens. However, since activated monocytes, despite their expression of B70, failed to prime naive T cells to these antigens, our results suggest that additional molecules contribute to the functions of DC in CD4+ T-cell activation.

  View details for Web of Science ID A1995RK30100018

  View details for PubMedID 7558137

• GENERATION OF ANTIGEN-SPECIFIC CD4(+) T-CELL LINES FROM NAIVE PRECURSORS EUROPEAN JOURNAL OF IMMUNOLOGY MEHTADAMANI, A., Markowicz, S., Engleman, E. G. 1995; 25 (5): 1206-1211

  Abstract

  The conditions required for sensitizing naive T cells to nominal antigen are poorly understood. In this report we describe an in vitro system for generating antigen-specific CD4+ T cells from previously unprimed individuals. Freshly isolated CD4+ T cells were cultured with keyhole limpet hemocyanin (KLH), sperm whale myoglobin (SWM), or human immunodeficiency virus (HIV) gp160, antigens to which most persons have not been sensitized, in the presence of either dendritic cells (DC) or macrophages (M phi). In short-term (< 8 days) cultures, CD4+ T cells or their CD4+, CD45RA (naive) subpopulation mounted significant proliferative responses to KLH, SWM, and HIV gp160, but only if the antigens were presented by DC. In contrast, CD4+, CD45RO (memory) T cells responded poorly to these antigens, although they responded vigorously to tetanus toxoid, a recall antigen, presented by either DC or M phi. KLH- and SWM-specific CD4+ T cell lines were established from the starting population that had been sensitized in vitro, following repeated stimulation with antigen and M phi in medium supplemented with interleukin-2 and interleukin-4. Despite the continued presence of these cytokines during T cell expansion, the expanded lines retained their ability to respond to the priming antigen in the absence of exogenous cytokines. When the CD45RA and CD45RO subpopulations were sensitized and expanded separately, the CD45RA cells alone gave rise to antigen-specific T cell lines, while the CD45RO cells proliferated nonspecifically. These results demonstrate that human naive CD4+ T cells can be sensitized in vitro to nominal antigens presented by DC and that the sensitized cells can be expanded into long-term lines that retain their antigen specificity.

  View details for Web of Science ID A1995RC85900010

  View details for PubMedID 7774624

• V7, A NOVEL LEUKOCYTE SURFACE PROTEIN THAT PARTICIPATES IN T-CELL ACTIVATION .2. MOLECULAR-CLONING AND CHARACTERIZATION OF THE V7 GENE JOURNAL OF IMMUNOLOGY Ruegg, C. L., Rivas, A., Madani, N. D., ZEITUNG, J., Laus, R., Engleman, E. G. 1995; 154 (9): 4434-4443

  Abstract

  V7 is a cell surface glycoprotein expressed on Ag-activated T cells, monocytes, and granulocytes, as well as subpopulations of T cells and accessory cells present in thymic medulla and tonsil. A mAb directed against V7 inhibits the proliferative response of T cells to allogeneic cells or immobilized anti-CD3 Ab, but not lectin mitogens, suggesting that V7 plays a role in TCR/CD3-mediated T cell activation. We have used the anti-V7 Ab in eukaryotic expression cloning experiments to isolate a cDNA clone containing a 3,340-bp insert that encodes V7 when transiently expressed in simian and murine fibroblastoid cells. DNA sequence analysis revealed a novel 1,021-amino acid open reading frame the structure of which conforms to the category of type I integral membrane proteins. The protein sequence includes a 20-residue putative hydrophobic signal sequence followed by a putative extracellular domain of 934 amino acids, a prototypic hydrophobic transmembrane spanning a domain of 25 residues, and finally a short and highly charged putative cytoplasmic domain of 42 residues. The extracellular domain contains seven pairs of regularly spaced cysteine residues, suggestive of Ig-like domains. On the basis of statistical analysis of the sequences of the putative cysteine loops, all seven of the Ig-like domains belong to the variable, or V-type, category. By using fluorescence in situ hybridization, we have mapped the V7 gene to human chromosome Ip13. Thus, the V7 glycoprotein represents a novel member of the Ig superfamily that is involved in critical intracellular signals essential for immune function.

  View details for Web of Science ID A1995QU82500022

  View details for PubMedID 7722300

• V7, A NOVEL LEUKOCYTE SURFACE PROTEIN THAT PARTICIPATES IN T-CELL ACTIVATION .1. TISSUE DISTRIBUTION AND FUNCTIONAL-STUDIES JOURNAL OF IMMUNOLOGY Rivas, A., Ruegg, C. L., ZEITUNG, J., Laus, R., Warnke, R., Benike, C., Engleman, E. G. 1995; 154 (9): 4423-4433

  Abstract

  Among a panel of mouse mAbs generated to a human T cell clone, one mAb, V7.1, inhibited T cell activation in the mixed lymphocyte reaction and was studied further. V7.1 reacted strongly with Ag-specific T cell clones, in addition to freshly isolated monocytes and granulocytes. However, the mAb reacted weakly with freshly isolated PBLs (T cells, B cells, and NK cells), T cells stimulated with phytohemagglutinin, or Con A, and did not stain the vast majority of transformed cell lines of hemopoietic origin. Stimulation of T cells with anti-CD3, or the combination of anti-CD3 and PMA, or anti-CD3, PMA and ionomycin, markedly increased V7.1 surface staining. The mAb precipitated a single polypeptide chain of approximately 135 kDa from alloactivated T cells or monocytes, which was reduced to approximately 110 kDa after treatment with N-glycanase. The proliferative response of T cells to allogeneic monocytes or B lymphoblastoid cells was inhibited by V7.1, and inhibition was maximal when the mAb was present at the initiation of culture. V7.1 also exhibited dose-dependent inhibition of the T cell response to immobilized anti-CD3 Ab in the absence of APCs, indicating that the inhibitory effect of this Ab occurs at the T cell level. Expression of CD25 (IL-2R) on anti-CD3-activated T cells and secretion of IL-2 induced with anti-CD3 and PMA were inhibited by V7.1, whereas the Ab had no effect on T cell proliferation induced by PHA or Con A or on T cell-mediated cytotoxicity. These results indicate that V7.1 recognizes a novel leukocyte surface glycoprotein, designated V7, that is up-regulated on Ag but not lectin-activated T cells, and appears to play a role in TCR/CD3-dependent T cell activation. In an accompanying study, the gene encoding the V7 Ag is described and the molecule is shown to be a novel member of the Ig superfamily.

  View details for Web of Science ID A1995QU82500021

  View details for PubMedID 7722299

• ANTIGEN-PULSED DENDRITIC CELLS IN THE TREATMENT OF PATIENTS WITH B-CELL LYMPHOMA Hsu, F. J., Engleman, E., Benike, C., Fagnoni, F., Czerwinski, D., LILES, T., Taidi, B., Levy, R. WILEY-BLACKWELL. 1995: 27–27

  View details for Web of Science ID A1995QT86400091

• LOW SERUM LEVELS OF DEHYDROEPIANDROSTERONE MAY CAUSE DEFICIENT IL-2 PRODUCTION BY LYMPHOCYTES IN PATIENTS WITH SYSTEMIC LUPUS-ERYTHEMATOSUS (SLE) CLINICAL AND EXPERIMENTAL IMMUNOLOGY Suzuki, T., Suzuki, N., Engleman, E. G., Mizushima, Y., Sakane, T. 1995; 99 (2): 251-255

  Abstract

  The principal cause of IL-2 deficiency, a common feature of both murine lupus and human SLE, remains obscure. Recent studies of our own as well as others have shown that dehydroepiandrosterone (DHEA), an intermediate compound in testosterone synthesis, significantly up-regulates IL-2 production of T cells, and that administration of exogenous DHEA or IL-2 via a vaccinia construct to murine lupus dramatically reverses their clinical autoimmune diseases. Thus, we have examined serum levels of DHEA in patients with SLE to test whether abnormal DHEA activity is associated with IL-2 deficiency of the patients. We found that nearly all of the patients examined have very low levels of serum DHEA. The decreased DHEA levels were not simply a reflection of a long term corticosteroid treatment which may cause adrenal atrophy, since serum samples drawn at the onset of disease, which are devoid of corticosteroid treatment, also contained low levels of DHEA. In addition, exogenous DHEA restored impaired IL-2 production of T cells from patients with SLE in vitro. These results indicate that defects of IL-2 synthesis of patients with SLE are at least in part due to the low DHEA activity in the serum.

  View details for Web of Science ID A1995QG10400018

  View details for PubMedID 7851019

• PREVENTION OF AIDS TRANSMISSION THROUGH SCREENING OF THE BLOOD-SUPPLY ANNUAL REVIEW OF IMMUNOLOGY Galel, S. A., Lifson, J. D., Engleman, E. G. 1995; 13: 201-227

  Abstract

  Shortly after the first cases of AIDS were reported in 1981, it became apparent that this disease was caused by a blood-borne infectious agent that could be transmitted by blood transfusion. Early reports documented a reduced ratio of CD4+ to CD8+ T cells not only in AIDS patients but also in likely carriers of the AIDS pathogen. On this basis, from July 1983 to June 1985, our blood center utilized flow cytometry to test each donated unit for the ratio of CD4 to CD8 T cells; we did not transfuse blood from donors with CD4/CD8 < 0.85. Despite available data supporting the utility of this or other surrogate blood tests to screen blood donors, the vast majority of blood banks did not initiate blood donor testing for AIDS until 1985, following the discovery of HIV and development of commercial HIV antibody tests. Retrospective analysis indicates that donor screening with surrogate markers would have removed the majority of AIDS-contaminated units from the blood supply and prevented a substantial fraction of the estimated 10,000 cases of transfusion-transmitted AIDS in the United States. In this report, we review the events that led to our decision to initiate blood donor testing prior to the identification of HIV, the results of such testing, the consequences of the decision by most blood banks not to initiate such testing, the current status of the blood supply with respect to HIV, and steps that can be taken in the future to protect the blood supply from new pathogens.

  View details for Web of Science ID A1995QV29000008

  View details for PubMedID 7612221

• Glucans as immunological adjuvants 8th International Symposium on the Immunobiology of Proteins and Peptides Mohagheghpour, N., Dawson, M., Hobbs, P., Judd, A., WINANT, R., Dousman, L., Waldeck, N., HOKAMA, L., Tuse, D., Kos, F., Benike, C., Engleman, E. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1995: 13–22

  View details for Web of Science ID A1995BE37X00003

  View details for PubMedID 8644497

• ROLE OF HIV ANTIGEN-PULSED DENDRITIC CELLS ON THE IMMUNOTHERAPY OF AIDS Benike, C. J., Hsu, F., Levy, R., Kundu, S., Merigan, T., Engleman, E. G. MARY ANN LIEBERT INC. 1995: S172–S172

  View details for Web of Science ID A1995RQ68900428

• CELLULAR AND MOLECULAR-BASIS OF HUMAN GAMMA-DELTA T-CELL ACTIVATION ROLE OF ACCESSORY MOLECULES IN ALLOACTIVATION JOURNAL OF CLINICAL INVESTIGATION Takamizawa, M., Fagnoni, F., MEHTADAMANI, A., Rivas, A., Engleman, E. G. 1995; 95 (1): 296-303

  Abstract

  Although gamma delta T cell receptor-bearing lymphocytes (gamma delta T cells) constitute a significant minority of circulating and tissue-associated T lymphocytes, the mechanism responsible for the activation of these cells is unknown. To address this question, resting gamma delta TCR+, CD3+, CD4-, CD8- cells isolated from the blood of healthy volunteers were cultured with allogeneic dendritic cells (DC) or monocytes, and their proliferative response measured. DC alone induced gamma delta T cells to proliferate, with a peak response on the sixth day of culture. Pretreatment of DC with an anti-HLA-DR mAb, but not anti-HLA class I or anti-CD1 mAbs, inhibited the response of gamma delta T cells. Antibodies to gamma delta T cell receptor, CD2, CD3, or CD11a were also inhibitory, whereas antibodies to alpha beta T cell receptor, CD4, CD5, and CD8 had no effect. Although only 40-60% of freshly isolated gamma delta T cells expressed CD28, mAbs directed against CD28 or its ligand, CD80, were markedly inhibitory. Moreover, removal of CD28+ cells from the gamma delta T cell population nearly abrogated the response to DC. These results demonstrate that resting gamma delta T cells recognize and respond to MHC class II determinants on allogeneic DC in a manner that is highly dependent on the CD28 activation pathway as well as molecules such as CD2 and CD11a that mediate cell-to-cell adhesion.

  View details for Web of Science ID A1995QA88200040

  View details for PubMedID 7814628

• ANTIGEN-PULSED DENDRITIC CELLS ARE EFFECTIVE IN INDUCING IMMUNE-RESPONSES IN PATIENTS WITH B-CELL LYMPHOMA Hsu, F. J., Engleman, E., Benike, C., FAGNORIL, F., Czerwinski, D., LILES, T., Taidi, B., Levy, R. AMER SOC HEMATOLOGY. 1994: A520–A520

  View details for Web of Science ID A1994PR75402061

• AN OPEN STUDY OF DEHYDROEPIANDROSTERONE IN SYSTEMIC LUPUS-ERYTHEMATOSUS ARTHRITIS AND RHEUMATISM VANVOLLENHOVEN, R. F., Engleman, E. G., McGuire, J. L. 1994; 37 (9): 1305-1310

  Abstract

  To determine if dehydroepiandrosterone (DHEA) has clinical benefits in patients with systemic lupus erythematosus (SLE).Ten female patients with mild to moderate SLE and various disease manifestations were given DHEA (200 mg/day orally) for 3-6 months. The patients were given other medications as clinically indicated, and followed with respect to overall disease activity and specific outcome parameters.After 3-6 months of DHEA treatment, indices for overall SLE activity including the SLEDAI (SLE Disease Activity Index) score and physician's overall assessment were improved, and corticosteroid requirements were decreased. Of 3 patients with significant proteinuria, 2 showed marked and 1 modest reductions in protein excretion. DHEA was well tolerated, the only frequently noted side effect being mild acneiform dermatitis.DHEA shows promise as a new therapeutic agent for the treatment of mild to moderate SLE. Further studies of DHEA in the treatment of SLE are warranted.

  View details for Web of Science ID A1994PJ93800005

  View details for PubMedID 7945493

• IDENTIFICATION OF A HUMAN OX-40 LIGAND, A COSTIMULATOR OF CD4+ T-CELLS WITH HOMOLOGY TO TUMOR-NECROSIS-FACTOR JOURNAL OF EXPERIMENTAL MEDICINE Godfrey, W. R., Fagnoni, F. F., HARARA, M. A., Buck, D., Engleman, E. G. 1994; 180 (2): 757-762

  Abstract

  The human OX-40 cell surface antigen is a CD4+ T cell activation marker that acts as a costimulatory receptor and is a member of the nerve growth factor receptor/tumor necrosis factor (TNF) receptor family. Using a soluble form of the receptor, the extracellular region fused with human immunoglobulin Fc, we expression cloned the human OX-40 ligand cDNA from a library derived from an activated B lymphoblastoid cell line MSAB. The encoded protein is identified as gp34, a type II transmembrane antigen previously known to be expressed only by human T cell lymphotropic virus 1-infected cells. We describe gp34 as a new member of the TNF family, and find that the recombinant ligand expressed in COS cells costimulates phorbol myristate acetate, phytohemagglutinin, and anti-CD3-induced CD4+ T cell proliferation.

  View details for Web of Science ID A1994NZ38500041

  View details for PubMedID 7913952

• GENERATION OF ANTIGEN-SPECIFIC CD8+ CTLS FROM NAIVE PRECURSORS JOURNAL OF IMMUNOLOGY MEHTADAMANI, A., Markowicz, S., Engleman, E. G. 1994; 153 (3): 996-1003

  Abstract

  Class I MHC-restricted CTLs are an important component of the host immune response against viral infections, and CTL effectors can often be isolated from infected individuals. However, the mechanism responsible for the induction of CTLs is incompletely understood because, in part, of the difficulty in generating such cells in vitro from naive precursors. In the present study we have used human peripheral blood dendritic cells (DCs), devoid of CD4+ T cells, to sensitize naive CD8+ T cells to exogenous Ags, resulting in the generation of Ag specific CTL effectors. With this system, Ag-specific CTL lines were generated to a complex glycoprotein, keyhole limpet hemocyanin, and to multiple small (9-15 amino acids) synthetic peptides derived from conserved regions of the HIV-1 gag and envelope proteins. The HIV-1-specific CTLs demonstrated potent HLA class I restricted killing of both Ag pulsed and virally infected target cells. In contrast to Ag-pulsed DCs, Ag-pulsed monocytes failed to sensitize CTL precursors although they could be used as feeders for purposes of CTL expansion and as target cells in cytolytic assays. With the use of the system described herein, a detailed analysis of the primary human T cell response to foreign Ags is now feasible, and CTL of desired specificity can be generated for potential clinical use in adoptive immunotherapy protocols.

  View details for Web of Science ID A1994NY34200010

  View details for PubMedID 8027569

• SIGNIFICANCE OF PERSISTENT CD4 LYMPHOCYTOPENIA IN VOLUNTEER BLOOD-DONORS Galel, S. A., Gonzalez, M., WOLLES, S., Lifson, J., Engleman, E. G. WILEY-BLACKWELL. 1993: S51–S51

  View details for Web of Science ID A1993LZ44700194

• TREATMENT OF SYSTEMIC LUPUS-ERYTHEMATOSUS WITH DEHYDROEPIANDROSTERONE - INTERIM ANALYSIS OF A DOUBLE-BLINDED, RANDOMIZED, PLACEBO-CONTROLLED, CLINICAL-TRIAL VANVOLLENHOVEN, R. F., Engleman, E. G., Lambert, R. E., Lee, Y. S., McGuire, J. L. WILEY-BLACKWELL. 1993: S92–S92

  View details for Web of Science ID A1993MB81600321

• TREATMENT OF SYSTEMIC LUPUS-ERYTHEMATOSUS WITH DEHYDROEPIANDROSTERONE FOLLOW-UP FROM AN OPEN-LABEL CLINICAL-TRIAL VANVOLLENHOVEN, R. F., Lambert, R. E., Lee, Y. S., McGuire, J. L., Engleman, E. G. WILEY-BLACKWELL. 1993: S228–S228

  View details for Web of Science ID A1993MB81601118

• HUMAN IMMUNODEFICIENCY VIRUS-INFECTED MONOCYTE-DERIVED MACROPHAGES EXPRESS SURFACE GP120 AND FUSE WITH CD4 LYMPHOID-CELLS INVITRO - A POSSIBLE MECHANISM OF LYMPHOCYTE-T DEPLETION INVIVO CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Crowe, S. M., Mills, J., Elbeik, T., Lifson, J. D., Kosek, J., Marshall, J. A., Engleman, E. G., McGrath, M. S. 1992; 65 (2): 143-151

  Abstract

  Monocyte-derived macrophages (MDM) infected in vitro with a macrophage-tropic strain of human immunodeficiency virus (HIV) fused with uninfected, CD4-expressing T lymphoblastoid cells, but not with a subclone of these cells lacking surface CD4. Infected MDM also fused with uninfected autologous and heterologous MDM. Recombinant soluble CD4 protein (rsCD4) (10 micrograms/ml) and full-length recombinant glycosylated gp120 (20 micrograms/ml) each inhibited fusion by 94-99%; the inhibition was dose-dependent. The N-terminal portion of gp120 did not inhibit syncytium formation. Fusion was also inhibited by a monoclonal antibody to an epitope which binds gp120 (S3.5), but not by antibody to an epitope not involved in gp120 binding (OKT4). HIV-infected MDM specifically bound fluorescein-conjugated rsCD4, and virus could be visualized budding from the surface of these cells. HIV-infected MDM express viral gp120 on their surface and fuse with CD4-bearing cells in a fashion similar to lymphoid cells. Macrophages may contribute to CD4 lymphocyte depletion in vivo by this fusion mechanism.

  View details for Web of Science ID A1992JU44000008

  View details for PubMedID 1356673

• DEGRADATION OF CD4 FOLLOWING PHORBOL-INDUCED INTERNALIZATION IN HUMAN LYMPHOCYTES-T - EVIDENCE FOR DISTINCT ENDOCYTIC ROUTING OF CD4 AND CD3 JOURNAL OF BIOLOGICAL CHEMISTRY Ruegg, C. L., Rajasekar, S., Stein, B. S., Engleman, E. G. 1992; 267 (26): 18837-18843

  Abstract

  Exposure of T lymphocytes to phorbol esters induces endocytosis of CD4 and the CD3/T-cell receptor complex. We compared the pathway of CD4 internalization to that of CD3 following activation of human T lymphocytes with phorbol 12,13-dibutyrate (PDBu). Both CD3 and CD4 were rapidly internalized in response to PDBu, but only CD3, and not CD4, was recycled to the cell surface after removal of PDBu. In support of a degradative fate for internalized CD4, radioimmuno-precipitation studies revealed that the total amount of cellular CD4 was reduced by greater than 90% after exposure to PDBu for 4 h, whereas total CD3 remained constant. PDBu induced CD4 capping and localization consistent with sequestration in intracellular vesicles, presumably lysosomes, prior to becoming degraded. Lysosomotropic agents, such as NH4Cl, chloroquine, and monensin inhibited CD4 degradation, consistent with a lysosomal fate for CD4. Internalization and degradation of CD4 was blocked by staurosporine, an inhibitor of protein kinase C suggestive of a role for protein kinase C in the endocytic fate of CD4. The results of this study demonstrate that CD3 and CD4 follow distinct endocytic pathways which may be relevant to their having distinct roles in T cell activation and function.

  View details for Web of Science ID A1992JN50200089

  View details for PubMedID 1527011

• TREATMENT OF SYSTEMIC LUPUS-ERYTHEMATOSUS WITH DEHYDROEPIANDROSTERONE - A PILOT-STUDY VANVOLLENHOVEN, R. F., Lee, Y. S., Lambert, R. E., Engleman, E. G., McDevitt, H. O., McGuire, J. L. WILEY-LISS. 1992: S55–S55

  View details for Web of Science ID A1992JR15800126

• DEHYDROEPIANDROSTERONE ENHANCES IL2 PRODUCTION AND CYTOTOXIC EFFECTOR FUNCTION OF HUMAN T-CELLS CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Suzuki, T., Suzuki, N., Daynes, R. A., Engleman, E. G. 1991; 61 (2): 202-211

  Abstract

  Dehydroepiandrosterone (DHEA) is the most abundant adrenal steroid hormone in humans. Although it is well established that DHEA serves as an intermediate in sex steroid synthesis, recent studies in mice suggest that DHEA may also be a physiologic regulator of IL2 secretion. To explore the effect of DHEA on the human immune system, T lymphocytes from healthy adults were exposed to DHEA followed by stimulation with mitogens or antigen. Upon activation with a variety of stimuli, T cells pretreated with 10(-8) to 10(-11) M DHEA produced significantly greater amounts of IL2 and mediated more potent cytotoxicity than T cells activated in the absence of this steroid hormone. The peak effect of DHEA was observed at 10(-9) M, the concentration of hormone present in the blood of normal adults. In contrast to its effect on murine T cells, the IL2 enhancing effect of DHEA on human lymphocytes was limited to fresh CD4+ T cells and CD4+ clones; neither fresh CD8+ cells nor CD8+ clones were directly affected by DHEA treatment, although CD8+ cells stimulated in the presence of CD4+ cells and DHEA demonstrated enhanced cytotoxicity. The enhancing effect of DHEA was also detected at the level of IL2 mRNA, suggesting that DHEA may act as a transcriptional enhancer of the IL2 gene in CD4+ T cells. These results corroborate and extend earlier studies in mice and suggest a physiologic role for DHEA in regulating the human immune response.

  View details for Web of Science ID A1991GM40100006

  View details for PubMedID 1833106

• INTERRELATIONSHIPS BETWEEN MEVALONATE METABOLISM AND THE MITOGENIC SIGNALING PATHWAY IN LYMPHOCYTE-T PROLIFERATION JOURNAL OF BIOLOGICAL CHEMISTRY Chakrabarti, R., Engleman, E. G. 1991; 266 (19): 12216-12222

  Abstract

  Upon stimulation with antigen or antibodies directed at the CD3.T cell receptor complex, T lymphocytes undergo a series of biochemical events that result in DNA synthesis and cellular proliferation. The purpose of the current study was to explore the role of mevalonic acid and its metabolites in this process. Stimulation of freshly isolated human T cells with immobilized anti-CD3 monoclonal antibody (mAb) results in the induction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase message, with maximum induction occurring at 24 h of culture, approximately 12 h before the onset of DNA synthesis. Protein kinase C (PKC) probably mediates this induction, as H7, which inhibits PKC and cyclic nucleotide-dependent protein kinases, but not HA1004, which inhibits all of these protein kinases except PKC, completely abrogates the appearance of HMG-CoA reductase message. The importance of HMG-CoA reductase induction and mevalonate production in cell cycle progression was demonstrated by the observation that either 25-hydroxycholesterol, which inhibits this induction, or lovastatin, a competitive inhibitor of HMG-CoA reductase, inhibited anti-CD3-induced T cell mitogenesis in a dose-dependent manner. The presence of lovastatin during the first 24-36 h of culture results in a progressive delay of cell cycle progression, whereas this agent, when present only for the first 12 h of culture, had no effect on T cell proliferation. These results suggest that mevalonate is required for cell cycle progression from mid-G1 into late G1. Exogenous mevalonate overcomes the antiproliferative effect of lovastatin but not of 25-hydroxycholesterol. Since 25-hydroxycholesterol suppresses the metabolism of mevalonic acid at multiple points, this result suggests that one or more metabolites of mevalonate, rather than mevalonate itself, plays an essential role in cell cycle progression. One metabolite of mevalonate, farnesol pyrophosphate, may play such a role, since free farnesol suppresses anti-CD3 mAb-induced T cell proliferation in a concentration-dependent manner. In mAb is associated with PKC-dependent induction of HMG-CoA reductase which, in turn, leads to the generation of mevalonic acid and its metabolites, one or more of which play a requisite role in cell cycle progression.

  View details for Web of Science ID A1991FV18000021

  View details for PubMedID 1712015

• EVIDENCE FOR THE INVOLVEMENT OF CD56 MOLECULES IN ALLOANTIGEN-SPECIFIC RECOGNITION BY HUMAN NATURAL-KILLER-CELLS JOURNAL OF EXPERIMENTAL MEDICINE Suzuki, N., Suzuki, T., Engleman, E. G. 1991; 173 (6): 1451-1461

  Abstract

  In recent reports we have described the generation of natural killer (NK) lines devoid of CD3/TCR structures but with apparent specificity for allogeneic target cells. Using one such NK line as an immunogen, we now report the generation of two monoclonal antibodies (mAbs), designated 2-13 and 5-38, which bind selectively to the majority of CD3-, CD16+, CD56+ lymphocytes and inhibit the lysis of specific allogeneic target cells by a panel of alloreactive NK lines. By contrast, these mAbs had no effect on classical NK cell mediated lysis of K562 cells or major histocompatibility-restricted T cell-mediated cytolysis. Immunoprecipitation of radiolabeled NK lines followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the target molecules of both mAbs have a molecular mass of approximately 180 kD. Leu 19, a well-described anti-CD56 mAb, precipitated a 180 kD protein from NK cells, and the binding of Leu 19 to NK cells was blocked by pretreatment with both 2-13 and 5-38. However, in contrast to these mAbs, Leu 19 had no effect on the cytolytic activity of allospecific NK cells. Sequential immunoprecipitation analysis revealed that all three mAbs recognized distinct molecular species of CD56. We interpret these findings as indicating that multiple isoforms of CD56 are differentially expressed on NK lines and play critical roles in the recognition/interaction of these cells with their specific allogeneic targets.

  View details for Web of Science ID A1991FN85900016

  View details for PubMedID 1709676

• EARLY ACTIVATION EVENTS RENDER T-CELLS SUSCEPTIBLE TO HIV-1-INDUCED SYNCYTIA FORMATION - ROLE OF PROTEIN-KINASE-C JOURNAL OF BIOLOGICAL CHEMISTRY Mohagheghpour, N., Chakrabarti, R., Stein, B. S., GOWDA, S. D., Engleman, E. G. 1991; 266 (11): 7233-7238

  Abstract

  In human immunodeficiency virus-1 (HIV-1)-infected cell cultures, cell-to-cell fusion and the formation of multinucleated giant cells (syncytia) are induced as a consequence of interactions between the viral envelope glycoprotein on infected cells and cell surface CD4 molecules on uninfected cells. Although activated CD4+ T cells rapidly form syncytia when cultured with HIV-1 envelope glycoprotein expressing (env+) cells, freshly isolated, unstimulated CD4+ T cells do so more slowly. In these studies, we sought to explore the role of T cell activation in rendering CD4+ T cells susceptible to HIV-1-mediated syncytia formation. Our results indicate that within 2 h of exposure to immunologic stimuli, CD4+ T cells acquire the ability to form syncytia with HIV-1 env+ cells. Both cholera toxin, an inhibitor of protein kinase C (PKC) through its effects on inositol triphosphate and diacylglycerol production, and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, a noncompetitive inhibitor (with respect to ATP) of PKC, prevented unstimulated but not previously stimulated CD4+ T cells from forming syncytia with HIV-1 env+ cells. 1-Oleoyl-2-acetyl glycerol, an analog of the PKC activator, diacylglycerol, enhanced syncytia formation whereas ionomycin, a calcium ionophore, had no effect. These results suggest that activation of PKC is essential for previously unstimulated CD4+ T cells to become fusogenic.

  View details for Web of Science ID A1991FG72700091

  View details for PubMedID 1826686

• MECHANISM OF HIV-1 ENTRY INTO CD4+ T-CELLS WORKSHOP ON MECHANISMS AND SPECIFICITY OF HIV ENTRY INTO HOST CELLS Stein, B. S., Engleman, E. G. PLENUM PRESS DIV PLENUM PUBLISHING CORP. 1991: 71–96

  Abstract

  Although the mechanism responsible for HIV-1 entry into susceptible CD4+ T cells is incompletely understood, a number of key components are now known. For example, the tropism of HIV-1 for cells expressing the CD4 membrane glycoprotein reflects the use of this protein as a specific viral receptor to which the HIV-1 gp120 envelope protein binds with high affinity. This binding apparently results in the exposure of hydrophobic domains of the gp41 transmembrane protein to apposing plasma membrane components, resulting in the fusion of viral and plasma membranes to one another which, in turn, releases HIV-1 RNA into the cytosol. This fusion event, which is requisite for viral entry as well as HIV-1 associated syncytia formation, occurs in a pH-independent fashion, but requires antecedent T cell activation. In the absence of T cell stimuli, resting CD4+ cells are resistant to HIV-1 entry, which may explain the observation that at any given time the vast majority of CD4+ T cells in HIV-1 seropositive patients are not infected despite the presence of relatively large quantities of free virus in the blood of such patients. The mechanism of HIV-1 entry into other CD4+ cell types, such as macrophages and dendritic cells, remains to be determined.

  View details for Web of Science ID A1991BU56P00006

  View details for PubMedID 1685857

• IMPAIRED IMMUNITY IN AIDS - THE MECHANISMS RESPONSIBLE AND THEIR POTENTIAL REVERSAL BY ANTIVIRAL THERAPY ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Ruegg, C. L., Engleman, E. G. 1990; 616: 307-317

  Abstract

  The inability of CD4+ T cells of HIV-1-infected patients to mount an effective immune response is widely believed to explain the increased susceptibility of these patients to opportunistic infections. Although the full explanation for T-cell dysfunction in HIV-1 infection is not yet understood, at least two fundamentally distinct mechanisms are thought to contribute: depletion of CD4+ T cells and qualitative CD4+ T-cell dysfunction independent of T-cell depletion. Many HIV-1-infected patients manifest reduced T-cell responses to recall antigens prior to measurable CD4+ T-cell depletion, and among the proposed explanations for this phenomenon are gp120-mediated interference with T-cell activation by way of inhibition of CD4-class II major histocompatibility complex (MHC) determinant interactions, gp41-mediated inhibition of protein kinase C-dependent T-cell activation, formation of gp41 cross-reactive antibodies that react with MHC class II determinants, transforming growth factor-beta (TGF-beta)-mediated immunosuppression, and decreased functions of antigen-presenting and antigen-processing cells (macrophages and bone marrow-derived dendritic cells). Despite their detection in most HIV-1-infected patients, these qualitative T-cell defects do not herald the onset of life-threatening disease. The appearance of severe clinical manifestations of AIDS, particularly opportunistic infections, occurs primarily in patients whose CD4+ T-cell count is significantly reduced. Depletion of CD4+ T cells may be a direct consequence of HIV-1 infection that occurs as a result of syncytia formation, autoantibody-mediated cytolysis, gp120-specific antibody-dependent cytolysis, and/or gp120-specific T-cell mediated cytolysis. The thymus is severely affected in patients with late-stage disease, and although there is no proof that the failure of the thymus to regenerate new T cells contributes to T-cell depletion in patients with AIDS, the likelihood seems high that this is the case. Indeed, if prolonged suppression of HIV-1 replication can be achieved with newer anti-HIV drugs or combinations of drugs, reconstitution of a normal immune system seems likely, provided that the capacity to regenerate T cells has not been irrevocably lost as a consequence of viral infection. In summary, available evidence indicates that HIV-1 uses a complex array of mechanisms to disrupt T-cell mediated immunity, but because most of these involve a direct role for HIV-1 proteins, such mechanisms are likely to be reversible if suppression of HIV-1 replication can be achieved.

  View details for Web of Science ID A1990FM70900030

  View details for PubMedID 1981824

• PEPTIDES DERIVED FROM THE CDR3-HOMOLOGOUS DOMAIN OF THE CD4 MOLECULE ARE SPECIFIC INHIBITORS OF HIV-1 AND SIV INFECTION, VIRUS-INDUCED CELL-FUSION, AND POSTINFECTION VIRAL TRANSMISSION INVITRO - IMPLICATIONS FOR THE DESIGN OF SMALL-PEPTIDE ANTI-HIV THERAPEUTIC AGENTS ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Rausch, D. M., Hwang, K. M., Padgett, M., VOLTZ, A. H., Rivas, A., Engleman, E., Gaston, I., McGrath, M., Fraser, B., Kalyanaraman, V. S., NARA, P. L., Dunlop, N., Martin, L., MURPHEYCORB, M., KIBORT, T., Lifson, J. D., Eiden, L. E. 1990; 616: 125-148

  Abstract

  Peptides 12-25 amino acids in length from the V1J1 region of the CD4 molecule (residues 1-120) were synthesized as randomly derivatized, deliberately derivatized, or pure peptide products, and tested for their ability to inhibit HIV-1-induced cell fusion, HIV-1 and SIV infection of CD4-positive human cells, HIV-1 envelope glycoprotein binding to the CD4 molecule, CD4-neutralizing antibody binding to the CD4 holoreceptor, and CD4-dependent cellular immune function in the mixed lymphocyte and cytotoxic T-cell bioassays. Only peptides derived from the complementarity-determining region 3 (CDR3)-homologous domain of CD4, in particular CD4(81-92) and CD4(81-101), were effective antiviral agents. Within the CD4(81-92) series, R-group derivatization of selective amino acid residues was an absolute requirement for biological activity. The prototype compound T1C4E5-tribenzyl-K10-acetyl-TYICEVEDQKEE inhibited HIV-1-induced cell fusion at 32 microM, HIV-1 infection of CEM-SS cells at 10 microM, SIV infection of CEM-174 cells at less than 125 microM, gp120/CD4 binding at 60 microM, and postinfection cell-mediated viral transmission at 10-15 microM. Compounds of identical structure and derivatization, but of altered primary sequence, were substantially less active, or without activity, in these assays. These data indicate that the effect of amino acid derivatization of the CD4(81-92) peptide was most likely restriction of the flexible underivatized peptide backbone to a conformation closely approximating that of the CDR3-homologous gp120 binding site of the native CD4 molecule. Peptide antiviral activity was specific, as judged by lack of cytotoxicity, lack of inhibition of HTLV-1-induced cell fusion, and lack of inhibition of CD4-dependent cellular immune function in vitro. Further derivatization of the prototype compound involving the production of cyclic congeners yielded peptides with submicromolar potency to block HIV-1 infection, strengthening the hypothesis that previous peptide derivations accomplished partial restriction of the conformation of CD4(81-92) to one favorable for interaction with gp120. Concentrations of the original prototype compound T1C4E5-tribenzyl-CD4(81-92) that inhibited infection in vitro more than 50% could be achieved for several hours by intravenous infusion in primates and were well-tolerated at these levels. The peptide was not efficacious to inhibit establishment of viral infection at these doses; however, peptide treatment did lower average viral antigenemia and delay the cumulative time to morbidity relative to the control group.

  View details for Web of Science ID A1990FM70900013

  View details for PubMedID 2078014

• T-CELL RECEPTOR-NEGATIVE NATURAL-KILLER-CELLS DISPLAY ANTIGEN-SPECIFIC CYTOTOXICITY FOR MICROVASCULAR ENDOTHELIAL-CELLS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bender, J. R., Pardi, R., Engleman, E. 1990; 87 (18): 6949-6953

  Abstract

  Based upon prior demonstrations that human microvascular endothelial cells (ECs) could serve as natural killer (NK) cell targets, we established NK cell lines and clones by repeated stimulation of highly purified CD16-positive, CD3/T-cell receptor (Ti)-negative cells with allogeneic ECs. After 3 weeks in culture these lymphoid cells, which neither expressed surface CD3/Ti molecules nor rearranged Ti beta- or gamma-chain genes and which lysed K562 human erythroleukemia cells, displayed specific cytotoxicity for the stimulating ECs. Furthermore, freshly isolated NK cells bound and then removed from each of several allogeneic EC lines displayed selective cytotoxicity for the adsorbing EC line. These results provide evidence for alloantigen-specific recognition of microvascular ECs by NK cells that appears to be determined, at least in part, at the level of adherence. We discuss the implications of these findings with respect to the rejection of vascularized organ allografts.

  View details for Web of Science ID A1990DZ45300005

  View details for PubMedID 2402485

• NATURAL-KILLER LINES AND CLONES WITH APPARENT ANTIGEN-SPECIFICITY JOURNAL OF EXPERIMENTAL MEDICINE Suzuki, N., Bianchi, E., Bass, H., Suzuki, T., Bender, J., Pardi, R., Brenner, C. A., Larrick, J. W., Engleman, E. G. 1990; 172 (2): 457-462

  Abstract

  Fresh CD3-, CD16+ lymphocytes that adhered to selected allogeneic lymphoblastoid cell lines (LCL) were cultured with LCL in the presence of IL-2-containing medium. The resulting lines as well as clones derived from these lines expressed CD16 and/or CD56, but lacked detectable CD3 or TCR-alpha/beta or TCR-gamma/delta complexes on the cell surface. Northern blot analysis failed to detect CD3 epsilon or TCR-beta transcripts, but revealed the presence of a TCR-gamma chain transcript in one of these lines. In addition to displaying potent cytolytic activity against K562 erythroleukemia cells (a classical NK target), the vast majority of these lines and clones lysed their specific stimulator LCL to a significantly greater extent than irrelevant LCL. This selective killing was inhibited by the addition of cold stimulator LCL or K562 cells, or anti-LFA 1 mAbs, but not by irrelevant LCL or mAbs to CD3, class I or class II MHC antigens. These results indicate that some CD3- lymphocytes, phenotypically indistinguishable from NK cells, can recognize and lyse allogeneic targets in a specific manner.

  View details for Web of Science ID A1990DQ70400006

  View details for PubMedID 2142719

• ALLOANTIGEN-SPECIFIC CYTOTOXIC CLONES BEARING THE ALPHA,BETA-T-CELL ANTIGEN RECEPTOR BUT NOT CD4 OR CD8 MOLECULES JOURNAL OF IMMUNOLOGY Rivas, A., Koide, J., Laus, R., Engleman, E. G. 1990; 145 (2): 470-476

  Abstract

  The vast majority of circulating lymphocytes that express the alpha,beta TCR in association with CD3 also express either CD4 or CD8 molecules, which are thought to act as important accessory structures in HLA class II- and I-restricted T cell functions, respectively. In the current study alpha,beta TCR+ clones devoid of detectable CD4 or CD8 were generated by repeated stimulation of fresh CD3+,CD4-,CD8- cells with an allogeneic lymphoblastoid cell line in the presence of conditioned medium containing IL-2. Except for the absence of CD4 and CD8, which was associated with undetectable levels of CD4 and CD8 mRNA, the clones were phenotypically indistinguishable from classical CD3+,alpha,beta TCR+ cells. Furthermore, they mediated potent cytolysis of their specific stimulator line but did not kill irrelevant LCL or NK-sensitive targets. mAb to CD3 and the alpha,beta TCR inhibited cytolysis, suggesting that the clones use the TCR/CD3 complex to recognize and respond to their targets. mAbs to CD2 and CD11a also inhibited cytolysis, indicating that the clones use these accessory molecules to interact with their targets. Finally, cytolysis was inhibited by an HLA-A,B,C framework-specific mAb (W6/32) as well as a mAb (MA2.1) specific for an HLA-A2 epitope. These results demonstrate that CD3+,alpha,beta TCR+,CD4-,CD8- cytotoxic clones can be generated from the peripheral blood of healthy adults, and use their TCR/CD3 complexes to function in an HLA class I-restricted manner.

  View details for Web of Science ID A1990DN53100009

  View details for PubMedID 2142178

• GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR PROMOTES DIFFERENTIATION AND SURVIVAL OF HUMAN PERIPHERAL-BLOOD DENDRITIC CELLS-INVITRO JOURNAL OF CLINICAL INVESTIGATION Markowicz, S., Engleman, E. G. 1990; 85 (3): 955-961

  Abstract

  Interest in human dendritic cells (DC) has been heightened recently by the discovery that this cell type is a primary target of the human immunodeficiency virus, the causative agent of AIDS. DC are bone marrow-derived cells with an extraordinarily potent ability to promote the immunological activity of T lymphocytes. Unfortunately, since DC constitute less than 0.5% of peripheral blood mononuclear cells and die within a few days of their isolation, they are not readily accessible to study. We report here that granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine with well-recognized effects on granulocyte and macrophage maturation, profoundly affects the morphology and viability of DC isolated from peripheral blood. GM-CSF not only promotes DC survival but also induces DC differentiation to mobile, reversibly adherent cells with long-branched projections. DC cultured in GM-CSF survive for up to 6 wk and retain their ability to stimulate the proliferation of T cells in allogeneic and autologous mixed leukocyte reactions.

  View details for Web of Science ID A1990CT38200044

  View details for PubMedID 2179270

• EFFECTIVE VACCINATION OF MICE AGAINST LEPROSY BACILLI WITH SUBUNITS OF MYCOBACTERIUM-LEPRAE INFECTION AND IMMUNITY Gelber, R. H., Brennan, P. J., Hunter, S. W., Munn, M. W., Monson, J. M., MURRAY, L. P., Siu, P., Tsang, M., Engleman, E. G., Mohagheghpour, N. 1990; 58 (3): 711-718

  Abstract

  Model vaccines against leprosy bacilli have consisted of nonvirulent, live, attenuated Mycobacterium bovis BCG and irradiated, heat-killed, or autoclaved intact M. leprae. We report that immunization with various cell wall fractions of M. leprae, progressively depleted of lipids, carbohydrates, and soluble proteins, as well as a partially purified protein(s) derived from a pellet fraction of sonicated M. leprae, conferred significant protection against subsequent infection with live leprosy bacilli. Moreover, lymphocytes from regional lymph nodes and spleens of mice immunized with these M. leprae-derived subunits responded by proliferation when stimulated with M. leprae in vitro. Our results provide the first evidence that vaccination with M. leprae-derived fractions protects mice against leprosy bacilli.

  View details for Web of Science ID A1990CQ17900020

  View details for PubMedID 2407654

  View details for PubMedCentralID PMC258523

• IDENTIFICATION OF AN IMMUNOSTIMULATING PROTEIN FROM MYCOBACTERIUM-LEPRAE INFECTION AND IMMUNITY Mohagheghpour, N., Munn, M. W., Gelber, R. H., Engleman, E. G. 1990; 58 (3): 703-710

  Abstract

  Despite the recent identification of a number of Mycobacterium leprae proteins, the major immunogenic determinants of this organism remain obscure. We isolated from M. leprae a potent immunostimulatory preparation, designated the MLP fraction, which contains a major protein of 35 kilodaltons (kDa). This protein was precipitated by monoclonal antibody ML03-A1, which recognizes a 35-kDa protein of M. leprae, and by sera obtained from patients with lepromatous leprosy. Neither sera from healthy controls nor sera from patients with pulmonary tuberculosis recognized the 35-kDa protein, and only one of four serum samples from patients with borderline tuberculoid leprosy reacted with this protein. The MLP fraction stimulated T-cell proliferation in patients with leprosy whose T cells proliferate in response to whole M. leprae cells. Apparently, the T-cell epitope associated with MLP is also expressed on M. tuberculosis and M. bovis BCG, since patients with pulmonary tuberculosis and BCG-vaccinated individuals demonstrated significant responses to the MLP fraction. The 35-kDa M. leprae protein, purified to homogeneity in the laboratory of P. J. Brennan, stimulated T-cell proliferative responses in all MLP-responsive subjects. These findings suggest that the 35-kDa protein present in MLP is an immunostimulatory component of M. leprae. In addition to serving as a useful probe for study of the T-cell anergy associated with lepromatous disease, this protein may ultimately be useful as a component of a vaccine designed to provide protection against infection with M. leprae.

  View details for Web of Science ID A1990CQ17900019

  View details for PubMedID 2407653

• INTRACELLULAR PROCESSING OF THE GP-160 HIV-1 ENVELOPE PRECURSOR - ENDOPROTEOLYTIC CLEAVAGE OCCURS IN A CIS OR MEDIAL COMPARTMENT OF THE GOLGI-COMPLEX JOURNAL OF BIOLOGICAL CHEMISTRY Stein, B. S., Engleman, E. G. 1990; 265 (5): 2640-2649

  Abstract

  The intracellular processing of the gp160 HIV-1 envelope precursor was characterized in acutely infected CD4+ T cells. Our data show that gp160 undergoes endoproteolytic cleavage by a nonacid dependent protease(s) in the rough endoplasmic reticulum-Golgi complex, within cis or medial cisternae, and is not transported to the cell surface. Two-dimensional electrophoretic pulse-chase analysis indicates that it takes greater than 2 h for gp160 to be transported from the rough endoplasmic reticulum to the site of action of sialyltransferases in the trans Golgi. Evidence is presented that gp160 is subject to mannose trimming in the Golgi complex, which is inhibited by 1-deoxymannojirimycin (a specific Golgi alpha-mannosidase I inhibitor). Preliminary data also suggest that gp120 is post-translationally modified by sialylated O-linked oligosaccharides.

  View details for Web of Science ID A1990CM84800040

  View details for PubMedID 2406237

• ABNORMAL T-SUPPRESSOR CELL-FUNCTION IN JUVENILE RHEUMATOID-ARTHRITIS ARTHRITIS AND RHEUMATISM Silverman, E. D., Somma, C., Khan, M. M., Melmon, K. L., Engleman, E. G. 1990; 33 (2): 205-211

  Abstract

  The purpose of this study was to analyze T suppressor cell function in juvenile rheumatoid arthritis (JRA). JRA is a chronic inflammatory childhood disease of unknown etiology that is characterized by arthritis and immunoregulatory abnormalities. T suppressor cell precursors (CD8+, CD28-) were purified from the peripheral blood of 24 JRA patients, using a combination of monoclonal antibodies. These cells were treated with histamine or concanavalin A, agents that are known to induce suppressor activity. They were also tested for their ability to inhibit the proliferative response of autologous T cells to phytohemagglutinin. In some experiments, the accumulation of intracellular cAMP following histamine treatment was also measured. Twelve of 13 patients with clinically active JRA showed abnormal histamine-inducible T suppressor cell function, characterized by the failure of CD8+, CD28- T cells to mediate any detectable suppression. The failure of these cells to accumulate intracellular cAMP after histamine treatment was observed in 5 of 5 patients tested who had active disease. In contrast, 11 of 11 patients with clinically inactive JRA, 5 of 5 patients with cystic fibrosis, and 9 of 9 pediatric control subjects had normal histamine- and concanavalin A-inducible T suppressor cell function, and a normal cAMP response to histamine. These results suggest that patients with clinically active JRA have a reversible defect in T suppressor cell function that is associated with a failure of T suppressor cell precursors to accumulate intracellular cAMP following their exposure to selected immune stimuli.

  View details for Web of Science ID A1990CP51800007

  View details for PubMedID 2154996

• ANTI-DNA ANTIBODY-PRODUCTION BY CD5+ AND CD5- B-CELLS OF PATIENTS WITH SYSTEMIC LUPUS-ERYTHEMATOSUS JOURNAL OF CLINICAL INVESTIGATION Suzuki, N., Sakane, T., Engleman, E. G. 1990; 85 (1): 238-247

  Abstract

  Although the presence of anti-DNA antibody is a hallmark of systemic lupus erythematosus (SLE), neither the subsets of B cells that secrete anti-DNA antibody nor the stimuli responsible for the induction of anti-DNA secretion is known. In particular, the role of CD5+ B cells in human SLE, a distinct subpopulation of antibody-secreting cells shown previously to be a source of anti-DNA antibody in murine models of SLE, is unknown. To approach these questions, we developed a sensitive enzyme-linked immunospot (ELIspot) assay to measure spontaneous secretion of antibody to single-stranded (ss) DNA, double-stranded (ds) DNA, tetanus toxoid, and polyclonal immunoglobulin (Ig) by purified CD5+ and CD5- B cells of 15 SLE patients and 15 healthy control subjects. The B cells of only 1 of 15 healthy subjects secreted a significant level of anti-ssDNA antibody, and none secreted anti-dsDNA. By contrast, in the majority of SLE patients both CD5+ and CD5- B cells secreted IgG and/or IgM anti-ssDNA as well as anti-dsDNA antibody. Further analysis of the anti-ssDNA response revealed that the level of IgG and IgM anti-DNA antibody secretion by CD5- B cells correlated closely with the level of polyclonal Ig production by the same subpopulation (r = 0.81 and 0.70, respectively). In contrast, production of anti-DNA by CD5+ B cells occurred independently of polyclonal Ig production by both CD5+ and CD5- B cell subpopulations. These results suggest that in human SLE there exist two anti-DNA antibody-producing B cell subpopulations with distinct induction mechanisms: one (CD5+), which independently secretes anti-DNA, and another (CD5-), which produces anti-DNA as an apparent consequence of polyclonal B cell activation.

  View details for Web of Science ID A1990CH52200034

  View details for PubMedID 1688569

• PEPTIDES DERIVED FROM THE CDR3-HOMOLOGOUS DOMAIN OF THE CD4 MOLECULE ARE SPECIFIC INHIBITORS OF HIV-1 AND SIV INFECTION, VIRUS-INDUCED CELL-FUSION, AND POSTINFECTION VIRAL TRANSMISSION INVITRO - IMPLICATIONS FOR THE DESIGN OF SMALL-PEPTIDE ANTI-HIV THERAPEUTIC AGENTS 2ND INTERNATIONAL CONF ON DRUG RESEARCH IN IMMUNOLOGIC AND INFECTIOUS DISEASES : ACQUIRED IMMUNE DEFICIENCY SYNDROME Rausch, D. M., Hwang, K. M., Padgett, M., VOLTZ, A. H., Rivas, A., Engleman, E., Gaston, I., McGrath, M., Fraser, B., Kalyanaraman, V. S., NARA, P. L., Dunlop, N., Martin, L., MURPHEYCORB, M., KIBORT, T., Lifson, J. D., Eiden, L. E. NEW YORK ACAD SCIENCES. 1990: 125–148

  View details for Web of Science ID A1990BT29L00013

• IMPAIRED IMMUNITY IN AIDS - THE MECHANISMS RESPONSIBLE AND THEIR POTENTIAL REVERSAL BY ANTIVIRAL THERAPY 2ND INTERNATIONAL CONF ON DRUG RESEARCH IN IMMUNOLOGIC AND INFECTIOUS DISEASES : ACQUIRED IMMUNE DEFICIENCY SYNDROME Ruegg, C. L., Engleman, E. G. NEW YORK ACAD SCIENCES. 1990: 307–317

  View details for Web of Science ID A1990BT29L00030

• HIV-1 ENTRY INTO CD4+ T-CELLS - ROLES OF ENDOSOMAL PH AND T-CELL ACTIVATION SYMP ON HUMAN RETROVIRUSES GOWDA, S. D., Stein, B. S., Engleman, E. G. WILEY-LISS, INC. 1990: 227–237

  View details for Web of Science ID A1990BR41Q00021

• DIFFERENCES IN SURFACE PHENOTYPE AND MECHANISM OF ACTION BETWEEN ALLOANTIGEN-SPECIFIC CD8+ CYTOTOXIC AND SUPPRESSOR T-CELL CLONES JOURNAL OF IMMUNOLOGY Koide, J., Engleman, E. G. 1990; 144 (1): 32-40

  Abstract

  We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.

  View details for Web of Science ID A1990CG62300005

  View details for PubMedID 1967266

• HETEROGENEOUS DISTRIBUTION AND TRANSMEMBRANE SIGNALING PROPERTIES OF LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN (LFA-1) IN HUMAN-LYMPHOCYTE SUBSETS JOURNAL OF IMMUNOLOGY Pardi, R., Bender, J. R., Dettori, C., GIANNAZZA, E., Engleman, E. G. 1989; 143 (10): 3157-3166

  Abstract

  The role of LFA-1, a member of the integrin supergene family, in intercellular adhesion, including lymphocyte-endothelial cell (EC) binding, has been established. We now demonstrate that differences in LFA-1 cell surface density are responsible for the variable adhesion efficiency of lymphocyte subsets to EC. Electrophoretic analysis revealed multiple glycosylated isoforms of both alpha and beta subunits, largely as a result of different degrees of sialylation, with variable expression among different lymphocyte subsets. Neuraminidase digestion before EC adhesion increased the binding efficiency of all lymphocyte subsets, although the relative increase in each subset was proportional to the initial LFA-1 sialic acid content. LFA-1 cross-linking resulted in phosphoinositide hydrolysis and a rise in [Ca2+]i when using anti-alpha but not anti-beta subunit antibodies. These findings indicate that the density of LFA-1 on lymphocyte subsets controls their adhesive properties, and that the LFA-1 alpha subunit has transmembrane signaling properties that may result in activation events after interaction with its natural ligands.

  View details for Web of Science ID A1989AZ28800007

  View details for PubMedID 2553807

• MESSAGE AMPLIFICATION PHENOTYPING (MAPPING) - A TECHNIQUE TO SIMULTANEOUSLY MEASURE MULTIPLE MESSENGER-RNAS FROM SMALL NUMBERS OF CELLS BIOTECHNIQUES Brenner, C. A., Tam, A. W., Nelson, P. A., Engleman, E. G., Suzuki, N., Fry, K. E., Larrick, J. W. 1989; 7 (10): 1096-1103

  Abstract

  A rapid and highly sensitive technique (MAPPing: message amplification phenotyping) has been developed to simultaneously analyze the array of messenger RNAs made by small numbers of cells. The technique incorporates a micro-procedure for isolating RNA, reverse transcription of total cellular RNA to produce cDNA, and enzymatic amplification of cytokine-specific DNA fragments using the polymerase chain reaction. In this study, the technique has been applied to the analysis of cytokines produced by lymphoid cells ranging in number from a single cell to 10(6) cells. The technique should be applicable to virtually any tissue or cell type.

  View details for Web of Science ID A1989CH01800002

  View details for PubMedID 2698190

• A SUBSET OF HUMAN ROSETTED LYMPHOCYTES CONTAINS PREVIOUSLY UNIDENTIFIED HISTAMINE CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Khan, M. M., Fishwild, D. M., Melmon, K. L., Coutre, S., Engleman, E. G., Bauer, J. A. 1989; 52 (2): 147-159

  Abstract

  By sequential solid-phase immunoadsorption (panning) steps, we have isolated a subset of lymphocytes (comprising 3-7% of rosetted cells) that contains high concentrations of histamine. We have used a radioenzymatic assay for the determination of histamine and have located 117 ng of histamine/1 x 10(6) cells in Leu-5+ (OKT-11), Leu-15+ cells. This subset did not contain basophils and was negative for Leu-4 (OKT-3), Leu-3 (OKT-4), Leu-2 (OKT-8), and 9.3 antigens. The function of this subset of rosetted cells has not been determined.

  View details for Web of Science ID A1989AD31000002

  View details for PubMedID 2736805

• Acquired immune tolerance to cadaveric renal allografts. A study of three patients treated with total lymphoid irradiation. New England journal of medicine Strober, S., Dhillon, M., Schubert, M., Holm, B., Engleman, E., Benike, C., Hoppe, R., Sibley, R., Myburgh, J. A., Collins, G. 1989; 321 (1): 28-33

  View details for PubMedID 2525231

• ACQUIRED IMMUNE TOLERANCE TO CADAVERIC RENAL-ALLOGRAFTS - A STUDY OF 3 PATIENTS TREATED WITH TOTAL LYMPHOID IRRADIATION NEW ENGLAND JOURNAL OF MEDICINE Strober, S., Dhillon, M., Schubert, M., Holm, B., Engleman, E., Benike, C., Hoppe, R., Sibley, R., Myburgh, J. A., Collins, G., Levin, B. 1989; 321 (1): 28-33

  View details for Web of Science ID A1989AD72900006

• NATURAL-KILLER (NK)-LIKE CYTO-TOXIC ACTIVITY OF ALLOSPECIFIC T-CELL RECEPTOR GAMMA,DELTA+ T-CELL CLONES - DISTINCT RECEPTOR-LIGAND INTERACTIONS MEDIATE NK-LIKE AND ALLOSPECIFIC CYTO-TOXICITY JOURNAL OF IMMUNOLOGY Koide, J., Rivas, A., Engleman, E. G. 1989; 142 (12): 4161-4168

  Abstract

  We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.

  View details for Web of Science ID A1989AA56700006

  View details for PubMedID 2470816

• EVIDENCE THAT CYTO-TOXIC LYMPHOCYTES ALTER AND TRAVERSE ALLOGENEIC ENDOTHELIAL-CELL MONOLAYERS TRANSPLANTATION Bender, J. R., Pardi, R., Kosek, J., Engleman, E. G. 1989; 47 (6): 1047-1053

  Abstract

  In an attempt to study the effects of allogeneic lymphocytes on endothelial cells (EC) and analyze the mechanism whereby such lymphocytes traverse an EC barrier, we have established human microvascular EC monolayers, in vitro, and analyzed the effects of lymphocyte subpopulations on such monolayers. Previous studies have shown that CD16+ (natural killer) and CD8+ (cytotoxic) lymphocytes but not CD4+ (helper) cells bind and induce the appearance of class II major histocompatibility complex antigens on allogeneic EC. The current findings indicate that these same lymphocyte subsets induce marked swirling and elongation of allogeneic EC, and traverse intact EC monolayers. In contrast, none of the functional consequences of the initial lymphocyte-EC adhesion were observed using autologous combinations, despite the presence of significant intercellular binding. Scanning and electron micrographs demonstrate extensive areas of lymphocyte-EC surface contact and EC-coated pit formation, whereas a panel of recombinant cytokines known to alter the surface phenotype of EC fail to induce the same morphologic changes whether used singly or in combination. We postulate that the cellular interactions observed here, in vitro, may represent the initial steps in the rejection of vascularized allografts in vivo.

  View details for Web of Science ID A1989AB98900026

  View details for PubMedID 2660343

• ROLE OF CD4 IN NORMAL IMMUNITY AND HIV INFECTION IMMUNOLOGICAL REVIEWS Lifson, J. D., Engleman, E. G. 1989; 109: 93-117

  Abstract

  In this report we have attempted to review our knowledge of the role(s) of CD4 in human T-cell function and the consequences of interactions between CD4 molecules and the human immunodeficiency virus (HIV). The observation in 1981 that antibodies to certain epitopes of CD4 inhibited the immune functions of CD4+ T cells led to the initial suggestion that CD4 molecules play a direct role in T-cell function. Although the precise functions of CD4 remain incompletely understood, a preponderance of evidence suggests that this molecule may in fact serve several critical roles. At least one such role is that of interacting directly with MHC class II molecules on antigen-presenting cells, presumably facilitating cell-to-cell interactions. On activated CD4+ T cells, CD4 molecules can also interact directly with the T-cell receptor complex to influence the immune response. Unfortunately, in addition to interacting with the T-cell receptor and class II MHC determinants, CD4 serves as a high affinity receptor for HIV, the causative agent of AIDS. Not only does interaction between the virus and CD4 initiate viral fusion to the cell membrane and HIV entry but, in addition, a similar molecular interaction initiates fusion between HIV-infected and uninfected CD4+ cells, resulting in the formation of multinucleated syncytia. Since uninfected CD4+ cells are, in effect, recruited into such syncytia, this mechanism may account in part for the depletion of CD4+ T cells in HIV-infected patients. Soluble forms of CD4 produced either by genetic engineering or solid phase peptide synthesis can completely block HIV infectivity and syncytia formation in vitro, remarkably without apparent effects on T-cell immunity. Such molecules are currently being explored for their possible therapeutic effects on HIV infection in vivo.

  View details for Web of Science ID A1989AC30900005

  View details for PubMedID 2475427

• IDENTIFICATION OF PROTEIN INTERMEDIATES IN THE PROCESSING OF THE P55 HIV-1 GAG PRECURSOR IN CELLS INFECTED WITH RECOMBINANT VACCINIA VIRUS JOURNAL OF BIOLOGICAL CHEMISTRY GOWDA, S. D., Stein, B. S., Engleman, E. G. 1989; 264 (15): 8459-8462

  Abstract

  We have used a recombinant vaccinia virus (VV) which expresses high levels of human immunodeficiency virus-1 (HIV-1) gag proteins to analyze the processing pathway of the gag p55 precursor. HIV-1 gag proteins were isolated from [3H]leucine-labeled VV:gag-infected H9 T lymphocytes by immunoprecipitation with either anti-p24, anti-p17, or anti-p6 antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that processing of the p55 precursor involves three major intermediates (p41a, p41b, and p39). The p41a and p39 proteins contain the p17 and p24 protein segments, and the p41b is comprised of p24 and p15 segments. On two-dimensional gels, each intermediate as well as the mature p24 and p17 proteins migrated as distinct species. [3H]Myristic acid labeling of the HIV-1 gag proteins revealed that in addition to p55 and p17, the p41a and p39 intermediates, but not p41b, are myristylated, confirming that myristylation occurs at the NH2 terminus before cleavage of the p55 precursor protein. We conclude that the myristylated HIV-1 gag p55 precursor is initially cleaved at random either at the p17/p24 junction or at two sites between p24 and p15 proteins, resulting in three intermediates (p41a, p41b, and p39) which are subsequently cleaved to yield mature gag proteins.

  View details for Web of Science ID A1989U743800005

  View details for PubMedID 2785991

• DIFFERENCES IN SURFACE PHENOTYPE BETWEEN CYTOLYTIC AND NON-CYTOLYTIC CD4+ T-CELLS - MHC CLASS-II-SPECIFIC CYTO-TOXIC LYMPHOCYTES-T LACK LEU-8 ANTIGEN AND EXPRESS CD2 IN HIGH-DENSITY JOURNAL OF IMMUNOLOGY Takada, S., Koide, J., Engleman, E. G. 1989; 142 (9): 3038-3044

  Abstract

  A number of cell surface molecules are differentially expressed on functionally distinct subsets of CD4+ T cells. However, to date CD4+ T cells capable of becoming CTL have not been shown to be phenotypically distinct from other CD4+ T cells, and in the current study we examined the ability of Leu 8+ and Leu 8- CD4+ subpopulations to become cytotoxic effectors after their stimulation with allogeneic lymphoblastoid cell lines. Although CD4+, Leu 8+ cells proliferated more vigorously than CD4+, Leu 8- cells in primary cultures stimulated with allogeneic LCL, the CD4+, Leu 8- population was the major source of cytotoxic effectors, killing targets with specificity for their class II MHC alloantigens. In most subjects, CD4+ precursors of CTL were distinguished not only by their lack of Leu 8 expression but also by their relatively high density of CD2, LFA-1, and LFA-3, molecules known to mediate non-specific cell-to-cell adhesion and postulated to be markers of immunologic memory. The absence of Leu 8 does not appear to be a reliable memory cell marker, however, because Leu 8+ as well as Leu 8-, CD4+ cells from PPD skin test positive subjects responded to the recall Ag, PPD. During 3 mo of continuous culture with allogeneic stimulators, Leu 8- cells retained their cytolytic activity and remained unreactive with anti-Leu 8 mAb, whereas Leu 8+ cells remained non-cytolytic and reactive with anti-Leu 8, suggesting that under the conditions used the Leu 8 phenotype is relatively stable. PHA or anti-CD3 mAb enhanced non-specific killing by alloantigen-stimulated CD4+,Leu 8- lines but failed to unmask any cytolytic potential in CD4+,Leu 8+ lines. We conclude that MHC class II-specific cytolytic CD4+ T cells can be distinguished from non-cytolytic CD4+ cells on the basis of their surface phenotype, and that most CD4+ CTL are contained within the Leu 8- subpopulation.

  View details for Web of Science ID A1989U275200010

  View details for PubMedID 2468710

• CYTO-TOXIC LYMPHOCYTES MORPHOLOGICALLY ALTER AND TRAVERSE ALLOGENEIC ENDOTHELIAL-CELL MONOLAYERS Bender, J. R., Pardi, R., Engleman, E. G. SLACK INC. 1989: A245–A245

  View details for Web of Science ID A1989U004400048

• EVIDENCE FOR INVOLVEMENT OF THE GAMMA,DELTA-T-CELL ANTIGEN RECEPTOR IN CYTO-TOXICITY MEDIATED BY HUMAN ALLOANTIGEN-SPECIFIC T-CELL CLONES JOURNAL OF IMMUNOLOGY Rivas, A., Koide, J., Cleary, M. L., Engleman, E. G. 1989; 142 (6): 1840-1846

  Abstract

  The vast majority of mature T cells express the alpha, beta TCR and use this receptor to recognize Ag in association with MHC determinants. Recent reports indicate that a small subpopulation of CD3+ T cells does not express alpha, beta TCR and lacks CD4 and CD8 molecules, but expresses another TCR heterodimer designated gamma, delta. To date, however, there are very few examples of gamma, delta TCR-bearing T cells which have Ag-specific functions and the precise role of the gamma, delta TCR remains unknown. In an attempt to explore the function of the gamma, delta TCR in man, we generated a panel of CD3+, CD4-, CD8- T cell clones from cultures of normal T cells stimulated with allogeneic lymphoblastoid cell line (LCL). All of the clones stained with mAb to gamma, delta TCR determinants, and Northern blot analysis of total cellular RNA revealed mature gamma- and delta-chain transcripts and immature beta-chain transcript. The clones displayed cytolytic activity against their specific stimulating LCL, but not irrelevant LCL, and killing was inhibited by a mAb to HLA class I (HLA-A,B,C) determinants, suggesting that these cells recognized class I MHC antigenic determinants on specific stimulator/target cells. Cytolysis by these clones was markedly inhibited by low concentrations (0.00001 to 0.001 microgram/ml) of mAb to CD3 or TCR delta-chain, but high concentrations (0.1 to 20 micrograms/ml) of the same antibodies enhanced cytotoxicity. When absorbed to plastic tissue culture wells, anti-CD3 mAb stimulated the clones to proliferate. These results indicate that alloantigen specific, gamma, delta TCR+, CD4-, CD8- T cell clones can be reproducibly generated in vitro, and that the gamma, delta TCR plays a direct role in the specific recognition and cytolysis displayed by these clones.

  View details for Web of Science ID A1989T588500009

  View details for PubMedID 2466078

• EXPRESSION AND PROCESSING OF HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 GAG AND POL GENES BY CELLS INFECTED WITH A RECOMBINANT VACCINIA VIRUS JOURNAL OF VIROLOGY GOWDA, S. D., Stein, B. S., Steimer, K. S., Engleman, E. G. 1989; 63 (3): 1451-1454

  Abstract

  Human cells infected with a recombinant vaccinia virus containing human immunodeficiency virus type 1 gag-pol genes produced large amounts of human immunodeficiency virus gag proteins beginning at 1 h and peaking at 48 h postinfection. We show that these polyproteins are processed accurately into mature forms and that the viral polymerase gene is encoded as a 160-kilodalton gag-pol fusion protein, most likely by translational frameshifting from the gag into the pol reading frame.

  View details for Web of Science ID A1989T291000057

  View details for PubMedID 2464705

• EVIDENCE THAT T-CELL ACTIVATION IS REQUIRED FOR HIV-1 ENTRY IN CD4+ LYMPHOCYTES JOURNAL OF IMMUNOLOGY GOWDA, S. D., Stein, B. S., Mohagheghpour, N., Benike, C. J., Engleman, E. G. 1989; 142 (3): 773-780

  Abstract

  The relationship of T cell activation to HIV entry and generation of viral DNA intermediates was studied in freshly isolated CD4+ T lymphocytes. Unstimulated cells exposed to infectious virus for up to 48 h did not synthesize any detectable unintegrated HIV DNA duplex forms or integrated genomic provirus. However, activation of these cells with either PHA or OKT3 (anti-CD3) mAb before viral exposure resulted in the generation of unintegrated HIV DNA after 6 h and integrated copies after 24 h. Cell-to-cell fusion studies showed significantly attenuated fusion between freshly isolated resting T cells and T cells constitutively expressing high levels of HIV envelope glycoprotein (HXB/gpt) compared with T cells first stimulated with either PHA or OKT3 mAb. The baseline fusion observed with resting T cells is believed to be a consequence of allogeneic stimulation by the HXB/gpt cell line. These results provide evidence that HIV entry and HIV envelope-dependent cell-to-cell fusion require T cell activation.

  View details for Web of Science ID A1989R950700007

  View details for PubMedID 2521508

• CD4 MOLECULES ARE ASSOCIATED WITH THE ANTIGEN RECEPTOR COMPLEX ON ACTIVATED BUT NOT RESTING T-CELLS JOURNAL OF IMMUNOLOGY Rivas, A., Takada, S., Koide, J., SONDERSTRUPMCDEVITT, G., Engleman, E. G. 1988; 140 (9): 2912-2918

  Abstract

  To explore the relationship between CD4 and CD3/Ti on the T cell surface, we have studied a panel of Ag-specific Th cell lines and clones, as well as resting and mitogen-activated CD4+ cells. Our results show that exposure of Th cells to their specific antigenic stimuli, but not to irrelevant stimuli, induced the rapid disappearance of approximately 20 to 35% of CD3 and CD4 molecules. The modulation of these molecules was detected in less than 1 h, became maximal at 12 h, and recovered thereafter in parallel. Treatment of Th cells with anti-CD4 antibody prevented Ag-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD4. In the absence of Ag, treatment of these cells with an antibody (WT-31) directed at a conformational determinant within CD3/Ti or with the combination of anti-CD3 antibody and goat anti-mouse Ig, also resulted in significant modulation of CD4. Similar treatment of PHA-activated CD4+ T cells with anti-CD3/Ti antibodies also induced CD4 modulation; however, the same antibodies failed to affect CD4 expression on fresh resting T cells. These results indicate that on activated, but not resting T cells, CD4 molecules can be physically associated with CD3/Ti. We postulate that this association is essential for efficient Th cell activation, and further that the ability of anti-CD4 antibodies to inhibit helper functions is due to their prevention of CD4-CD3/Ti interaction on the T cell surface.

  View details for Web of Science ID A1988N048700010

  View details for PubMedID 2966201

• ACTIVATION OF HLA-RESTRICTED EBV-SPECIFIC CYTO-TOXIC T-CELLS DOES NOT REQUIRE CD4+ (HELPER) T-CELLS OR EXOGENOUS CYTOKINES JOURNAL OF IMMUNOLOGY Fishwild, D. M., Benike, C. J., Engleman, E. G. 1988; 140 (6): 1994-1998

  Abstract

  MHC-restricted, viral Ag-specific "memory" CTL are thought to play a decisive role in the defense against pathogenic viruses. However, the requirements for activating such CTL remain controversial. In particular, the role of CD4+ helper cells and their soluble products (e.g., IL-2) are uncertain. To approach these questions as they relate to EBV-specific CTL, highly purified CD8+ T cells from healthy EBV-seropositive individuals were cultured with autologous irradiated EBV-transformed B lymphoblastoid cell lines (LCL), in the presence or absence of autologous CD4+ cells or 1 to 10 U/ml purified rIL-2. The results indicate that the induction of CTL requires neither Th cells nor exogenous IL-2. The CTL generated from isolated CD8+ cells were HLA class I restricted as demonstrated by their ability to lyse targets sharing at least one HLA-A or -B Ag with the stimulating autologous LCL. Furthermore, a mAb (W6/32) to a common determinant on HLA class I Ag blocked both the generation and effector phases of killing, whereas an HLA class II directed mAb had no effect. Addition of an IL-2R-specific antibody (anti-Tac) to the culture medium blocked induction of CTL, suggesting that endogenously produced IL-2 plays an obligatory role in this system. Paraformaldehyde fixation of LCL abrogated their ability to function as stimulator cells; however, addition of 2 U/ml exogenous IL-2 to fixed LCL cultured with CD8+ cells allowed for the induction of highly specific CTL. These results indicate that EBV-specific memory CTL can be activated in the absence of CD4+ helper cells or their soluble products, but nonetheless require Ag and IL-2.

  View details for Web of Science ID A1988M523200042

  View details for PubMedID 2831276

• CLINICAL AND IMMUNOLOGICAL STUDIES OF CADAVERIC RENAL-TRANSPLANT RECIPIENTS GIVEN TOTAL-LYMPHOID IRRADIATION AND MAINTAINED ON LOW-DOSE PREDNISONE TRANSPLANTATION Saper, V., Chow, D., ENGLEMAN, E. D., Hoppe, R. T., Levin, B., Collins, G., Strober, S. 1988; 45 (3): 540-546

  Abstract

  Twenty-five recipients of cadaveric renal transplants were given total lymphoid irradiation (TLI), perioperative antithymocyte globulin, and low-dose prednisone as the sole maintenance immunosuppressive drug. Nine patients were diabetic, and follow-up was between 19 and 37 months. One-year graft and patient survival was 76% and 87%, respectively, Serious complications included four deaths from cardiovascular disorders, and two deaths from viral infections. Studies of peripheral blood T cell subsets showed a prolonged reduction in the absolute number of helper (Leu-3+) cells, and a rapid recovery of cytotoxic/suppressor (Leu-2+) cells. Analysis of the latter subset, using the monoclonal antibody 9.3, showed that the ratio of suppressor/cytotoxic cells was approximately 10:1. The normal ratio is 1:1. The mean mixed leukocyte reaction remained below 30% of the pre-TLI value for 6 months, and approached 80% at two years. Similar kinetics were observed in the proliferative response to mitogens. The results show that maintenance immunosuppressive drug therapy can be reduced after TLI as compared with conventional drug regimens that use prednisone in combination with cyclosporine and/or azathioprine.

  View details for Web of Science ID A1988M597000008

  View details for PubMedID 3279577

• HUMAN T-CELL LEUKEMIA VIRUS-I-ASSOCIATED T-SUPPRESSOR CELL-INHIBITION OF ERYTHROPOIESIS IN A PATIENT WITH PURE RED-CELL APLASIA AND CHRONIC T-GAMMA-LYMPHOPROLIFERATIVE DISEASE JOURNAL OF CLINICAL INVESTIGATION Levitt, L. J., Reyes, G. R., Moonka, D. K., Bensch, K., MILLER, R. A., Engleman, E. G. 1988; 81 (2): 538-548

  Abstract

  Human retroviruses have recently been linked with T cell lymphoproliferative disorders and with the acquired immune deficiency syndrome. We investigated the mechanisms for acquired pure red cell aplasia and cutaneous anergy in a patient with the chronic T gamma-lymphoproliferative disease (T gamma-LPD) syndrome. Patient marrow erythroid progenitors (BFU-E) were 17 +/- 9% of control and were selectively increased to 88-102% of control after marrow T cell depletion. Patient Leu 2+ suppressor T cells spontaneously produced high titers of human gamma-interferon and resulted in a concentration-dependent selective inhibition (74-91%) of BFU-E when co-cultured with autologous or allogeneic marrow. Conditioned media (CM) derived from patient Leu 2+ T cells similarly inhibited growth of autologous or allogeneic marrow BFU-E. The inhibitory factor derived from patient CM was acid-labile (pH 2) and sensitive to trypsin; prior treatment of patient T cells with anti-HLA-DR monoclonal antibody plus complement abrogated the suppressive effect of T cell-derived CM. Patient peripheral blood mononuclear cells (PBMC) were unable to support growth of cultured interleukin 2 (IL 2)-dependent T cells, but responded to exogenous IL 2 in vitro with a 16-21-fold augmentation, relative to control, in mitogen-induced proliferation. Antibodies to HTLV-I core proteins p19 and p24 but not to HTLV-III proteins were detected in patient serum by Western blotting; patient cultured PBMC stained (7-11%) with antibodies to p19 and p24. Patient cultured PBMC demonstrated integrated HTLV-I genomic sequences by the Southern technique and expressed both specific HTLV-I genomic sequences by RNA dot blot plus reverse transcriptase activity. Utilizing a cloned DNA probe for the beta chain of the T cell receptor gene, patient PMBC demonstrated gene rearrangements providing presumptive evidence for clonality. The presence in serum of HTLV-I p19 and p24 antibodies, the expression of p19 and p24 core antigens on patient mononuclear cells, the evidence of HTLV-I proviral integration sequences and the expression of HTLV-I genomic sequences in patient cells, indicates infection with HTLV-I and raises the possibility of an etiologic link between human retrovirus infection and some instances of large granular lymphocytic leukemia (T gamma-LPD).

  View details for Web of Science ID A1988L939600035

  View details for PubMedID 2892860

• CHARACTERIZATION AND EPITOPE MAPPING OF A HUMAN MONOCLONAL-ANTIBODY REACTIVE WITH THE ENVELOPE GLYCOPROTEIN OF HUMAN-IMMUNODEFICIENCY-VIRUS JOURNAL OF IMMUNOLOGY Banapour, B., Rosenthal, K., Rabin, L., Sharma, V., Young, L., Fernandez, J., Engleman, E., McGrath, M., Reyes, G., Lifson, J. 1987; 139 (12): 4027-4033

  Abstract

  A human monoclonal antibody (IgG2, lambda), 1B8.env, was produced, reactive with the envelope glycoprotein of human immunodeficiency virus (HIV). The antibody specifically stains cells infected with HIV, as assessed by indirect immunofluorescence analysis and reacts with determinants displayed on the surface of infected cells. In Western blot analysis, the antibody reacts with bands of 160 and 41 kD, consistent with the precursor and transmembrane forms of the HIV envelope glycoprotein. The antibody also reacts specifically in immunofluorescence and Western blot analysis with cells infected with the recombinant vaccinia virus VSC-25, which contains the envelope gene of HIV. With the lambda gt11 expression vector, the epitope recognized by 1B8.env was mapped to a region of 11 amino acids in the coding region of gp41. This domain is highly conserved between several otherwise highly variable HIV isolates. In addition, this epitope appears to be recognized by the vast majority of HIV seropositive individuals. Although antibody IB8.env does not neutralize HIV virion infectivity or virally mediated cell fusion, the results presented here demonstrate the feasibility of generating and characterizing human monoclonal antibodies to HIV with these techniques. Additional antibodies produced in this manner will help to further characterize the humoral response to HIV infection, define biologically significant determinants on HIV proteins, and may be useful in clinical applications.

  View details for Web of Science ID A1987L283500019

  View details for PubMedID 2447158

• T-CELL REGULATORY DISTURBANCES IN THE RHEUMATIC DISEASES RHEUMATIC DISEASE CLINICS OF NORTH AMERICA ALPERT, S. D., Koide, J., Takada, S., Engleman, E. G. 1987; 13 (3): 431-445

  Abstract

  Significant immunoregulatory abnormalities have been described in both SLE and RA. In SLE, deficient suppressor T cell activity may result from depletion of CD8+ suppressor precursors, depletion of CD4+ suppressor-inducer cells, or impaired lymphokine production and deficient CD4+ cell activation of suppression by CD4+ cells. The net result is an apparent failure to inhibit antibody synthesis. The defects in RA are less well defined, although in RA there also is evidence that either deficient suppression or deficient suppression-induction plays a role in the pathogenesis of disease. There is evidence for local lymphocyte activation in the synovium, with possibly impaired local immunoregulation. However, the precise nature of the immune reactions in the synovium and their relationship to systemic immunoregulatory abnormalities remain unclear.

  View details for Web of Science ID A1987L302800003

  View details for PubMedID 2893438

• EVIDENCE FOR AN ASSOCIATION BETWEEN CD8 MOLECULES AND THE T-CELL RECEPTOR COMPLEX ON CYTOTOXIC T-CELLS JOURNAL OF IMMUNOLOGY Takada, S., Engleman, E. G. 1987; 139 (10): 3231-3235

  Abstract

  The T cell differentiation molecule CD8 is thought to play an important role in class I major histocompatibility complex-restricted T cell activities but the precise function of this molecule is unknown. To explore this question, we have studied several CD3+, CD8+ class I alloantigen-specific cytotoxic T lymphocyte (CTL) lines and clones. The ability of these CTL to proliferate as well as to lyse specific targets was inhibited by either anti-CD3 or anti-CD8 monoclonal antibodies. Exposure of CTL to relevant but not irrelevant target cells induced the rapid (less than 1 hr) disappearance of approximately 20 to 30% of CD3 and CD8 molecules from the cell surface. The modulation of these molecules became maximal at 6 to 12 hr and recovered thereafter in parallel. Treatment of CTL with anti-CD8 prevented alloantigen-induced modulation of CD3, and treatment with anti-CD3 blocked modulation of CD8. Incubation of CTL with the combination of anti-CD3 and goat anti-mouse Ig also resulted in modulation of CD8. In contrast, the expression of other CTL surface antigens, such as CD2 (Leu-5, T11) and HLA-DR, was not reduced by any of these manipulations. These results suggest that CD8 molecules are associated with the CD3/antigen receptor complex on the surface of CTL, and may play a direct role in antigen-induced modulation and cross-linking of the T cell receptor.

  View details for Web of Science ID A1987K794600009

  View details for PubMedID 3500208

• LYMPHOCYTE SUBSETS DIFFERENTIALLY INDUCE CLASS-II HUMAN-LEUKOCYTE ANTIGENS ON ALLOGENEIC MICROVASCULAR ENDOTHELIAL-CELLS JOURNAL OF IMMUNOLOGY Pardi, R., Bender, J. R., Engleman, E. G. 1987; 139 (8): 2585-2592

  Abstract

  Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.

  View details for Web of Science ID A1987K320700013

  View details for PubMedID 3116086

• ANTIBODY TO HUMAN-IMMUNODEFICIENCY-VIRUS CORRELATES WITH DECREASED T-HELPER LYMPHOCYTES IN ASYMPTOMATIC INDIVIDUALS JOURNAL OF MEDICAL VIROLOGY Weiser, B., Burger, H., Steimer, K., Lifson, J., Engleman, E., Grimson, R., Robinson, W. S. 1987; 22 (3): 237-244

  Abstract

  To examine the relationship between human immunodeficiency virus (HIV) seropositivity and T lymphocyte subsets in a clinically well population, we assayed HIV antibody and analyzed T lymphocyte subsets in 30 people at increased risk for acquired immunodeficiency syndrome (AIDS) who were clinically well. Seventy-six percent of the HIV-seropositive individuals had abnormally low numbers of T helper lymphocytes, and HIV seropositivity was strongly correlated with an abnormally low number of T helper cells (p less than 0.00002). Among these clinically well subjects at increased risk for AIDS, HIV-sero-positive individuals had a significant decrease in mean T helper lymphocytes and mean T helper:T suppressor ratios as compared to those who were seronegative (483 cells/mm3 vs 915 cells/mm3, p less than 0.002; and 0.80 vs 1.7, p less than 0.002, respectively). Because of the strong correlation of HIV seropositivity and abnormally low numbers of T helper lymphocytes in this asymptomatic population, these findings suggest that asymptomatic seropositive individuals should be followed closely for development of AIDS-related disease and should be considered for future antiviral therapy when it becomes available.

  View details for Web of Science ID A1987H925100006

  View details for PubMedID 2957464

• PH-INDEPENDENT HIV ENTRY INTO CD4-POSITIVE T-CELLS VIA VIRUS ENVELOPE FUSION TO THE PLASMA-MEMBRANE CELL Stein, B. S., GOWDA, S. D., Lifson, J. D., Penhallow, R. C., Bensch, K. G., Engleman, E. G. 1987; 49 (5): 659-668

  Abstract

  CD4 functions as the cell-surface receptor for human immunodeficiency virus (HIV); however, the mechanism of virus entry into susceptible cells is unknown. To explore this question we used a human T lymphoblastic cell line (VB) expressing high levels of surface CD4. Neutralization of endosomal compartments (pH greater than 6.4) with lysosomotropic agents did not effectively inhibit HIV nucleocapsid entry into the cytoplasm, and virus treated at low pH (5.5) failed to induce rapid cell-to-cell fusion in uninfected cells. Electron microscopy of VB cells acutely exposed to HIV at neutral pH revealed direct fusion of the virus envelope with the plasma membrane within minutes at 4 degrees C. No endocytosed virions were visualized upon rewarming the HIV-exposed cells to 37 degrees C for as long as 60 min. These results indicate that HIV penetrates CD4-positive T cells via pH-independent membrane fusion.

  View details for Web of Science ID A1987H684900012

  View details for PubMedID 3107838

• PHENOTYPIC AND FUNCTIONAL-CHARACTERIZATION OF LYMPHOCYTES THAT BIND HUMAN MICROVASCULAR ENDOTHELIAL-CELLS INVITRO - EVIDENCE FOR PREFERENTIAL BINDING OF NATURAL-KILLER-CELLS JOURNAL OF CLINICAL INVESTIGATION Bender, J. R., Pardi, R., Karasek, M. A., Engleman, E. G. 1987; 79 (6): 1679-1688

  Abstract

  The microvascular endothelium has been postulated to be a critical target in the rejection of vascularized allografts. This study was undertaken to examine the ability of human sheep erythrocyte rosette forming lymphocytes (E-RFC) to form stable conjugates with microvascular endothelial cells (EC), and to assess whether a receptor-ligand interaction mediates this event. Human foreskin microvascular EC monolayers were used as targets of chromium-51-labeled E-RFC in a quantitative adherence assay. Binding was saturable, displaceable by unlabeled E-RFC, augmented by recombinant interleukin 1 (rIL-1) and inhibited by anti-LFA1 antibody. The Leu-11+ lymphocyte subset, known to be enriched for natural killer (NK) cells, bound preferentially. Only the EC-adherent lymphocyte fraction contained NK effectors, which lysed EC and classical NK targets. Thus, NK cells adhere to microvascular EC via a specific receptor-ligand interaction. The possibility exists that such binding occurs in recipients of vascularized allografts, representing the initial stage of graft rejection.

  View details for Web of Science ID A1987H575600020

  View details for PubMedID 3495552

• PHENOTYPIC IDENTIFICATION OF SUPPRESSOR-EFFECTOR, SUPPRESSOR-AMPLIFIER AND SUPPRESSOR-INDUCER T-CELLS OF B-CELL DIFFERENTIATION IN MAN EUROPEAN JOURNAL OF IMMUNOLOGY Kansas, G. S., Engleman, E. G. 1987; 17 (4): 453-457

  Abstract

  Using monoclonal anti-Leu8 antibody to isolate subpopulations of human helper/inducer (CD4+) and suppressor/cytotoxic (CD8+) T cells, we have investigated the role of these subpopulations in the regulation of B cell differentiation in the human autologous mixed leukocyte reaction (AMLR). Whereas AMLR-activated CD8+,Leu8- cells were capable of suppressing fresh AMLR cultures in the absence of fresh CD8+ cells, CD8+,Leu8+ cells suppressed only those cultures containing fresh CD8+ cells. On the other hand, CD8+,Leu8- cells became suppressor cells only when cultured in the presence of CD8+,Leu8+ cells. Finally, the development of CD8+ suppressor cells was dependent on the presence of CD4+,Leu8+ cells; CD4+,Leu8- cells were incapable of acting as suppressor-inducer cells, but have been shown previously to mediate T cell help for B cell differentiation. Thus, at least 3 phenotypically distinct subsets of T cells interact sequentially to generate suppression of B cell differentiation induced in the AMLR: CD4+,Leu8+ suppressor/inducer cells, CD8+,Leu8+ suppressor-amplifier cells and CD8+,Leu8- suppressor-effector cells.

  View details for Web of Science ID A1987H594900002

  View details for PubMedID 2952513

• T-CELL DEFECT IN LEPROMATOUS LEPROSY IS REVERSIBLE INVITRO IN THE ABSENCE OF EXOGENOUS GROWTH-FACTORS JOURNAL OF IMMUNOLOGY Mohagheghpour, N., GELBER, R. R., Engleman, E. G. 1987; 138 (2): 570-574

  Abstract

  T lymphocytes from patients with lepromatous leprosy (LL) characteristically fail to respond to Mycobacterium leprae. This specific immunologic defect is thought to contribute to the aggressive clinical course that typifies patients with LL. We report that although fresh CD4+ (helper) T cells from most LL patients are specifically unresponsive to M. leprae, after culture in medium alone for 48 hr the same cells respond to M. leprae antigens. The recovery of T cell function is specific for M. leprae, occurs at the level of responder CD4+ T cells, and is not affected by monocytes or CD8+ (suppressor) T cells. Recovery of T cell reactivity is blocked by the presence of M. leprae bacilli in the preculture medium. These findings indicate that despite the apparent specific anergy seen in patients with LL, the T cells of most LL patients can respond to M. leprae. Their failure to do so, in vivo, may be due to the persistence of antigen, which renders antigen-reactive T cells nonresponsive either directly or via activation of CD4+ suppressor cells.

  View details for Web of Science ID A1987F617900037

  View details for PubMedID 2947952

• HUMAN MONOCLONAL-ANTIBODIES TO RH0(D) VOX SANGUINIS Foung, S. K., Blunt, J. A., Wu, P. S., AHEARN, P., WINN, L. C., Engleman, E. G., GRUMET, F. C. 1987; 53 (1): 44-47

  Abstract

  B lymphocytes from Rh negative donors with serum anti-D antibodies were isolated and fused with the mouse-human heteromyeloma, SBC-H20, to produce hybridomas secreting IgM or IgG1 human monoclonal antibodies to D antigen. The IgM antibody in hybridoma supernatant agglutinates all normal D positive cells at the immediate spin phase of reactivity. Using concentrated IgM hybridoma supernatant of approximately 50 micrograms/ml, Du cells were also agglutinated. The IgG1 antibody reacts by indirect hemagglutination with all D and Du cells. Against Rh mosaics, different reactivity was noted for each antibody. Furthermore, D positive cells precoated with the IgG1 antibody inhibit the IgM direct hemagglutination, suggesting that the antibodies identify closely associated epitopes. These human monoclonal antibodies will be useful diagnostic reagents and, ultimately, should be useful in the prevention of Rh hemolytic disease of the newborn.

  View details for Web of Science ID A1987J436100009

  View details for PubMedID 3116771

• HUMAN MONOCLONAL ANTI-T-CELL ANTIBODY FROM A PATIENT WITH JUVENILE RHEUMATOID-ARTHRITIS JOURNAL OF IMMUNOLOGY ALPERT, S. D., Turek, P. J., Foung, S. K., Engleman, E. G. 1987; 138 (1): 104-108

  Abstract

  Antibody JRAI is a human monoclonal IgM antibody derived from a patient with juvenile rheumatoid arthritis that is cytotoxic to a subpopulation of normal T lymphocytes. JRAI recognizes approximately 80% of normal peripheral blood T cells, 90% of CD4+ cells, and 75% of CD8+ cells, as determined by complement-mediated cytotoxicity. Within the CD8+ population, JRAI preferentially spares OKM1+ and Leu-11+ cells. These CD8+ cells retain suppressor-effector potential and show enriched natural killer cell activity. Within the CD4+ population, JRAI preferentially kills cells within the Leu-8+ subset, which contains suppressor-inducer cells, and spares the Leu-8- subset, which contains the helpers for immunoglobulin synthesis. JRAI appears to recognize a previously undefined human lymphocyte surface molecule expressed differentially on phenotypically and functionally distinct subsets of human T cells.

  View details for Web of Science ID A1987F377900018

  View details for PubMedID 3097140

• ROLE OF ENVELOPE GLYCOPROTEIN CARBOHYDRATE IN HUMAN-IMMUNODEFICIENCY-VIRUS (HIV) INFECTIVITY AND VIRUS-INDUCED CELL-FUSION JOURNAL OF EXPERIMENTAL MEDICINE Lifson, J., Coutre, S., Huang, E., Engleman, E. 1986; 164 (6): 2101-2106

  Abstract

  Human immunodeficiency virus (HIV) envelope glycoprotein interactions with cell surface CD4 are involved in both virion infectivity and virally mediated cell fusion. D-mannose-specific lectins such as Con A specifically blocked virion infectivity and cell fusion. Studies with a recombinant vaccinia virus containing the HIV envelope gene demonstrated that Con A-mediated inhibition of HIV-induced fusion involved lectin binding to the viral envelope glycoprotein. These results indicate the importance of envelope glycosylation in the pathobiology of HIV infection, and suggest potential mechanisms for interfering with HIV infectivity and cytopathology.

  View details for Web of Science ID A1986F117600024

  View details for PubMedID 3640800

• THE IMMUNOHISTOLOGY OF THE PERSISTENT GENERALIZED LYMPHADENOPATHY SYNDROME (PGL) AMERICAN JOURNAL OF CLINICAL PATHOLOGY Garcia, C. F., Lifson, J. D., Engleman, E. G., Schmidt, D. M., Warnke, R. A., Wood, G. S. 1986; 86 (6): 706-715

  Abstract

  The authors employed a large panel of monoclonal antibodies to characterize and quantitate lymphoid subpopulations within the paracortex, mantle, and germinal centers of frozen sections of lymph nodes from 18 patients with the persistent generalized lymphadenopathy (PGL) syndrome and five heterosexual controls. The authors' data indicate that Leu-3+ phenotypic T-helper cells (TH) are reduced within all three compartments, while T-cytotoxic-suppressor cells (Tcs) are increased. Using the antibody 9.3, which allows dissection of the Leu-2+ Tcs subset into 9.3+ cytotoxic cells (Tc) and 9.3- suppressor cells (Ts), the authors found that the Ts subset is increased in the lymph nodes of these patients. In contrast to acquired immune deficiency syndrome (AIDS) patients, paracortical total T-cells and Leu-8+ cells appear to be preserved in patients with PGL. Study of TH and Tcs subpopulations in peripheral blood in 12 of these patients revealed inverted ratios (mean, 0.59), which did not correlate with those seen in the lymph nodes. Although the paracortical TH/Tcs ratios were significantly reduced (mean, 1.44) they were not inverted, in contrast to some other reported series. In aggregate, these data suggest that, relative to AIDS, there is preservation of the paracortical T-cell microenvironment in PGL. Clinically, this correlates with more intact cell-mediated immunity and the absence of opportunistic infections and Kaposi's sarcoma in this patient group. Follicle lysis was present in 11 patients. Increased HLA-DR+ paracortical cells, aggregates of Leu-6+ dendritic cells, decreased TAC+ cells, increased OKT-10+ plasma cells, and increased interstitial immunoglobulin were among the other features observed in these patients.

  View details for Web of Science ID A1986F110100002

  View details for PubMedID 3491535

• INDUCTION OF CD4-DEPENDENT CELL-FUSION BY THE HTLV-III/LAV ENVELOPE GLYCOPROTEIN NATURE Lifson, J. D., Feinberg, M. B., Reyes, G. R., Rabin, L., Banapour, B., Chakrabarti, S., Moss, B., WONGSTAAL, F., Steimer, K. S., Engleman, E. G. 1986; 323 (6090): 725-728

  Abstract

  Formation of syncytia, with progression to cell death, is a characteristic feature of in vitro cultures of susceptible cells infected with human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). Viral antigen-positive multinucleated giant cells have also been observed in histological sections from infected individuals. In vitro, formation of these multinucleated giant cells occurs through cell fusion which is dependent on cell-surface expression of the differentiation antigen CD4. Utilizing a recombinant vaccinia virus containing the gene for the envelope glycoprotein of HTLV-III/LAV, we demonstrate that cell-surface expression of this protein, in the absence of other HTLV-III/LAV structural or regulatory proteins, is sufficient to induce CD4-dependent cell fusion, leading to cell death, one of the characteristic manifestations of AIDS (acquired immune deficiency syndrome) virus cytopathology. This process may contribute to the loss of CD4+ T cells seen in AIDS.

  View details for Web of Science ID A1986E529300060

  View details for PubMedID 3095663

• TRANSMISSION OF LYMPHADENOPATHY-ASSOCIATED VIRUS HUMAN LYMPHOTROPIC-T VIRUS TYPE-III IN SEXUAL PARTNERS - SEROPOSITIVITY DOES NOT PREDICT INFECTIVITY IN ALL CASES AMERICAN JOURNAL OF MEDICINE Burger, H., Weiser, B., Robinson, W. S., Lifson, J., Engleman, E., Rouzioux, C., BRUNVEZINET, F., BarreSinoussi, F., MONTAGNIER, L., CHERMANN, J. C. 1986; 81 (1): 5-10

  Abstract

  To investigate transmission of lymphadenopathy-associated virus (LAV)/human T lymphotropic virus type III (HTLV-III) in long-term sexual partners, and the relationship between lymphadenopathy-associated virus seropositivity and transmission, nine couples (five heterosexual and four homosexual) at increased risk for acquired immune deficiency syndrome (AIDS) were studied. In two heterosexual couples, transmission of lymphadenopathy-associated virus from a seropositive man at increased risk to his monogamous wife occurred. In one couple, the wife of a man with hemophilia had lymphadenopathy-associated virus antibody and decreased T helper cells; in the other couple, the wife of a bisexual intravenous drug-user had AIDS. Neither woman had a recognized AIDS risk except marriage to a seropositive man at increased risk. However, study of the other couples revealed that regular sexual contact with seropositive persons over long periods did not always lead to evidence of lymphadenopathy-associated virus infection. This study suggests that presence of lymphadenopathy-associated virus antibody does not always indicate a high degree of infectivity.

  View details for Web of Science ID A1986D368200001

  View details for PubMedID 3014878

• AIDS RETROVIRUS INDUCED CYTOPATHOLOGY - GIANT-CELL FORMATION AND INVOLVEMENT OF CD4 ANTIGEN SCIENCE Lifson, J. D., Reyes, G. R., McGrath, M. S., Stein, B. S., Engleman, E. G. 1986; 232 (4754): 1123-1127

  Abstract

  The formation of multinucleated giant cells with progression to cell death is a characteristic manifestation of the cytopathology induced by the AIDS retrovirus in infected T lymphoid cells. The mechanism of giant cell formation was studied in the CD4 (T4/Leu 3) positive T cell lines JM (Jurkat) and VB and in variants of these lines that are negative for cell surface CD4 antigen. By means of a two-color fluorescent labeling technique, multinucleated giant cells in infected cultures were shown to form through cell fusion. Antibody to CD4 specifically inhibited fusion, and uninfected CD4 negative cells, in contrast to uninfected CD4 positive cells, did not undergo fusion with infected cells, suggesting a direct role for the CD4 antigen in the process of syncytium formation. These results suggest that, in vivo, cell fusion involving the CD4 molecule may represent a mechanism whereby uninfected cells can be incorporated into AIDS virus infected syncytia. Because the giant cells die soon after they are formed, this process may contribute to the depletion of helper/inducer T cells characteristically observed in AIDS.

  View details for Web of Science ID A1986C427200029

  View details for PubMedID 3010463

• BETA-ADRENERGIC RECEPTORS ON HUMAN SUPPRESSOR, HELPER, AND CYTOLYTIC LYMPHOCYTES BIOCHEMICAL PHARMACOLOGY Khan, M. M., Sansoni, P., Silverman, E. D., Engleman, E. G., Melmon, K. L. 1986; 35 (7): 1137-1142

  Abstract

  Using the radioligand beta-adrenergic blocker [125I]cyanopindolol (ICYP), we have characterized the beta-adrenergic receptors on Leu 3+ (T helper [TH]), Leu 2+, 9.3- (T suppressor [Ts]) and Leu 2+, 9.3+ (T cytolytic [Tc]) subsets of human lymphocytes. Peripheral blood T cells were isolated by rosetting, and then subsets were purified by their affinities to monoclonal antibodies against their Leu 3 and 9.3 markers. ICYP binding to the subsets was saturable with time and with concentration; the binding was stereoselective and reversible by beta-adrenergic antagonists. A biological response produced by beta agonists increased intracellular concentrations of cAMP and corresponded to the number of binding sites. Each subset of cells had a number of binding sites, which was characteristic for the given subset. The data indicate that the density of distribution of beta-adrenergic receptors was not homogeneous on the precursors of phenotypically and functionally distinct T cells (Ts approximately 2900, Tc approximately 1800 and TH approximately 750 binding sites). The displacement studies using beta-adrenergic agonists were performed on the cytolytic and suppressor T cell subsets, suggesting that the receptors were mainly of the beta-2 type. The immunobiological significance of such selective distribution of numbers and subtypes of beta-adrenergic receptors on distinct T cell subsets is under investigation.

  View details for Web of Science ID A1986A899200011

  View details for PubMedID 3008755

• GENERATION OF HUMAN MONOCLONAL-ANTIBODIES BY FUSION OF EBV-ACTIVATED B-CELLS TO A HUMAN MOUSE HYBRIDOMA METHODS IN ENZYMOLOGY Foung, S. K., Engleman, E. G., GRUMET, F. C. 1986; 121: 168-174

  View details for Web of Science ID A1986D161200013

  View details for PubMedID 3014266

• UTILITY OF FORMALDEHYDE FIXATION FOR FLOW-CYTOMETRY AND INACTIVATION OF THE AIDS ASSOCIATED RETROVIRUS JOURNAL OF IMMUNOLOGICAL METHODS Lifson, J. D., Sasaki, D. T., Engleman, E. G. 1986; 86 (1): 143-149

  Abstract

  To maximize safety in the setting of an increasing number of requests for flow cytometric analysis of specimens potentially contaminated with the AIDS retrovirus, we evaluated some commonly used fixatives for their ability to inactivate the infectious potential of the virus. We found that both formaldehyde (0.37% v/v) and paraformaldehyde (0.5% w/v) completely inactivated the infectious activity of both free and cell-associated lymphadenopathy associated virus (LAV), the etiologic agent for the acquired immunodeficiency syndrome (AIDS). Based on encouraging preliminary results we formally evaluated the effect of formaldehyde fixation on flow cytometric parameters. In addition to inactivating LAV, 0.37% formaldehyde in phosphate buffered saline preserved light scatter and fluorescence properties of cells stained with fluorescein isothiocyanate (FITC) and beta-phycoerythrin (PE) conjugated monoclonal antibodies. These findings suggest that formalin fixation may be useful for laboratories performing flow cytometric analysis of specimens potentially contaminated with the AIDS virus.

  View details for Web of Science ID A1986AYM1500022

  View details for PubMedID 3003200

• PURIFIED INTERLEUKIN-2 INDUCES PROLIFERATION OF FRESH HUMAN-LYMPHOCYTES IN THE ABSENCE OF EXOGENOUS STIMULI JOURNAL OF BIOLOGICAL RESPONSE MODIFIERS Lifson, J., Raubitschek, A., Benike, C., Koths, K., Ammann, A., Sondel, P., Engleman, E. 1986; 5 (1): 61-72

  Abstract

  Highly purified interleukin-2 (IL-2) induced proliferation of fresh human peripheral blood mononuclear leukocytes (PBML) in the absence of identifiable exogenous mitogenic or antigenic stimuli. Dose dependent proliferation was observed with three IL-2 preparations, including two preparations purified from natural sources and a preparation produced by recombinant DNA techniques. Both T and non-T cells proliferated. Purified helper/inducer and suppressor/cytotoxic T cells cultured in the absence of non-T cells proliferated only weakly; the proliferative response to IL-2 was restored by the addition of irradiated non-T cells. Proliferation to IL-2 was not blocked by the monoclonal antibody anti-Leu 4, which reacts with a component of the T-cell receptor complex for antigen and blocks mitogen and antigen-induced T-cell responses. Monoclonal antibody to HLA-DR also failed to significantly block the proliferation of resting cells to IL-2. The IL-2 induced proliferative response thus appears to be dependent on interactions between different subpopulations of PBML but probably does not simply reflect augmentation by IL-2 of antigen-driven or autoreactive processes.

  View details for Web of Science ID A1986A012300007

  View details for PubMedID 3007680

• PRODUCTION OF HUMAN-ANTIBODIES TO MYCOBACTERIUM-LEPRAE Mohagheghpour, N., Cho, S. N., Gelber, R. H., Foung, S. K., Engleman, E. G. AMER LEPROSY MISSION. 1985: 717–18

  View details for Web of Science ID A1985A045900040

• HUMAN T-CELL SUBPOPULATIONS DISTINGUISHED BY MONOCLONAL-ANTIBODIES INTERNATIONAL OPHTHALMOLOGY CLINICS Fishwild, D., Engleman, E. G. 1985; 25 (2): 55-62

  View details for Web of Science ID A1985AJK6600006

  View details for PubMedID 3159705

• MATURATIONAL AND FUNCTIONAL DIVERSITY OF HUMAN LYMPHOCYTES-B DELINEATED WITH ANTI-LEU-8 JOURNAL OF IMMUNOLOGY Kansas, G. S., Wood, G. S., Engleman, E. G. 1985; 134 (5): 3003-3006

  Abstract

  Human B lymphocyte subpopulations distinguished by their expression of the Leu-8 antigen were studied and found to differ in their respective maturational state and functional repertoire. The absence of Leu-8 expression correlated with an early step in antigen-driven B cell differentiation in that 1) as presented in the companion paper, germinal center B lymphocytes were uniformly Leu-8-, whereas mantle zone B cells were virtually all Leu-8+; 2) treatment of Leu-8+ B cells with the combination of rabbit anti-human mu-chain and B cell growth factor (BCGF), but not with either reagent alone, caused the loss of Leu-8 expression in addition to causing these cells to proliferate; and 3) only the Leu-8- B cell subset contained cells expressing the 4F2 activation antigen. Functional studies of peripheral blood B cells revealed that B cells giving rise to antibody-forming cells in the presence of T cells and pokeweed mitogen (PWM) were found almost exclusively in the Leu-8- subset of B cells, even though this subset comprised a minority of circulating B lymphocytes. By contrast, Leu-8+ B cells proliferated more vigorously than Leu-8- B cells to formalinized Staphylococcus aureus Cowan I (SAC). These data demonstrate that Leu-8 is an important maturational and functional marker for human B lymphocytes.

  View details for Web of Science ID A1985AFX6000032

  View details for PubMedID 3920308

• TREATMENT OF INTRACTABLE LUPUS NEPHRITIS WITH TOTAL LYMPHOID IRRADIATION ANNALS OF INTERNAL MEDICINE Strober, S., Field, E., Hoppe, R. T., Kotzin, B. L., Shemesh, O., Engleman, E., Ross, J. C., Myers, B. D. 1985; 102 (4): 450-458

  Abstract

  Ten patients with lupus nephritis and marked proteinuria (3.9 g or more/d) that did not respond adequately to treatment with prednisone alone or prednisone in combination with azathioprine were treated with total lymphoid irradiation in an uncontrolled feasibility study. Within 6 weeks after the start of total lymphoid irradiation, the serum albumin level rose in all patients in association with a reduction in the serum level of anti-DNA antibodies, an increase in the serum complement level, or both. Improvement in these variables persisted in eight patients followed for more than 1 year, with the stabilization or reduction of the serum creatinine level. Urinary leakage of albumin was substantially reduced in all patients. Side effects associated with radiotherapy included transient constitutional complaints in ten patients, transient blood element depressions in three, localized viral and bacterial infections in four, and ovarian failure in one. The results suggest that total lymphoid irradiation may provide an alternative to cytotoxic drugs in the treatment of lupus nephritis.

  View details for Web of Science ID A1985AEV5700002

  View details for PubMedID 3872090

• EFFICACY OF TOTAL LYMPHOID IRRADIATION IN INTRACTABLE RHEUMATOID-ARTHRITIS - A DOUBLE-BLIND, RANDOMIZED TRIAL ANNALS OF INTERNAL MEDICINE Strober, S., Tanay, A., Field, E., Hoppe, R. T., Calin, A., Engleman, E. G., Kotzin, B., Brown, B. W., KAPLAN, H. S. 1985; 102 (4): 441-449

  Abstract

  Twenty-six patients participated in a randomized, double-blind study of the efficacy of total lymphoid irradiation in the treatment of intractable rheumatoid arthritis. All 26 patients, for whom therapy with gold compounds and penicillamine had failed, would ordinarily have been considered candidates for cytotoxic or antimetabolite drug therapy. Thirteen patients randomly assigned to receive full-dose total lymphoid irradiation (2000 rad) and 11 patients assigned to receive control low-dose total lymphoid irradiation (200 rad) completed radiotherapy. Alleviation of joint disease activity was significantly greater in the high-dose group as judged by morning stiffness, joint tenderness, and functional assessment (global composite score) at 3 and 6 months after radiotherapy. The high-dose group had a marked reduction in both T-lymphocyte function and numbers, but this finding was not observed in the low-dose group. Complications seen in the high-dose but not low-dose group included transient neutropenia, thrombocytopenia, pericarditis, and pleurisy.

  View details for Web of Science ID A1985AEV5700001

  View details for PubMedID 3883868

• HUMAN-BONE MARROW AND PERIPHERAL-BLOOD LYMPHOCYTE-T DEPLETION - EFFICACY AND EFFECTS OF BOTH T-CELLS AND MONOCYTES ON GROWTH OF HEMATOPOIETIC PROGENITORS BLOOD Levitt, L., Kipps, T. J., Engleman, E. G., Greenberg, P. L. 1985; 65 (3): 663-679

  Abstract

  The efficacy of four separate methods of human bone marrow T lymphocyte depletion was assessed, and the effect of T cells and monocytes on in vitro growth of marrow (CFU-GEMM, BFU-E, and CFU-GM) and peripheral blood (BFU-E) hematopoietic progenitors was determined. Extent of T cell depletion was assessed by multiparameter fluorescent cell sorter (FACS) analysis and by functional studies. Cells staining positively by FACS analysis for one or more of three separate fluorescent pan-T cell monoclonal antibodies (MCAbs) comprised 8.4% to 9.5% of control marrow mononuclear cells (MNCs). T cells constituted 3.2% to 5.1% of marrow following single, sequential, or combination treatment with two different pan-T cell MCAbs (Leu 1 and TM1) plus complement, 1.5% to 2.2% of marrow following solid-phase immunoabsorption ("panning"), 0.2% of marrow after sheep cell rosetting, and only 0.05% of marrow after FACS selective cell sorting and gated separation. T cells made up 59% to 73% of control peripheral blood MNCs and 0.8% to 2.8% of peripheral MNCs following sheep cell rosetting plus treatment with Leu 1 MCAb and complement. Mitogen (PHA, Con A) and allogeneic MLC-induced blastogenic responses (stimulation indices, experimental/control or E/C) revealed a concordant decrement in marrow T cell function after MCAb plus complement (E/C of 3.9 to 9.0), after panning (E/C of 1.6 to 3.5) and after sheep cell rosetting (E/C of 0.7 to 1.3), compared with control marrow (E/C of 5.3 to 15.7). After T cell depletion, marrow BFU-E growth was 95% to 120% of control, CFU-GM growth was 90% to 108% of control, and CFU-GEMM growth was 89% to 111% of control. Marrow T cell and/or monocyte depletion did not alter erythropoietin-dependent BFU-E growth in the absence of Mo-conditioned medium (81% to 95% of control), and the addition of as many as 50 to 100 X 10(3) purified marrow monocytes or T cells to 10(5) autologous nonadherent T cell-depleted marrow target cells had a negligible (P greater than .1) effect on marrow BFU-E growth in vitro. Peripheral blood (PB) BFU-E/10(5) T-depleted target cells were 106% +/- 19% of expected; PB BFU-E growth was significantly diminished after monocyte depletion alone (7% +/- 6% of expected) or after monocyte plus T cell depletion (8% +/- 4% of expected).(ABSTRACT TRUNCATED AT 400 WORDS)

  View details for Web of Science ID A1985ADE3300023

  View details for PubMedID 3871644

• RECOMBINANT INTERLEUKIN-2 ENHANCED NATURAL-KILLER CELL-MEDIATED CYTO-TOXICITY IN HUMAN-LYMPHOCYTE SUBPOPULATIONS EXPRESSING THE LEU-7 AND LEU-11 ANTIGENS JOURNAL OF IMMUNOLOGY Lanier, L. L., Benike, C. J., Phillips, J. H., Engleman, E. G. 1985; 134 (2): 794-801

  Abstract

  Highly purified recombinant human interleukin 2 (rIL 2) markedly augments the natural killer (NK) cell-mediated cytotoxicity of peripheral blood lymphocytes. In this study, we examined the cellular and metabolic basis of rIL 2-mediated activation of human lymphocyte subpopulations expressing the NK cell-associated surface antigens Leu 7 and Leu 11. All rIL 2-responsive cytotoxic NK cells were found within the subset of lymphocytes expressing the Leu 11 marker, an antigen associated with the Fc-IgG receptor on human NK cells. Cells lacking the Leu 11 antigen, including cells expressing another NK cell-associated marker, Leu 7, did not express NK cell-mediated cytotoxicity either before or after rIL 2 treatment. By contrast, rIL 2 augmented the NK activity of both Leu7-,11+ and Leu 7+,11+ subpopulations. Activation of Leu 11+ NK cells resulted from a direct effect of rIL 2 on these cells and neither required nor was amplified by the presence of T lymphocytes. Enhanced NK cell-mediated cytotoxicity occurred within 4 hr after exposure to rIL 2, and was blocked by the protein synthesis inhibitor cyclohexamide, but not by the DNA synthesis inhibitor mitomycin C or 1500 rad of x-irradiation. Neither Tac antigen, a high-affinity receptor for IL 2, nor other activation markers, such as transferrin receptor or HLA-DR antigen, were detectable on a significant proportion of Leu 11+ cells, either before or after incubation with rIL 2 for 48 hr. In addition, saturating concentrations of antibodies to each of these markers had no effect on the enhancement of NK activity by rIL 2. Finally, preliminary experiments with neutralizing antibodies to gamma- and alpha-interferons also failed to prevent rIL 2 enhancement of NK cell-mediated cytotoxicity, suggesting that rIL 2 does not mediate its effect via release of these cytokines.

  View details for Web of Science ID A1985AAL9200024

  View details for PubMedID 3871216

• DEFECTIVE CELL-MEDIATED-IMMUNITY IN LEPROSY - FAILURE OF T-CELLS FROM LEPROMATOUS LEPROSY PATIENTS TO RESPOND TO MYCOBACTERIUM-LEPRAE IS ASSOCIATED WITH DEFECTIVE EXPRESSION OF INTERLEUKIN-2 RECEPTORS AND IS NOT RECONSTITUTED BY INTERLEUKIN-2 JOURNAL OF IMMUNOLOGY Mohagheghpour, N., Gelber, R. H., Larrick, J. W., Sasaki, D. T., Brennan, P. J., Engleman, E. G. 1985; 135 (2): 1443-1449

  Abstract

  Patients with lepromatous leprosy (LL) but not borderline tuberculoid leprosy (BT) have defective cell-mediated immune responses to Mycobacterium leprae, despite normal responses to other stimuli, as judged by in vivo skin testing and in vitro lymphocyte transformation. To investigate the basis of the immune defect in LL patients, we studied the ability of patient mononuclear leukocytes to produce interleukin 1 (IL 1) and interleukin 2 (IL 2) upon stimulation with M. leprae, and determined the ability of exogenous IL 1 and IL 2 to reconstitute the LL patient response to this antigen in vitro. Equal numbers of adherent non-T cells from LL and BT patients produced similar amounts of IL 1 upon challenge with M. leprae, and addition of IL 1 to the culture medium failed to reconstitute the response of lymphocytes from LL patients to M. leprae. On the other hand, T cells of LL patients failed to express receptors for IL 2 or to produce IL 2 in response to M. leprae, whereas similarly treated T cells of BT patients both expressed IL 2 receptors and produced IL 2. Finally, recombinant human IL 2 purified to homogeneity as well as crude supernatants of mitogen-activated lymphocytes failed to reconstitute the response of LL patients to M. leprae. These results suggest that T cells of LL patients fail to respond to M. leprae despite an ability to produce IL 1 and that their failure to express receptors for IL 2 may explain both defective proliferation and the failure of exogenous IL 2 to reconstitute the response.

  View details for Web of Science ID A1985AQP9100091

  View details for PubMedID 2989366

• RESPONSES OF SUBPOPULATIONS OF HUMAN HELPER T-CELLS TO AUTACOIDS PROCEEDINGS OF THE WESTERN PHARMACOLOGY SOCIETY Khan, M. M., Silverman, E., Engleman, E. G., Melmon, K. L. 1985; 28: 225-228

  View details for Web of Science ID A1985AKA5700056

  View details for PubMedID 2866535

• FUNCTIONAL-CHARACTERIZATION OF HUMAN LYMPHOCYTE-T SUBSETS DISTINGUISHED BY MONOCLONAL ANTI-LEU-8 JOURNAL OF IMMUNOLOGY Kansas, G. S., Wood, G. S., Fishwild, D. M., Engleman, E. G. 1985; 134 (5): 2995-3002

  Abstract

  Previous studies have shown that monoclonal anti-Leu-8 antibody identifies functionally distinct subpopulations within both the Leu-2 (T8+) and Leu-3 (T4+) lineages of human T lymphocytes. We now report in detail on the tissue distribution of the Leu-8 antigen and on extensive functional studies of T cells subsets distinguished by their expression or lack of expression of this marker. Leu-8 is present on a wide variety of hematologic cells, including granulocytes, T and B lymphocytes, monocytes, and null or NK cells. Within lymph nodes and tonsils, Leu-8 is absent from both B and T cells within germinal centers but is present on nearly all paracortical lymphocytes. Leu-8 is present on most but not all EBV-transformed B cell lines, reflecting its presence on a subset of normal peripheral blood B cells. None of six malignant T cell lines tested were Leu-8+, whereas most circulating T cells are Leu-8+. Although standard immunoprecipitation techniques failed to demonstrate any specific bands on SDS polyacrylamide gels, the antigenic determinant recognized by anti-Leu-8 is protein or protein-associated, because brief treatment of target cells with pronase abrogated binding of anti-Leu-8. Both Leu-3+8+ and Leu-3+8- cells proliferated in response to several soluble antigens and to autologous and allogeneic non-T cells. Nonetheless, nearly all of the helper T cells for PWM- and AMLR-induced PFC were contained within the Leu3+8- subset. Optimal suppression of the PWM-induced PFC response required both Leu-2+8+ and Leu-2+8- cells, and irradiation of either subset with 3000 R abrogated the capacity of the recombined subsets to effect suppression. In contrast to help for B cell differentiation, both Leu-3+8+ and Leu-3+8- cells were capable of amplifying the development of allospecific T killer cells; precursor and effector T killer cells could be found within both Leu-2+8+ and Leu-2+8- subpopulations. The correlation between Leu-8 phenotype and selected immune functions of T cells (and B cells; see companion paper) indicates that anti-Leu-8 distinguishes important immunoregulatory T and B lymphocyte subsets in man.

  View details for Web of Science ID A1985AFX6000031

  View details for PubMedID 3156924

• VARIABLES AFFECTING LYMPHOCYTE-T SUBSETS IN A VOLUNTEER BLOOD-DONOR POPULATION CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Lifson, J. D., Finch, S. L., Sasaki, D. T., Engleman, E. G. 1985; 36 (2): 151-160

  Abstract

  Inversion of the normal ratio of helper/inducer (Th) to suppressor/cytotoxic (Ts) T lymphocytes is a characteristic finding in symptomatic and presymptomatic patients with acquired immunodeficiency syndrome (AIDS). As an interim measure to prevent transmission of AIDS via blood transfusion, the Stanford Blood Center performed T-lymphocyte subset analysis as a screening procedure. In this report we present results from 8715 consecutive volunteer blood donors, aged 17-77. The mean Th:Ts ratio was 2.00 +/- 0.70 with a significant trend for increasing Th:Ts with increasing age. In all age groups, donors with antibody to cytomegalovirus (CMV) had lower Th:Ts ratios than CMV-seronegative donors. 1.9% of the donors had Th:Ts values less than or equal to 0.85 and blood from these donors was not used for clinical purposes. Compared to the overall donor population, individuals with Th:Ts less than or equal to 0.85 tended to be male, (79 vs 53%) and CMV sero-positive (71 vs 36%); a majority (83%) had a low absolute number of Th cells as well as a low Th:Ts value. Follow-up of donors with low Th:Ts values revealed that some belonged to AIDS high-risk populations, despite denials at the time of donation. One such donor was diagnosed with disseminated Kaposi's sarcoma 8 months after a markedly abnormal T-lymphocyte subset profile was noted during screening. These results suggest that T-lymphocyte subset analysis is capable of identifying at least some blood donors at risk for transmitting AIDS and also point to variable affecting T-cell subsets in apparently healthy blood donors.

  View details for Web of Science ID A1985ALS7600003

  View details for PubMedID 2988842

• IMMUNOREGULATORY T-CELL IN MAN - HISTAMINE-INDUCED SUPPRESSOR T-CELLS ARE DERIVED FROM A LEU 2+ (T8+) SUBPOPULATION DISTINCT FROM THAT WHICH GIVES RISE TO CYTO-TOXIC T-CELLS JOURNAL OF CLINICAL INVESTIGATION Sansoni, P., Silverman, E. D., Khan, M. M., Melmon, K. L., Engleman, E. G. 1985; 75 (2): 650-656

  Abstract

  One mechanism of histamine-mediated inhibition of the immune response in man is to activate T suppressor cells that bear the Leu 2 (OKT8) marker. The current study was undertaken to characterize the histamine-induced suppressor cell using a monoclonal antibody (9.3) shown previously to distinguish cytotoxic T cells from antigen-specific suppressor T cells. Leu 2+ cells isolated from peripheral blood were further separated with antibody 9.3 into Leu 2+, 9.3+, and Leu 2+, 9.3- subsets and each subset was incubated with different concentrations of histamine before determining their ability to suppress immune responses in vitro. The results indicate that the Leu 2+, 9.3- subpopulation includes all histamine-induced suppressor cells, that 10(-4) M histamine is the optimal concentration for suppressor cell induction, and that exposure of Leu 2+, 9.3- cells to histamine for 30 s is sufficient to initiate the induction process. After treatment with histamine these cells inhibit both phytohemagglutinin-induced T cell proliferation and pokeweed mitogen-induced B cell differentiation. The suppression of phytohemagglutinin-induced proliferation was resistant to x-irradiation with 1,200 rad, either before or after histamine exposure, suggesting that Leu 2+, 9.3- cells need not proliferate to become suppressor cells or exert suppression. Moreover, suppression by these cells was not due to altered kinetics of the response. Finally, a histamine type 2 receptor antagonist (cimetidine) but not a type 1 receptor antagonist (mepyramine) blocked the induction of suppressor cells. On the basis of these results and our previous studies of antigen specific suppressor cells, we conclude that Leu 2+ suppressor cells in man are derived from a precursor pool that is phenotypically distinct from cells that can differentiate into cytotoxic T cells.

  View details for Web of Science ID A1985ACA2500040

  View details for PubMedID 3156150

• FUNCTIONAL AND QUANTITATIVE ALTERATIONS IN LYMPHOCYTE-T SUBPOPULATIONS IN ACUTE TOXOPLASMOSIS JOURNAL OF INFECTIOUS DISEASES Luft, B. J., Kansas, G., Engleman, E. G., Remington, J. S. 1984; 150 (5): 761-767

  Abstract

  The cellular immune response to Toxoplasma gondii has been studied in 23 patients with acute toxoplasma infection. Abnormalities of T cell subpopulations included a marked and significant elevation in suppressor (Leu 2) T cells in patients with prolonged symptoms due to acute infection and either a decrease in the number of T helper cells or an increase in the number of suppressor cells--or both--in patients with asymptomatic lymphadenopathy. There was no significant difference in lymphocyte proliferation to phytohemagglutinin or pokeweed mitogen among the various groups tested. The peak lymphocyte response to toxoplasma antigen, however, was significantly depressed in patients with acute infection compared with that in chronically infected control patients. The kinetics of the depression were consistent with the induction of a non-Leu 2 suppressor cell. These results demonstrate marked quantitative alterations in T lymphocyte subpopulations and functional alterations of T cells to toxoplasma antigen during infection with T. gondii.

  View details for Web of Science ID A1984TP54200019

  View details for PubMedID 6238107

• SUSTAINED IMPROVEMENT OF INTRACTABLE RHEUMATOID-ARTHRITIS AFTER TOTAL LYMPHOID IRRADIATION ARTHRITIS AND RHEUMATISM Field, E. H., Strober, S., Hoppe, R. T., Calin, A., Engleman, E. G., Kotzin, B. L., TANAY, A. S., CALIN, H. J., TERRELL, C. P., KAPLAN, H. S. 1983; 26 (8): 937-946

  Abstract

  Total lymphoid irradiation (TLI) was administered to 11 patients who had intractable rheumatoid arthritis that was unresponsive to conventional medical therapy, including aspirin, multiple nonsteroidal antiinflammatory drugs, gold salts, and D-penicillamine. Total lymphoid irradiation was given as an alternative to cytotoxic drugs such as azathioprine and cyclophosphamide. After radiotherapy, 9 of the 11 patients showed a marked improvement in clinical disease activity as measured by morning stiffness, joint tenderness, joint swelling, and overall functional abilities. The mean improvement of disease activity in all patients ranged from 40-70 percent and has persisted throughout a 13-28 month followup period. This improvement permitted the mean daily steroid dose to be reduced by 54%. Complications included severe fatigue and other constitutional symptoms during radiotherapy, development of Felty's syndrome in 1 patient, and an exacerbation of rheumatoid lung disease in another. After therapy, all patients exhibited a profound T lymphocytopenia, and a reversal in their T suppressor/cytotoxic cell to helper cell ratio. The proliferative responses of peripheral blood mononuclear cells to phytohemagglutinin, concanavalin A, and allogeneic leukocytes (mixed leukocyte reaction) were markedly reduced, as was in vitro immunoglobulin synthesis after stimulation with pokeweed mitogen. Alterations in T cell numbers and function persisted during the entire followup period, except that the mixed leukocyte reaction showed a tendency to return to normal values.

  View details for Web of Science ID A1983RC91100001

  View details for PubMedID 6882488

• SELECTIVE ENRICHMENT OF HUMAN EPIDERMAL-CELL SUBPOPULATIONS USING MONOCLONAL-ANTIBODIES JOURNAL OF INVESTIGATIVE DERMATOLOGY Morhenn, V. B., Wood, G. S., Engleman, E. G., Oseroff, A. R. 1983; 81 (1): S127-S131

  View details for Web of Science ID A1983QZ98700023

• INHIBITION OF A LANGERHANS CELL-MEDIATED IMMUNE-RESPONSE BY TREATMENT MODALITIES USEFUL IN PSORIASIS JOURNAL OF INVESTIGATIVE DERMATOLOGY Morhenn, V. B., Orenberg, E. K., Kaplan, J., PFENDT, E., Terrell, C., Engleman, E. G. 1983; 81 (1): 23-27

  Abstract

  Neither the pathogenesis of psoriasis nor the mechanism whereby seemingly diverse therapies alter the disease is understood. In this study, several antipsoriatic agents were tested for their effects on the skin cell lymphocyte reaction (SLR), an immunologic assay in which HLA-DR antigens on Langerhans cells (LC) stimulate proliferation of allogeneic lymphocytes. Every agent tested (cortisol, methotrexate, hyperthermia, anthralin) inhibited the SLR at therapeutic dose levels. By contrast, a variety of antibiotics, an anti-inflammatory agent, and lithium carbonate and propranolol, two drugs known to be ineffective in psoriasis, failed to inhibit the SLR. Finally, we have shown that hyperthermia and anthralin treatments are toxic for LC whereas they have little or no effect on keratinocyte viability. These results suggest that antipsoriatic agents may act in psoriasis by alteration or killing of LC.

  View details for Web of Science ID A1983QZ18600007

  View details for PubMedID 6863976

• CORRELATION OF FUNCTIONAL-PROPERTIES OF HUMAN LYMPHOID-CELL SUBSETS AND SURFACE MARKER PHENOTYPES USING MULTIPARAMETER ANALYSIS AND FLOW-CYTOMETRY IMMUNOLOGICAL REVIEWS Lanier, L. L., Engleman, E. G., Gatenby, P., Babcock, G. F., Warner, N. L., Herzenberg, L. A. 1983; 74: 143-160

  View details for Web of Science ID A1983RJ69300007

  View details for PubMedID 6226586

• CHANGES IN T-CELL SUBSETS IN PATIENTS WITH RHEUMATOID-ARTHRITIS TREATED WITH TOTAL LYMPHOID IRRADIATION CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Kotzin, B. L., Kansas, G. S., Engleman, E. G., Hoppe, R. T., KAPLAN, H. S., Strober, S. 1983; 27 (2): 250-260

  Abstract

  Patients with intractable rheumatoid arthritis (RA) were treated with total lymphoid irradiation (TLI, 2000 rads). We previously reported long-lasting clinical improvement associated with marked suppression of in vitro lymphocyte function in this group. In an attempt to better understand the mechanism of immunosuppression and clinical changes observed after TLI, we studied in greater detail changes in peripheral blood T-cell subsets identified by monoclonal antibodies. Before TLI, RA patients had a higher percentage of Leu-3 (helper subset) cells and a lower percentage of Leu-2 (suppressor/cytotoxic subset) cells than normals. Immediately after TLI, the absolute numbers of both Leu-2 and Leu-3 cells were reduced by at least 90%. Within 6-12 weeks, the number of Leu-2 cells returned to the pretreatment levels, but the levels of Leu-3 cells remained depressed for months thereafter. The lack of repopulation of Leu-3 cells resulted in a marked increase in the ratio of Leu-2 to Leu-3 cells as compared to pretreatment values (1.73 +/- 0.23 vs 0.39 +/- 0.06), and in a decrease in the percentage and absolute number of total T (Leu-1 and Leu-4) cells. The failure of Leu-3 cells (which mediate predominantly helper/inducer functions) to repopulate the peripheral blood may contribute to the prolonged clinical immunosuppression observed after TLI. Similar changes in T-cell subsets were not observed in RA patients given remittive drugs or low doses (200 rads) of radiotherapy. Thus, TLI differs from other treatment modalities with regard to its prolonged selective effect on the Leu-3 subset.

  View details for Web of Science ID A1983QM43600011

  View details for PubMedID 6409481

• SEPARATION OF HUMAN-SKIN CELLS BY VELOCITY SEDIMENTATION INTO FUNCTIONALLY DISTINCT FRACTIONS JOURNAL OF INVESTIGATIVE DERMATOLOGY Morhenn, V. B., STARR, E. D., Terrell, C., Cox, A. J., Engleman, E. G. 1982; 78 (4): 319-322

  Abstract

  The epidermis consists of a heterogeneous population of cells including Langerhans cells, Merkel cells, melanocytes, and keratinocytes in various stages of differentiation. The current study was undertaken to determine if skin cell suspensions can be separated into morphologically and/or functionally distinct fractions. Skin cells were suspended by trypsinization and separated into multiple fractions by velocity sedimentation. Certain fractions reproducibly stimulated proliferation of allogeneic lymphocytes in the skin cell lymphocyte reaction, whereas other fractions, containing larger cells, supported growth of keratinocyte colonies in cell cultures. These results indicate that stimulation in the skin cell lymphocyte reaction and growth of keratinocyte colonies are mediated by distinct cells, separable by velocity sedimentation.

  View details for Web of Science ID A1982NK42300015

  View details for PubMedID 7069211

• DISSECTION OF IMMUNOREGULATORY SUB-POPULATIONS OF LYMPHOCYTES-T WITHIN THE HELPER AND SUPPRESSOR SUB-LINEAGES IN MAN JOURNAL OF IMMUNOLOGY Gatenby, P. A., Kansas, G. S., Xian, C. Y., Evans, R. L., Engleman, E. G. 1982; 129 (5): 1997-2000

  Abstract

  The helper/inducer (Leu-3) and suppressor/cytotoxic (Leu-2) sublineages of human peripheral blood T cells can both the subdivided into functionally distinct subsets with anti-Leu-8, a new monoclonal antibody that identifies 75 +/- 10% of the Leu-3 cells and 60 +/- 10% of the Leu-2 cells. Using the autologous MLR as a T cell-dependent stimulus for immunoglobulin synthesis, we have shown that the major helper effect for antibody formation lies within the numerically minor Leu-3,8- subset. In addition, neither Leu-2,8- cells alone suppress Leu-3-induced immunoglobulin synthesis, but in combination these subsets are markedly inhibitory. These results indicate that at least two phenotypically distinct cell types of suppressor lineage interact to produce suppression of an immune response in man.

  View details for Web of Science ID A1982PM14700035

  View details for PubMedID 6214586

• IMMUNOGLOBULIN SECRETION IN THE HUMAN AUTOLOGOUS MIXED LEUKOCYTE REACTION - DEFINITION OF A SUPPRESSOR-AMPLIFIER CIRCUIT USING MONOCLONAL-ANTIBODIES JOURNAL OF EXPERIMENTAL MEDICINE Gatenby, P. A., Kotzin, B. L., Kansas, G. S., Engleman, E. G. 1982; 156 (1): 55-67

  Abstract

  The induction of immunoglobulin (Ig) synthesis in the autologous MLR has an absolute requirement for helper/inducer (Leu-3) T cells, whereas an excess of suppressor/cytotoxic (leu-2) cells suppresses the response. The current study was an effort to assess the immunoregulatory potential to T cells activated in the autologous mixed-leukocyte response (MLR). T cells were cultured with autologous non-T cells for 8-9 d, after which the activated T cells were fractionated into subsets with monoclonal antibodies to T cell markers and HLA-DR antigen. Each population was co-cultured in fresh autologous MLR, and on the 8th day of culture, Ig-secreting cells were measured in a reverse hemolytic plaque assay. The results show that activated Leu-2, DR+ T cells, but neither Leu-2, DR- nor Leu-3 T cells, were at least 50 times more potent as suppressors of IgM and IgG synthesis than fresh Leu-2 cells alone. The activation of this Leu-2, DR+ subpopulation required Leu-3 cells in the primary culture. Furthermore, in the absence of Leu-2 cells in the second culture, little or no suppression was observed, suggesting that the Leu-2, DR+ cells act to amplify or induce suppressor effects of fresh Leu-2 cells. This indicates that at least two distinct subpopulations of Leu-2 cells are required for maximal suppression of an immune response, and that immunoregulatory circuits analogous to those described in the mouse exist in man.

  View details for Web of Science ID A1982NV93500005

  View details for PubMedID 6211500

  View details for PubMedCentralID PMC2186740

• TREATMENT OF INTRACTABLE RHEUMATOID-ARTHRITIS WITH TOTAL LYMPHOID IRRADIATION NEW ENGLAND JOURNAL OF MEDICINE Kotzin, B. L., Strober, S., Engleman, E. G., Calin, A., Hoppe, R. T., Kansas, G. S., TERRELL, C. P., KAPLAN, H. S. 1981; 305 (17): 969-976

  Abstract

  Eleven patients with intractable rheumatoid arthritis were treated with total lymphoid irradiation (total dose, 2000 rad) in an uncontrolled feasibility study, as an alternative to long-term therapy with cytotoxic drugs such as cyclophosphamide and azathioprine. During a follow-up period of five to 18 months after total lymphoid irradiation, there was a profound and sustained suppression of the absolute lymphocyte count and in vitro lymphocyte function, as well as an increase in the ratio of Leu-2 (suppressor/cytotoxic) to Leu-3 (helper) T cells in the blood. Persistent circulating suppressor cells of the mixed leukocyte response and of pokeweek mitogen-induced immunoglobulin secretion developed in most patients. In nine of the 11 patients, these changes in immune status were associated with relief of joint tenderness and swelling and with improvement in function scores. Maximum improvement occurred approximately six months after irradiation and continued for the remainder of the observation period. Few severe or chronic side effects were associated with the radiotherapy.

  View details for Web of Science ID A1981ML04900002

  View details for PubMedID 6456414

• INDUCTION OF IMMUNOGLOBULIN SECRETING CELLS IN THE HUMAN AUTOLOGOUS MIXED LEUKOCYTE REACTION - REGULATION BY HELPER AND SUPPRESSOR LYMPHOCYTE SUBSETS DEFINED WITH MONOCLONAL-ANTIBODIES JOURNAL OF IMMUNOLOGY Gatenby, P. A., Kotzin, B. L., Engleman, E. G. 1981; 127 (5): 2130-2135

  Abstract

  The generation of IgM- and IgG-secreting cells in the human autologous MLR has been confirmed by using a microwell culture system and a reverse hemolytic plaque assay. The regulation of this response by T cell subsets was examined by using monoclonal antibodies that have previously allowed the definition of a Leu-3 helper/inducer subpopulation and a Leu-2 suppressor/cytotoxic subpopulation. These subsets were isolated by "panning" with monoclonal antibodies and were then cultured with autologous non-T cells, which serve as both stimulators of the reaction and a B cell source. The induction of antibody-forming cells was found to be dependent upon the Leu-3 subset whereas the Leu-2 subset not only failed to support C cell maturation, it actively suppressed the Leu-3 induced response. The Leu-3 response was found to be partially sensitive to radiation doses of 1000 rad or greater, whereas Leu-2 mediated suppression, even at high doses of cells, was totally ablated by 1500 rad. Thus, the generation of antibody-forming cells in the human autologous MLR is regulated by a balance of Leu-3 cell help and Leu-2 cell suppression. Such a finding may prove to be of relevance to immunoregulation in both health and disease.

  View details for Web of Science ID A1981MP76800078

  View details for PubMedID 6457864

• AUTOLOGOUS MIXED LYMPHOCYTE-REACTION IN PATIENTS WITH HODGKINS-DISEASE - EVIDENCE FOR A T-CELL DEFECT JOURNAL OF CLINICAL INVESTIGATION Engleman, E. G., Benike, C. J., Hoppe, R. T., KAPLAN, H. S., Berberich, F. R. 1980; 66 (1): 149-158

  Abstract

  The proliferative response of T lymphocytes cultured with autologous non-T lymphocytes is known as the autologous mixed lymphocyte reaction (MLR). This reaction can be demonstrated reproducibly in healthy individuals and has been shown to generate specific cytotoxic T cells, as well as T cells that regulate antibody synthesis and cell-mediated immunity. In this study, we demonstrate that the autologous MLR is impaired or absent in most patients with Hodgkin's disease regardless of age, sex, pathologic stage, or histologic classification. In 64 patients, the mean autologous MLR was 3,084+/-1,878 cpm compared to 16,552+/-6,532 in 29 healthy donors. A defect in autologous MLR was observed in newly diagnosed patients before the initiation of therapy, but was also found in patients without evidence of recurrent disease up to 15 yr after treatment. These findings could not be explained by abnormal kinetics or poor viability of stimulator or responder cells. The possibility that suppressor cells are responsible for the reduction of T cell autoreactivity was examined by comparing the autologous MLR of a healthy HLA-identical sibling in the presence and absence of T or non-T cells of an affected sibling. No inhibitory effects were observed. Similarly, substitution of patient plasma for pooled AB serum failed to inhibit the autologous responses of normal donors. Increasing the number of responder T cells in the culture or removing adherent cells from the stimulator population enhanced autoreactivity in some patients, indicating that the defect is not absolute. In two families, T cells of healthy HLA-A, B, and DR-identical siblings of patients responded normally to the non-T cells of their affected siblings, whereas patients' T cells failed to respond both to their own stimulator cells and those of their healthy HLA-identical siblings. These data indicate that the impairment of autologous MLR in some patients is due to a reduction or dysfunction of responder T cell activity and not to a defect of autologous stimulator cells.

  View details for Web of Science ID A1980JX70500019

  View details for PubMedID 6447164

• SUPPRESSOR CELLS OF THE MIXED LYMPHOCYTE-REACTION IN PATIENTS WITH HODGKINS-DISEASE TRANSPLANTATION PROCEEDINGS Engleman, E. G., Benike, C., Hoppe, R. T., KAPLAN, H. S. 1979; 11 (4): 1827-1829

  View details for Web of Science ID A1979JB52500038

  View details for PubMedID 161096

• SUPPRESSOR T-CELL OF MIXED LYMPHOCYTE-REACTION IN MAN SPECIFIC FOR STIMULATING ALLOANTIGEN - EVIDENCE THAT IDENTITY AT HLA-D BETWEEN SUPPRESSOR AND RESPONDER IS REQUIRED FOR SUPPRESSION JOURNAL OF EXPERIMENTAL MEDICINE Engleman, E. G., McMichael, A. J., BATEY, M. E., McDevitt, H. O. 1978; 147 (1): 137-146

  Abstract

  It has previously been shown that J.H., a human leukocyte antigen (HLA)-Dw2 homozygous multiparous woman, fails to respond in a mixed lymphocyte reaction (MLR) to her Dw1 homozygous husband W.H., and that her T cells suppress the responses of HLA matched responders to W.H. The present studies take advantage of the observation that J.H. suppressor cells resist a dose of gamma-irradiation which functionally eliminates her MLR responder cells. J.H. cells, depleted of alloreactive cells, suppress the responses of Dw2 heterozygous or homozygous cells to W.H., regardless of their associated HLA-A or B antigens. Only when W.H. or a few other cells are present as the irradiated stimulator is J.H. suppression of Dw2 responses detected. Thus, the J.H. suppressor T cell recognizes determinants in the irradiated stimulator cells as well as D locus products in the responder.

  View details for Web of Science ID A1978EF34700012

  View details for PubMedID 75232

  View details for PubMedCentralID PMC2184102

• MIXED LYMPHOCYTE-REACTION IN HEALTHY WOMEN WITH RHEUMATOID-FACTOR - LACK OF ASSOCIATION WITH HLA-DW4 ARTHRITIS AND RHEUMATISM Engleman, E. G., SPONZILLI, E. E., BATEY, M. E., Ramcharan, S., McDevitt, H. O. 1978; 21 (6): 690-693

  Abstract

  In independent studies, 51% and 36% of patients with rheumatoid arthritis have been shown to possess HLA-Dw4 compared to 7% and 13% of controls. In one study Dw4 positive patients more frequently had rheumatoid factor in their sera than did Dw4 negative patients. In order to determine if the Dw4 specificity is associated with the disease or with the presence of rheumatoid factor, the frequency of this HLA antigen has been determined in 24 healthy women known to have rheumatoid factor (median titer 1:160). Only 3 were found to have the Dw4 specificity, suggesting that this specificity is not associated with rheumatoid factor in the absence of rheumatoid arthritis.

  View details for Web of Science ID A1978FL76500012

  View details for PubMedID 153750

• SUPPRESSOR T-CELL OF MIXED LYMPHOCYTE-REACTION SPECIFIC FOR HLA-D REGION IN MAN JOURNAL OF CLINICAL INVESTIGATION Engleman, E. G., McDevitt, H. O. 1978; 61 (3): 828-838

  Abstract

  The mixed lymphocyte reaction (MLR) is the proliferative response of one individual's lymphocytes cultured in the presence of another individual's lymphocytes. In man, the MLR is elicited by cell surface antigens coded for by the HLA-D gene locus. This locus is among a cluster of genes which are located on the sixth chromosome and which include genes coding for the major histocompatibility antigens HLA-A, B, and C as well as HLA-D. If the stimulator cell possesses D locus antigens not present in the responder, the lymphocytes of the latter will undergo blast transformation resulting in DNA synthesis which can be measured. A vigorous response in the MLR to allogeneic cells is the rule among healthy individuals. We describe studies of a 23-yr-old man whose lymphocytes respond normally to mitogens and soluble antigens but fail to respond to allogeneic cells in the MLR. His medical history is unremarkable except that he received thymic irradiation as an infant. HLA typing revealed that he is homozygous for HLA-A2, B12, and Cw5 as well as for the D locus antigen Dw4. When his lymphocytes were added to the responder lymphocytes of other persons homozygous for the same HLA antigens, their responses to allogeneic cells but not mitogens were suppressed by 50-95%. Their responses to a soluble antigen, tetanus toxoid, were suppressed to a lesser degree. These inhibitory effects were mediated by a relatively radioresistant thymus-derived (T) lymphocyte. Further studies of the requirements for MLR suppression revealed that only persons heterozygous or homozygous for the Dw4 antigen were inhibited by the suppressor T cell. This effect was not altered by differences in the HLA-A, B, or C antigens between the suppressor and responder. It is concluded that genes in or near the HLA-D locus code not only for antigens (primarily on bone marrow-derived (B) cells), that elicit the MLR, but also for structures on T cells, or possibly macrophages, which are recognized by MLR suppressor T cells.

  View details for Web of Science ID A1978EQ35500033

  View details for PubMedID 147885

  View details for PubMedCentralID PMC372598

• SUPPRESSION OF MIXED LYMPHOCYTE-REACTION IN MAN BY A SOLUBLE T-CELL FACTOR - SPECIFICITY OF FACTOR FOR BOTH RESPONDER AND STIMULATOR JOURNAL OF EXPERIMENTAL MEDICINE Engleman, E. G., McMichael, A. J., McDevitt, H. O. 1978; 147 (4): 1037-1043

  Abstract

  J.H., an HLA-Dw2 homozygous multiparous woman, fails to respond to her husband, W.H. (HLA Dw1,-) in the unidirectional mixed lymphocyte reaction. T cells from J.H. were previously shown to suppress the responses of Dw2-positive cells but not Dw2-negative cells to W.H. We now report that a soluble factor released into the supernate of the mixed lymphocyte reaction by J.H. T cells, mediates this suppression. Like the cell from which it is derived, the factor is highly specific for HLA Dw2 in the responder cell and partially specific for the stimulatory alloantigen.

  View details for Web of Science ID A1978ET46700008

  View details for PubMedID 148486

  View details for PubMedCentralID PMC2184242