Alan M. Krensky, M.D.

All Publications

• KLF13 sustains thymic memory-like CD8(+) T cells in BALB/c mice by regulating IL-4-generating invariant natural killer T cells JOURNAL OF EXPERIMENTAL MEDICINE Lai, D., Zhu, J., Wang, T., Hu-Li, J., Terabe, M., Berzofsky, J. A., Clayberger, C., Krensky, A. M. 2011; 208 (5): 1093-1103

  Abstract

  "Memory-like T cells" are a subset of thymic cells that acquire effector function through the maturation process rather than interaction with specific antigen. Disruption of genes encoding T cell signaling proteins or transcription factors have provided insights into the differentiation of such cells. In this study, we show that in BALB/c, but not C57BL/6, mice, a large portion of thymic CD4(-)CD8(+) T cells exhibit a memory-like phenotype. In BALB/c mice, IL-4 secreted by invariant natural killer T (iNKT) cells is both essential and sufficient for the generation of memory-like T cells. In C57BL/6 mice, iNKT cells are less abundant, producing IL-4 that is insufficient to induce thymic memory-like CD8(+) T cells. BALB/c mice deficient in the transcription factor Kruppel-like factor (KLF) 13 have comparable numbers of iNKT cells to C57BL/6 mice and extremely low levels of thymic memory-like CD8(+) T cells. This work documents the impact of a small number of KLF13-dependent iNKT cells on the generation of memory-like CD8(+) T cells.

  View details for DOI 10.1084/jem.20101527

  View details for Web of Science ID 000291425400019

  View details for PubMedID 21482696

• 15 kDa Granulysin versus GM-CSF for monocytes differentiation: analogies and differences at the transcriptome level JOURNAL OF TRANSLATIONAL MEDICINE Castiello, L., Stroncek, D. F., Finn, M. W., Wang, E., Marincola, F. M., Clayberger, C., Krensky, A. M., Sabatino, M. 2011; 9

  Abstract

  Granulysin is an antimicrobial and proinflammatory protein with several isoforms. While the 9 kDa isoform is a well described cytolytic molecule with pro-inflammatory activity, the functions of the 15 kDa isoform is less well understood. Recently it was shown that 15 kDa Granulysin can act as an alarmin that is able to activate monocytes and immature dendritic cells. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) is a growth factor widely used in immunotherapy both for in vivo and ex vivo applications, especially for its proliferative effects.We analyzed gene expression profiles of monocytes cultured with 15 kDa Granulysin or GM-CSF for 4, 12, 24 and 48 hours to unravel both similarities and differences between the effects of these stimulators.The analysis revealed a common signature induced by both factors at each time point, but over time, a more specific signature for each factor became evident. At all time points, 15 kDa Granulysin induced immune response, chemotaxis and cell adhesion genes. In addition, only 15 kDa Granulsyin induced the activation of pathways related to fundamental dendritic cell functions, such as co-stimulation of T-cell activation and Th1 development. GM-CSF specifically down-regulated genes related to cell cycle arrest and the immune response. More specifically, cytokine production, lymphocyte mediated immunity and humoral immune response were down-regulated at late time points.This study provides important insights on the effects of a novel agent, 15 kDa granulysin, that holds promise for therapeutic applications aimed at the activation of the immune response.

  View details for DOI 10.1186/1479-5876-9-41

  View details for Web of Science ID 000290444300001

  View details for PubMedID 21501511

• Monocyte-derived DC maturation strategies and related pathways: a transcriptional view CANCER IMMUNOLOGY IMMUNOTHERAPY Castiello, L., Sabatino, M., Jin, P., Clayberger, C., Marincola, F. M., Krensky, A. M., Stroncek, D. F. 2011; 60 (4): 457-466

  Abstract

  Ex vivo production of highly stimulator mature dendritic cells (DCs) for cellular therapy has been used to treat different pathological conditions with the aim of inducing a specific immune response. In the last decade, several protocols have been developed to mature monocyte-derived DCs: each one has led to the generation of DCs showing different phenotypes and stimulatory abilities, but it is not yet known which one is the best for inducing effective immune responses. We grouped several different maturation protocols according to the downstream pathways they activated and reviewed the shared features at a transcriptomic level to reveal the potential of DCs matured by each protocol to develop Th-polarized immune responses.

  View details for DOI 10.1007/s00262-010-0954-6

  View details for Web of Science ID 000288556700001

  View details for PubMedID 21258790

• Granulysin Delivered by Cytotoxic Cells Damages Endoplasmic Reticulum and Activates Caspase-7 in Target Cells JOURNAL OF IMMUNOLOGY Saini, R. V., Wilson, C., Finn, M. W., Wang, T., Krensky, A. M., Clayberger, C. 2011; 186 (6): 3497-3504

  Abstract

  Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.

  View details for DOI 10.4049/jimmunol.1003409

  View details for Web of Science ID 000287923500025

  View details for PubMedID 21296981

• Expression and purification of 15 kDa granulysin utilizing an insect cell secretion system PROTEIN EXPRESSION AND PURIFICATION Finn, M. W., Clayberger, C., Krensky, A. M. 2011; 75 (1): 70-74

  Abstract

  Granulysin is an antimicrobial and proinflammatory protein expressed in activated human T cells and natural killer cells. A single mRNA produces the 15 kDa isoform which is then cleaved at the amino and carboxy termini to produce the 9 kDa isoform. Recombinant 9 kDa granulysin has been studied in detail but little is known about the function of the 15 kDa isoform, and no protocol has been published describing expression and purification of this form. Two commercially available preparations of the recombinant 15 kDa granulysin contain tags that may affect function. Here we describe for the first time a method to produce 15 kDa granulysin as a secreted protein from insect cells. The 15 kDa granulysin is purified using a HiTrap Heparin column and a Resource S column. A typical a yield of purified 15 kDa granulysin is 0.6 mg/L of insect cell supernatant.

  View details for DOI 10.1016/j.pep.2010.07.009

  View details for Web of Science ID 000284452600009

  View details for PubMedID 20674748

• Induction of Granulysin and Perforin Cytolytic Mediator Expression in 10-Week-Old Infants Vaccinated with BCG at Birth CLINICAL & DEVELOPMENTAL IMMUNOLOGY Semple, P. L., Watkins, M., Davids, V., Krensky, A. M., Hanekom, W. A., Kaplan, G., Ress, S. 2011

  Abstract

  While vaccination at birth with Mycobacterium bovis Bacilli Calmette-Guérin (BCG) protects against severe childhood tuberculosis, there is no consensus as to which components of the BCG-induced immune response mediate this protection. However, granulysin and perforin, found in the granules of cytotoxic T lymphocytes and Natural Killer (NK) cells, can kill intracellular mycobacteria and are implicated in protection against Mycobacterium tuberculosis.We compared the cellular expression of granulysin and perforin cytolytic molecules in cord blood and peripheral blood from 10-week-old infants vaccinated at birth with either Japanese or Danish BCG, administered either intradermally or percutaneously.In cord blood, only CD56+ NK cells expressed granulysin and perforin constitutively. These cytolytic mediators were upregulated in CD4+ and CD8+ cord blood cells by ex vivo stimulation with BCG but not with PPD. Following BCG vaccination of neonates, both BCG and PPD induced increased expression of granulysin and perforin by CD4+ and CD8+ T cells. There was no difference in expression of cytolytic molecules according to vaccination route or strain.Constitutive expression of perforin and granulysin by cord blood NK-cells likely provides innate immunity, while BCG vaccination-induced expression of these cytolytic mediators may contribute towards protection of the neonate against tuberculosis.

  View details for DOI 10.1155/2011/438463

  View details for Web of Science ID 000286248400001

  View details for PubMedID 21234358

• Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin BLOOD Tewary, P., Yang, D., de la Rosa, G., Li, Y., Finn, M. W., Krensky, A. M., Clayberger, C., Oppenheim, J. J. 2010; 116 (18): 3465-3474

  Abstract

  Granulysin (GNLY), an antimicrobial protein present in the granules of human cytotoxic T lymphocytes and natural killer (NK) cells, is produced as an intact 15-kDa form that is cleaved to yield a 9-kDa form. Alarmins are endogenous mediators that can induce recruitment and activation of antigen-presenting cells (APCs) and consequently promote the generation of immune response. We hypothesized that GNLY might function as an alarmin. Here, we report that both 9- and 15-kDa forms of recombinant GNLY-induced in vitro chemotaxis and activation of both human and mouse dendritic cells (DCs), recruited inflammatory leucocytes, including APCs in mice, and promoted antigen-specific immune responses upon coadministration with an antigen. GNLY-induced APC recruitment and activation required the presence of Toll-like receptor 4. The observed activity of recombinant GNLY was not due to endotoxin contamination. The capability of the supernatant of GNLY-expressing HuT78 cells to activate DC was blocked by anti-GNLY antibodies. Finally we present evidence that supernatants of degranulated human NK92 or primary NK cells also activated DCs in a GNLY- and Toll-like receptor 4-dependent manner, indicating the physiologic relevance of our findings. Thus, GNLY is the first identified lymphocyte-derived alarmin capable of promoting APC recruitment, activation, and antigen-specific immune response.

  View details for DOI 10.1182/blood-2010-03-273953

  View details for Web of Science ID 000283853200016

  View details for PubMedID 20660289

• Granulysin Production and Anticryptococcal Activity Is Dependent upon a Far Upstream Enhancer That Binds STAT5 in Human Peripheral Blood CD4(+) T Cells JOURNAL OF IMMUNOLOGY Xing, J., Wu, F., Wang, S., Krensky, A. M., Mody, C. H., Zheng, C. 2010; 185 (9): 5074-5081

  Abstract

  Previous studies have demonstrated that STAT5 is critical for expression of granulysin and antimicrobial activity. Because the signaling pathway and the resultant microbicidal activity are defective in HIV-infected patients, the mechanism by which STAT5 leads to granulysin expression is of great interest. In the current study, IL-2-stimulated CRL-2105 CD4(+) T cells expressed granulysin and killed Cryptococcus neoformans similar to primary CD4(+) T cells. The enhancer activity of the upstream element of the granulysin promoter was analyzed in primary CD4(+) T cells and CRL-2105 T cells with a luciferase reporter assay, and a STAT5 binding site, 18,302 to 18,177 bp upstream of the transcription start site, was identified as an enhancer. Additionally, the enhancer functioned in the context of heterologous SV40 promoter irrespective of its transcriptional orientation. Chromatin immunoprecipitation and EMSAs demonstrated that the enhancer element bound STAT5 both in vivo and in vitro, and mutation of the STAT5 binding site abrogated its enhancer activity. Furthermore, overexpression of a dominant negative STAT5a abolished the enhancer activity of the STAT5 binding site and abrogated the anticryptococcal activity of IL-2-stimulated primary CD4(+) T cells. Taken together, these data provide details about the complex regulation leading to granulysin expression and anticryptococcal activity in primary CD4(+) T cells.

  View details for DOI 10.4049/jimmunol.1001725

  View details for Web of Science ID 000283248700020

  View details for PubMedID 20889547

• National Institutes of Health Center for Human Immunology Conference, September 2009. Annals of the New York Academy of Sciences Nussenblatt, R. B., Bielekova, B., Childs, R., Krensky, A., Strober, W., Trinchieri, G. 2010; 1200: E1-23

  Abstract

  Although studies of the laboratory mouse model have laid the groundwork for our rich understanding of immunobiology, they have fallen short in deciphering human disease and providing much needed therapeutic modalities. Indeed, bench-to-bedside approaches have not been a particularly effective means of developing translational research.(1) Recently, a symposium was held at the National Institutes of Health (NIH) entitled "Meeting the Human Immunology Challenge," highlighting the opportunities for the new Intramural NIH Center for Human Immunology, Autoimmunity, and Inflammation (http://www.nhlbi.nih.gov/resources/chi/); among other things it has become clear that a broader definition of the human immune spectrum in health and disease is needed. The human immunology meeting was held in the Clinical Center of the National Institutes of Health, Bethesda, Maryland, on September 3 and 4, 2009.

  View details for DOI 10.1111/j.1749-6632.2010.05682.x

  View details for PubMedID 20633145

• Ten Years of the Immune Tolerance Network: An Integrated Clinical Research Organization SCIENCE TRANSLATIONAL MEDICINE Bluestone, J. A., Krensky, A. M., Turka, L. A., Rotrosen, D., Matthews, J. B. 2010; 2 (19)

  Abstract

  The U.S. National Institutes of Health Roadmap and the U.S. Food and Drug Administration's Critical Path Initiative have endorsed the establishment of large academic clinical research networks as part of the solution to the growing divide between increased R&D spending and the lagging number of new drugs making it to market. Clearly, the role of these networks as translational science incubators that complement industry-sponsored programs is laudable and much-needed. However, the path to success for such organizations is less clear. Here, drawing on the experiences of the Immune Tolerance Network, a multidisciplinary clinical research network founded in 1999, we discuss some of the barriers inherent in developing such consortia and offer firsthand insight into the planning, resources, and organizational infrastructure required for a successful research program.

  View details for DOI 10.1126/scitranslmed.3000672

  View details for Web of Science ID 000277303600002

  View details for PubMedID 20371484

• A HUMAN T CELL-SPECIFIC MOLECULE IS A MEMBER OF A NEW GENE FAMILY (Reprinted from vol 141, pg 1018-1025, 1988) JOURNAL OF IMMUNOLOGY Schall, T. J., Jongstra, J., Dyer, B. J., Jorgensen, J., Clayberger, C., Davis, M. M., Krensky, A. M. 2009; 182 (7): 3947-3954

  View details for Web of Science ID 000264574600004

  View details for PubMedID 19299689

• Biology and clinical relevance of granulysin TISSUE ANTIGENS Krensky, A. M., CLAYBERGER, C. 2009; 73 (3): 193-198

  Abstract

  Granulysin is a cytolytic and proinflammatory molecule first identified by a screen for genes expressed 'late' (3-5 days) after activation of human peripheral blood mononuclear cells. Granulysin is present in cytolytic granules of cytotoxic T lymphocytes and natural killer cells. Granulysin is made in a 15-kDa form that is cleaved into a 9-kDa form at both the amino and the carboxy termini. The 15-kDa form is constitutively secreted, and its function remains poorly understood. The 9-kDa form is released by receptor-mediated granule exocytosis. Nine kiloDalton granulysin is broadly cytolytic against tumors and microbes, including gram-positive and gram-negative bacteria, fungi/yeast and parasites. It kills the causative agents of both tuberculosis and malaria. Granulysin is also a chemoattractant for T lymphocytes, monocytes and other inflammatory cells and activates the expression of a number of cytokines, including regulated upon activation T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage inflammatory protein (MIP)-1 alpha, interleukin (IL)-10, IL-1, IL-6 and interferon (IFN)-alpha. Granulysin is implicated in a myriad of diseases including infection, cancer, transplantation, autoimmunity, skin and reproductive maladies. Small synthetic forms of granulysin are being developed as novel antibiotics. Studies of the full-length forms may give rise to new diagnostics and therapeutics for use in a wide variety of diseases.

  View details for DOI 10.1111/j.1399-0039.2008.01218.x

  View details for Web of Science ID 000263522500001

  View details for PubMedID 19254247

• XCL1 enhances regulatory activities of CD4(+)CD25(high)CD127(low/-) T cells in human allergic asthma JOURNAL OF IMMUNOLOGY Nguyen, K. D., Fohner, A., Booker, J. D., Dong, C., Krensky, A. M., Nadeau, K. C. 2008; 181 (8): 5386-5395

  Abstract

  Chemokine-mediated recruitment of regulatory cell subsets to the airway during inflammation and enhancement of their activities are potential strategies for therapeutic development in allergic asthma (AA). In this study, we aim to explore the role of XCL1, a chemokine associated with immune suppression and allergy, on CD4(+)CD25(high)CD127(low/-) regulatory T cell (Treg) function in AA. Flow cytometry and PCR analysis showed a reduction in XCL1 and XCR1 expression in AA Treg compared with healthy control and nonallergic asthmatic counterparts. This reduction in XCL1 expression was associated with the suboptimal regulatory function of Treg in AA. Interestingly, incubation with recombinant human XCL1 significantly increased Treg-mediated suppression and cytotoxicity by up-regulating expression of XCL1 and chief effector molecules of Treg function. Altogether, these results suggest an association between dysregulated XCL1 expression and reduced Treg activities in AA, as well as a potential role of XCL1 in reversing defective Treg function in the disease.

  View details for Web of Science ID 000260025300030

  View details for PubMedID 18832695

• Late expression of granulysin by microbicidal CD4(+) T cells requires PI3K- and STAT5-dependent expression of IL-2R beta that is defective in HIV-infected patients JOURNAL OF IMMUNOLOGY Zheng, C. F., Jones, G. J., Shi, M., Wiseman, J. C., Marr, K. J., Berenger, B. M., Huston, S. M., Gill, M. J., Krensky, A. M., Kubes, P., Mody, C. H. 2008; 180 (11): 7221-7229

  Abstract

  Granulysin is a cytolytic effector molecule used by lymphocytes to kill tumor and microbial cells. Regulation of granulysin production is complex. A significant delay (5 days) following stimulation of CD4(+) T cells with IL-2 occurs before granulysin is produced. Unfortunately, the mechanisms responsible for this delay are unknown. We have recently demonstrated that granulysin-mediated killing of Cryptococcus neoformans by CD4(+) T cells is defective during HIV infection. This is because CD4(+) T cells from HIV-infected patients fail to produce granulysin in response to IL-2 activation. The present studies examined the mechanism of delayed production of granulysin and the mechanism of the defect in HIV patients. We demonstrate that IL-2 initially requires both STAT5 and PI3K activation to increase expression of IL-2Rbeta, produce granulysin, and kill C. neoformans. The increased expression of IL-2Rbeta precedes granulysin, and preventing the increased expression of IL-2Rbeta using small interfering RNA knockdown abrogates granulysin expression. Moreover, following the increased expression of IL-2Rbeta, blocking subsequent signaling by IL-2 using IL-2Rbeta-specific blocking Abs abrogates expression of granulysin. Finally, CD4(+) T cells from HIV-infected patients, who are defective in both STAT5 and PI3K signaling, fail to express IL-2Rbeta and fail to produce granulysin. These results suggest that IL-2 signals via PI3K and STAT5 to increase expression of IL-2Rbeta, which in turn is required for production of granulysin. These results provide a mechanism to explain the "late" production of granulysin during normal T cell responses, as well as for defective granulysin production by CD4(+) T cells in HIV-infected patients.

  View details for Web of Science ID 000257507300018

  View details for PubMedID 18490721

  View details for PubMedCentralID PMC2661617

• In vitro and in vivo antimicrobial activity of granulysin-derived peptides against Vibrio cholerae JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY Galvao da Silva, A. P., Unks, D., Lyu, S., Ma, J., Zbozien-Pacamaj, R., Chen, X., Krensky, A. M., Clayberger, C. 2008; 61 (5): 1103-1109

  Abstract

  To determine the antibacterial activity of synthetic peptides derived from the cationic antimicrobial peptide granulysin against Vibrio cholerae.The antibacterial activity of granulysin-derived peptides was assessed in vitro by microtitre and cfu assays. Toxicity against human peripheral blood mononuclear cells (PBMCs) was measured by propidium iodide uptake and haemolysis by measuring the levels of haemoglobin released after incubation of red blood cells (RBCs) with granulysin peptides. The ability of granulysin peptides to control bacterial growth in vivo was tested by the treatment of suckling mice infected with V. cholerae with granulysin peptides, administered by gavage 1 h after infection and determining the number of bacteria in the small and large intestines 24 h after infection.All peptides tested inhibited V. cholerae growth in vitro, and they were more effective against stationary phase cells. Two peptides, G12.21 and G14.15, effectively controlled bacterial growth in vivo. The peptides did not lyse RBCs and, with the exception of two peptides, exhibited very little toxicity against human PBMCs.These results suggest that granulysin-derived peptides are candidates for the development of new agents for the treatment of V. cholerae infection.

  View details for DOI 10.1093/jac/dkn058

  View details for Web of Science ID 000254955300024

  View details for PubMedID 18310138

  View details for PubMedCentralID PMC2664651

• IL-24 modulates IFN-gamma expression in patients with tuberculosis IMMUNOLOGY LETTERS Wu, B., Huang, C., Kato-Maeda, M., Hopewell, P. C., Daley, C. L., Krensky, A. M., Clayberger, C. 2008; 117 (1): 57-62

  Abstract

  IL-24 is a newly described member of the IL-10 family. We previously demonstrated that PBMC from TB patients exhibited low levels of IL-24 and IFN-gamma compared to subjects with latent tuberculosis infection (LTBI). In order to investigate the role of IL-24 in IFN-gamma expression in TB patients, we stimulated PBMC from individuals with LTBI or TB patients with the Mtb-specific antigen, early secretory antigenic target-6 (ESAT-6) and measured cytokine expression using quantitative real-time PCR (qPCR). Exogenous IL-24 increased IFN-gamma expression in PBMC obtained from TB patients while neutralization of IL-24 reduced IFN-gamma expression in PBMC from subjects with LTBI. Exogenous IL-24 enhanced IFN-gamma expression by increasing expression of IL-12 family cytokines, including IL-12alpha, IL-12beta, IL-23alpha and IL-27, and by reducing FOXP3 expression in PBMC from TB patients. This is the first demonstration that IL-24 may play an important role in IFN-gamma expression following infection with Mtb.

  View details for DOI 10.1016/j.imlet.2007.11.018

  View details for Web of Science ID 000255183400008

  View details for PubMedID 18199488

  View details for PubMedCentralID PMC2679252

• IL-9 is associated with an impaired Th1 immune response in patients with tuberculosis CLINICAL IMMUNOLOGY Wu, B., Huang, C., Kato-Maeda, M., Hopewell, P. C., Daley, C. L., Krensky, A. M., Clayberger, C. 2008; 126 (2): 202-210

  Abstract

  Although a defective Th1 response has been demonstrated in patients infected with Mycobacterium tuberculosis (Mtb), the mechanisms leading to this defect are not well understood. To study the immune response to Mtb infection, we stimulated PBMC from individuals with latent tuberculosis infection (LTBI) or patients with tuberculosis (TB) with the Mtb specific antigen early secretory antigenic target-6 (ESAT-6). mRNAs for a panel of cytokines were measured using quantitative real-time PCR (qPCR). PBMC from TB patients exhibited low levels of IFN-gamma, IL-12alpha, IL-12beta, and IL-23 mRNA but high levels of IL-9 mRNA. Sera from TB patients blocked the differentiation and function of dendritic cells from TST negative (TST-) donors. Exogenous IL-9 reduced IFN-gamma mRNA expression in PBMC from LTBI by 30% (n=4) and neutralization of IL-9 restored the IFN-gamma mRNA expression in PBMC from TB patients by 66% (n=8). Thus, increased expression of IL-9 may contribute to the development of TB.

  View details for DOI 10.1016/j.clim.2007.09.009

  View details for Web of Science ID 000252607500010

  View details for PubMedID 18032114

• Visiting a forest, but not a city, increases human natural killer activity and expression of anti-cancer proteins INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY Li, Q., Morimoto, K., Kobayashi, M., Inagaki, H., Katsumata, M., Hirata, Y., Hirata, K., Suzuki, H., Li, Y. J., Wakayama, Y., Kawada, T., Park, B. J., Ohira, T., MATSU, N., Kagawa, T., Miyazak, Y., KRENSKY, A. M. 2008; 21 (1): 117-127

  Abstract

  We previously reported that a forest bathing trip enhanced human NK activity, number of NK cells, and intracellular anti-cancer proteins in lymphocytes. In the present study, we investigated how long the increased NK activity lasts and compared the effect of a forest bathing trip on NK activity with a trip to places in a city without forests. Twelve healthy male subjects, age 35-56 years, were selected with informed consent. The subjects experienced a three-day/two-night trip to forest fields and to a city, in which activity levels during both trips were matched. On day 1, subjects walked for two hours in the afternoon in a forest field; and on day 2, they walked for two hours in the morning and afternoon, respectively, in two different forest fields; and on day 3, the subjects finished the trip and returned to Tokyo after drawing blood samples and completing the questionnaire. Blood and urine were sampled on the second and third days during the trips, and on days 7 and 30 after the trip, and NK activity, numbers of NK and T cells, and granulysin, perforin, and granzymes A/B-expressing lymphocytes in the blood samples, and the concentration of adrenaline in urine were measured. Similar measurements were made before the trips on a normal working day as the control. Phytoncide concentrations in forest and city air were measured. The forest bathing trip significantly increased NK activity and the numbers of NK, perforin, granulysin, and granzyme A/B-expressing cells and significantly decreased the concentration of adrenaline in urine. The increased NK activity lasted for more than 7 days after the trip. In contrast, a city tourist visit did not increase NK activity, numbers of NK cells, nor the expression of selected intracellular anti-cancer proteins, and did not decrease the concentration of adrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene were detected in forest air, but almost not in city air. These findings indicate that a forest bathing trip increased NK activity, number of NK cells, and levels of intracellular anti-cancer proteins, and that this effect lasted at least 7 days after the trip. Phytoncides released from trees and decreased stress hormone may partially contribute to the increased NK activity.

  View details for Web of Science ID 000254686700013

  View details for PubMedID 18336737

• Altered phosphorylated signal transducer and activator of transcription profile of CD4(+)CD161(+) T cells in asthma: Modulation by allergic status and oral corticosteroids JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY Gernez, Y., Tirouvanziam, R., Nguyen, K. D., Herzenberg, L. A., Krensky, A. M., Nadeau, K. C. 2007; 120 (6): 1441-1448

  Abstract

  Asthma is a complex immunologic disorder linked to altered cytokine signaling.We tested whether asthmatic patients showed any change in cytokine-dependent signal transducer and activator of transcription (STAT) levels, focusing on the central/effector-memory CD4(+)CD161(+) subset, which represents 15% to 25% of circulating T cells.We quantified intracellular levels of active phosphorylated STAT (phospho-STAT) 1, 3, 5, and 6 by means of flow cytometry, without any activation or expansion.Baseline phospho-STAT1 and phospho-STAT6 levels were increased in CD4(+)CD161(+) T cells from asthmatic patients compared with those from healthy control subjects (by 10- and 8-fold, respectively). This asthma-associated alteration was both subset specific because no change was seen in CD4(+)CD161(-)CD25(+) (regulatory T cells) and CD4(+)CD161(-)CD25(-) subsets and isoform specific because phospho-STAT5 and phospho-STAT3 levels were unchanged. Among asthmatic patients, phospho-STAT1 and phospho-STAT6 levels correlated negatively with each other, suggesting antagonistic regulation. Oral corticosteroid (OCS) treatment significantly decreased phospho-STAT6 and IL-4 levels but not phospho-STAT1 levels. Disease parameters showing significant correlations with phospho-STAT1, phospho-STAT6, or both included age at onset, plasma IgE levels, and levels of the T(H)2 cytokines IL-4 and IL-10 and the T(H)1 cytokine IL-2. Overall, combined phospho-STAT1 and phospho-STAT6 measurements showed excellent predictive value for identifying (1) asthmatic patients versus healthy control subjects, (2) allergic versus nonallergic asthmatic patients, and (3) asthmatic patients taking versus those not taking OCSs.Baseline changes in phospho-STAT1 and phospho-STAT6 levels in blood CD4(+)CD161(+) T cells identify asthmatic patients and mirror their allergic status and response to OCSs.These results confirm the pathologic importance of activated STAT1 and STAT6 in asthma and suggest their potential use as clinical biomarkers.

  View details for DOI 10.1016/j.jaci.2007.08.012

  View details for Web of Science ID 000251653800029

  View details for PubMedID 17919711

  View details for PubMedCentralID PMC2679255

• Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Chen, X., Howe, J., Andrae, J., Roessle, M., Richter, W., da Silva, A. P., Krensky, A. M., Clayberger, C., Brandenburg, K. 2007; 1768 (10): 2421-2431

  Abstract

  To combat infections by Gram-negative bacteria, it is not only necessary to kill the bacteria but also to neutralize pathogenicity factors such as endotoxin (lipopolysaccharide, LPS). The development of antimicrobial peptides based on mammalian endotoxin-binding proteins is a promising tool in the fight against bacterial infections, and septic shock syndrome. Here, synthetic peptides derived from granulysin (Gra-pep) were investigated in microbiological and biophysical assays to understand their interaction with LPS. We analyzed the influence of the binding of Gra-pep on (1) the acyl chain melting of the hydrophobic moiety of LPS, lipid A, by Fourier-transform spectroscopy, (2) the aggregate structure of LPS by small-angle X-ray scattering and cryo-transmission electron microscopy, and 3) the enthalpy change by isothermal titration calorimetry. In addition, the influence of Gra-pep on the incorporation of LPS and LPS-LBP (lipopolysaccharide-binding protein) complexes into negatively charged liposomes was monitored. Our findings demonstrate a characteristic change in the aggregate structure of LPS into multilamellar stacks in the presence of Gra-pep, but little or no change of acyl chain fluidity. Neutralization of LPS by Gra-pep is not due to a scavenging effect in solution, but rather proceeds after incorporation into target membranes, suggesting a requisite membrane-bound step.

  View details for DOI 10.1016/j.bbamem.2007.05.001

  View details for Web of Science ID 000250663400009

  View details for PubMedID 17555705

  View details for PubMedCentralID PMC2679260

• Alan Krensky interview. Drawing a map for the twenty-seven divisions in NIH's army. Science Krensky, A., Kaiser, J. 2007; 317 (5840): 887-?

  View details for PubMedID 17702920

• Unique gene expression profiles in infants vaccinated with different strains of Mycobacterium bovis bacille Calmette-Guerin INFECTION AND IMMUNITY Wu, B., Huang, C., Garcia, L., Ponce de Leon, A., Sifuentes Osornio, J., Bobadilla-del-Valle, M., Ferreira, L., Canizales, S., Small, P., Kato-Maeda, M., Krens, A. M., Clayberger, C. 2007; 75 (7): 3658-3664

  Abstract

  Vaccination with Mycobacterium bovis bacille Calmette-Guérin (BCG) has variable efficacy in preventing tuberculosis. We hypothesized that some of this variation might be due to differences among BCG strains. To test this, neonates in Orizaba, Mexico, were vaccinated with one of three different BCG strains (BCG-Brazil [BBCG], BCG-Denmark [DBCG], or BCG-Japan [JBCG]). One year after vaccination, peripheral blood mononuclear cells (PBMC) were obtained and recall immune responses to culture filtrate proteins (CFP) of Mycobacterium tuberculosis were evaluated using quantitative real-time PCR. CFP-activated PBMC from BBCG- and DBCG-immunized children expressed high levels of cytokines characteristic of an adaptive immune response (gamma interferon, interleukin-2beta [IL-12beta], and IL-27), while those from children immunized with JBCG did not. In contrast, vaccination with JBCG resulted in significantly greater expression of cytokines characteristic of a proinflammatory immune response (IL-1alpha, IL-1beta, IL-6, and IL-24) in PBMC activated with CFP compared to PBMC from children vaccinated with BBCG or DBCG. Thus, different strains of BCG can activate different immune pathways, which may affect long-term vaccine efficacy.

  View details for DOI 10.1128/IAI.00244-07

  View details for Web of Science ID 000247707600048

  View details for PubMedID 17502394

  View details for PubMedCentralID PMC1932948

• Decreased serum granulysin levels in childhood tuberculosis which reverse after therapy TUBERCULOSIS Di Liberto, D., Buccheri, S., Caccamo, N., Meraviglia, S., Romano, A., Di Carlo, P., Titone, L., Dieli, F., Krensky, A. M., Salerno, A. 2007; 87 (4): 322-328

  Abstract

  Granulysin is a cytolytic protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Serum levels of granulysin are related to host cellular immunity. We used an ELISA to quantify granulysin serum levels in children with tuberculosis (TB), before and after chemotherapy. The study involved children affected by different clinical forms of TB (n=72) and healthy control children (n=150) from the same geographical area and of similar socio-economic background. Serum granulysin levels before the initiation of TB therapy were significantly lower in children with TB compared to controls, with the lowest levels being found in TB patients who were PPD skin test negative. No statistically significant differences were found between serum granulysin levels and clinical severity (mild/moderate or advanced pulmonary TB) or the clinical form (pulmonary or extra-pulmonary) of TB. At four months after completion of therapy, serum granulysin levels in children treated for TB were not significantly different to those observed in control children. This finding was paralleled by the increased in vitro mycobactericidal activity of sera from TB patients after completion of therapy. We propose that serum granulysin levels may provide a marker of disease activity in childhood TB and might be useful for monitoring improvement after chemotherapy.

  View details for DOI 10.1016/j.tube.2007.01.003

  View details for Web of Science ID 000248157000008

  View details for PubMedID 17379576

  View details for PubMedCentralID PMC2692947

• CD96 is a leukemic stem cell-specific marker in human acute myeloid leukemia PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hosen, N., Park, C. Y., Tatsumi, N., Oji, Y., Sugiyama, H., Gramatzki, M., Krensky, A. M., Weissman, I. L. 2007; 104 (26): 11008-11013

  Abstract

  Permanent cure of acute myeloid leukemia (AML) by chemotherapy alone remains elusive for most patients because of the inability to effectively eradicate leukemic stem cells (LSCs), the self-renewing component of the leukemia. To develop therapies that effectively target LSC, one potential strategy is to identify cell surface markers that can distinguish LSC from normal hematopoietic stem cells (HSCs). In this study, we employ a signal sequence trap strategy to isolate cell surface molecules expressed on human AML-LSC and find that CD96, which is a member of the Ig gene superfamily, is a promising candidate as an LSC-specific antigen. FACS analysis demonstrates that CD96 is expressed on the majority of CD34(+)CD38(-) AML cells in many cases (74.0 +/- 25.3% in 19 of 29 cases), whereas only a few (4.9 +/- 1.6%) cells in the normal HSC-enriched population (Lin(-)CD34(+)CD38(-)CD90(+)) expressed CD96 weakly. To examine whether CD96(+) AML cells are enriched for LSC activity, we separated AML cells into CD96(+) and CD96(-) fractions and transplanted them into irradiated newborn Rag2(-/-) gamma(c)(-/-) mice. In four of five samples, only CD96(+) cells showed significant levels of engraftment in bone marrow of the recipient mice. These results demonstrate that CD96 is a cell surface marker present on many AML-LSC and may serve as an LSC-specific therapeutic target.

  View details for DOI 10.1073/pnas.0704271104

  View details for Web of Science ID 000247641900048

  View details for PubMedID 17576927

  View details for PubMedCentralID PMC1904175

• Interaction of PRP4 with Kruppel-like factor 13 regulates CCL5 transcription JOURNAL OF IMMUNOLOGY Huang, B., Ahn, Y., McPherson, L., Clayberger, C., Krensky, A. M. 2007; 178 (11): 7081-7087

  Abstract

  Activation of resting T lymphocytes initiates differentiation into mature effector cells over 3-7 days. The chemokine CCL5 (RANTES) and its major transcriptional regulator, Krüppel-like factor 13 (KLF13), are expressed late (3-5 days) after activation in T lymphocytes. Using yeast two-hybrid screening of a human thymus cDNA library, PRP4, a serine/threonine protein kinase, was identified as a KLF13-binding protein. Specific interaction of KLF13 and PRP4 was confirmed by reciprocal coimmunoprecipitation. PRP4 is expressed in PHA-stimulated human T lymphocytes from days 1 and 7 with a peak at day 3. Using an in vitro kinase assay, it was found that PRP4 phosphorylates KLF13. Furthermore, although phosphorylation of KLF13 by PRP4 results in lower binding affinity to the A/B site of the CCL5 promoter, coexpression of PRP4 and KLF13 increases nuclear localization of KLF13 and CCL5 transcription. Finally, knock-down of PRP4 by small interfering RNA markedly decreases CCL5 expression in T lymphocytes. Thus, PRP4-mediated phosphorylation of KLF13 plays a role in the regulation of CCL5 expression in T lymphocytes.

  View details for Web of Science ID 000246896300049

  View details for PubMedID 17513757

• Kruppel-like transcription factor 13 regulates T lymphocyte survival in vivo JOURNAL OF IMMUNOLOGY Zhou, M., McPherson, L., Feng, D., Song, A., Dong, C., Lyu, S., Zhou, L., Shi, X., Ahn, Y., Wang, D., Clayberger, C., Krensky, A. M. 2007; 178 (9): 5496-5504

  Abstract

  Krüppel-like transcription factor (KLF)13, previously shown to regulate RANTES expression in vitro, is a member of the Krüppel- like family of transcription factors that controls many growth and developmental processes. To ascertain the function of KLF13 in vivo, Klf13-deficient mice were generated by gene targeting. As expected, activated T lymphocytes from Klf13(-/-) mice show decreased RANTES expression. However, these mice also exhibit enlarged thymi and spleens. TUNEL, as well as spontaneous and activation-induced death assays, demonstrated that prolonged survival of Klf13(-/-) thymocytes was due to decreased apoptosis. Microarray analysis suggests that protection from apoptosis-inducing stimuli in Klf13(-/-) thymocytes is due in part to increased expression of BCL-X(L), a potent antiapoptotic factor. This finding was confirmed in splenocytes and total thymocytes by real-time quantitative PCR and Western blot as well as in CD4+CD8- single-positive thymocytes by real-time quantitative PCR. Furthermore, EMSA and luciferase reporter assays demonstrated that KLF13 binds to multiple sites within the Bcl-X(L) promoter and results in decreased Bcl-X(L) promoter activity, making KLF13 a negative regulator of BCL-X(L).

  View details for Web of Science ID 000246054400013

  View details for PubMedID 17442931

• Forest bathing enhances human natural killer activity and expression of anti-cancer proteins. International journal of immunopathology and pharmacology Li, Q., Morimoto, K., Nakadai, A., Inagaki, H., Katsumata, M., Shimizu, T., Hirata, Y., Hirata, K., Suzuki, H., Miyazaki, Y., Kagawa, T., Koyama, Y., Ohira, T., Takayama, N., KRENSKY, A. M., Kawada, T. 2007; 20 (2): 3-8

  Abstract

  In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

  View details for PubMedID 17903349

• Messenger RNA expression of IL-8, FOXP3, and IL-12 beta differentiates latent tuberculosis infection from disease JOURNAL OF IMMUNOLOGY Wu, B., Huang, C., Kato-Maeda, M., Hopewell, P. C., Daley, C. L., Krensky, A. M., Clayberger, C. 2007; 178 (6): 3688-3694

  Abstract

  Differentiation of active from latent tuberculosis (TB) is a major challenge in the control of TB. In this study, PBMC from latent TB-infected subjects, TB patients, and tuberculin skin test-negative donors stimulated with the Mycobacterium tuberculosis (Mtb)-specific Ag, early secretory antigenic target 6, and mRNA for 45 immune-related genes was measured by quantitative real-time PCR. Univariate analysis showed significant differences in the expression of 10 genes (IFN-gamma, FOXP3, IL-1alpha, IL-1beta, IL-2, IL-6, IL-8, IL-12alpha, IL-12beta, and IL-24) in PBMC from TB patients vs latent TB-infected subjects (p < 0.01). Multivariate logistic regression and classification and regression tree analyses revealed that expression of three genes, IL-8, FOXP3, and IL-12beta, is predictive for TB vs latent Mtb infection. Thus, measurement of Ag-specific expression of these three genes may offer a specific and noninvasive means of differentiating between latent Mtb infection and TB.

  View details for Web of Science ID 000244942400041

  View details for PubMedID 17339466

• Cytotoxic CD4(+) T cells use granulysin to kill Cryptococcus neoformans, and activation of this pathway is defective in HIV patients BLOOD Zheng, C. F., Ma, L. L., Jones, G. J., Gill, M. J., Krensky, A. M., Kubes, P., Mody, C. H. 2007; 109 (5): 2049-2057

  Abstract

  An important mechanism of host defense to Cryptococcus neoformans involves the direct microbicidal activity of lymphocytes. The importance of CD4+ T cells is illustrated by the incidence of this infection in the acquired immunodeficiency syndrome (AIDS) patients; however, the relative activity of microbicidal CD4+ T cells compared with CD8+ T cells and natural killer (NK) cells has not been established. Further, although NK cells and CD8+ T cells use perforin or granulysin, respectively, to kill C neoformans, the effector molecule used by CD4+ T cells is not known. Experiments demonstrated that IL-2-activated peripheral blood lymphocytes from healthy adults acquire anticryptococcal activity, and surprisingly, that CD4+ T cells had the most profound effect on this activity. Using SrCl(2)induced degranulation and siRNA knockdown, granulysin was shown to be the effector molecule. Although activation by anti-CD3 + IL-2 resulted in the additional expression of perforin, this did not improve the anticryptococcal activity. Cryptococcal killing by CD4+ T cells was defective in human immunodeficiency virus (HIV)-infected patients due to dysregulated granulysin and perforin production in response to IL-2 or anti-CD3 + IL-2. In conclusion, CD4+ T cells are the major subset of cells responsible for killing C neoformans in peripheral blood. These cells use granulysin as the effector molecule, and priming is dysregulated in HIV-infected patients, which results in defective microbicidal activity.

  View details for DOI 10.1182/blood-2006-03-009720

  View details for Web of Science ID 000244641100044

  View details for PubMedID 17038537

• Mechanisms of disease: regulation of RANTES (CCL5) in renal disease NATURE CLINICAL PRACTICE NEPHROLOGY Krensky, A. M., Ahn, Y. 2007; 3 (3): 164-170

  Abstract

  Chemokines (chemoattractant cytokines) are fundamental regulators of immune cell movement from the bloodstream into tissues. Regulating expression of chemokines might, therefore, alleviate inflammation in autoimmune diseases and transplant rejection, or augment immune responses in cancer and immunodeficiency. RANTES (regulated upon activation, normal T cell expressed and secreted [also known as CCL5]) is a model chemokine of relevance to a myriad of diseases. Regulation of RANTES expression is complex. In fibroblasts and monocytes, rel proteins alone suffice to induce transcription of RANTES. By contrast, expression of RANTES in T lymphocytes 3-5 days after activation requires the development of a molecular complex (enhancesome) including KLF13 (Krueppel-like factor 13), rel proteins p50 and p65, and scaffolding proteins. This complex recruits enzymes involved in acetylation, methylation and phosphorylation of chromatin, and ultimately in the expression of RANTES. In addition, KLF13-the lynchpin for recruitment of this molecular complex-is itself translationally regulated. Such complex regulation of biological systems has major implications for the rational design of drugs aimed at increasing or decreasing inflammatory responses in patients.

  View details for DOI 10.1038/ncpneph0418

  View details for Web of Science ID 000244421300012

  View details for PubMedID 17322928

  View details for PubMedCentralID PMC2702760

• Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes PREVENTIVE MEDICINE Li, Q., Morimoto, K., Nakadai, A., Qu, T., Matsushima, H., Katsumata, M., Shimizu, T., Inagaki, H., Hirata, Y., Hirata, K., Kawada, T., Lu, Y., Nakayama, K., Krensky, A. M. 2007; 44 (2): 117-123

  Abstract

  It is well documented that natural killer (NK) cells provide host defense against tumors and viruses. We previously showed that lifestyle affects human NK and LAK activities. In order to explore the underlying mechanism, we investigated the effect of lifestyle on intracellular perforin, granulysin, and granzymes A/B in peripheral blood lymphocytes (PBL).114 healthy male subjects, aged 20-59 years, from a large company in Osaka, Japan were selected with informed consent. The subjects were divided into groups reporting good, moderate, and poor lifestyles according to their responses on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress). Peripheral blood was taken, and numbers of NK, T, perforin, granulysin, and granzymes A/B-expressing cells in PBL were measured by flow cytometry.Subjects with good or moderate lifestyle showed significantly higher numbers of NK, and perforin, granulysin, and granzymes A/B-expressing cells and a significantly lower number of T cells in PBL than subjects with poor lifestyle. Among the eight health practices, cigarette smoking, physical exercise, eating breakfast, and balanced nutrition significantly affect the numbers of NK, T cells, perforin, granulysin, and/or granzymes A/B-expressing cells, and alcohol consumption significantly affects the number of granzyme A-expressing cells. On the other hand, mental stress, sleeping, and working hours had no effect on those parameters.Taken together, these findings indicate that poor lifestyle significantly decreases the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells in PBL.

  View details for DOI 10.1016/j.ypmed.2006.08.017

  View details for Web of Science ID 000244842200005

  View details for PubMedID 17030356

• Dynamic interplay of transcriptional machinery and chromatin regulates "late" expression of the chemokine RANTES in T lymphocytes MOLECULAR AND CELLULAR BIOLOGY Ahn, Y., Huang, B., McPherson, L., Clayberger, C., Krensky, A. M. 2007; 27 (1): 253-266

  Abstract

  The chemokine RANTES (regulated upon activation normal T cell expressed and secreted) is expressed "late" (3 to 5 days) after activation in T lymphocytes. In order to understand the molecular events that accompany changes in gene expression, a detailed analysis of the interplay between transcriptional machinery and chromatin on the RANTES promoter over time was undertaken. Krüppel-like factor 13 (KLF13), a sequence-specific DNA binding transcription factor, orchestrates the induction of RANTES expression in T lymphocytes by ordered recruitment of effector molecules, including Nemo-like kinase, p300/cyclic AMP response element binding protein (CBP), p300/CBP-associated factor, and Brahma-related gene 1, that initiate sequential changes in phosphorylation and acetylation of histones and ATP-dependent chromatin remodeling near the TATA box of the RANTES promoter. These events recruit RNA polymerase II to the RANTES promoter and are responsible for late expression of RANTES in T lymphocytes. Therefore, KLF13 is a key regulator of late RANTES expression in T lymphocytes.

  View details for DOI 10.1128/MCB.01071-06

  View details for Web of Science ID 000243136800021

  View details for PubMedID 17074812

  View details for PubMedCentralID PMC1800668

• Granulysin-mediated tumor rejection in transgenic mice JOURNAL OF IMMUNOLOGY Huang, L. P., Lyu, S., Clayberger, C., Krensky, A. M. 2007; 178 (1): 77-84

  Abstract

  Granulysin (GNLY) is a cytolytic molecule expressed by human CTL and NK cells with activity against a variety of tumors and microbes, including Mycobacterium tuberculosis. Although the molecular mechanism of GNLY-induced apoptosis of Jurkat T cells is well defined in vitro, no direct evidence for its in vivo effects has been demonstrated. Because there is no murine homologue of GNLY, we generated mice expressing GNLY using a bacterial artificial chromosome containing the human GNLY gene and its 5' and 3' flanking regions. GNLY is expressed in leukocytes from transgenic mice with similar kinetics as in PBMC from humans: GNLY is constitutively expressed in NK cells and, following stimulation through the TCR, appears in T lymphocytes 8-10 days after activation. Both forms of GNLY (9 and 15 kDa) are produced by activated T cells, whereas the 15-kDa form predominates in freshly isolated NK cells from transgenic animals. GNLY mRNA is highest in spleen, with detectable expression in thymus and lungs, and minimal expression in heart, kidney, liver, muscle, intestine, and brain. Allospecific cell lines generated from GNLY transgenic animals showed enhanced killing of target cells. In vivo effects of GNLY were evaluated using the syngeneic T lymphoma tumor C6VL. GNLY transgenic mice survived significantly longer than nontransgenic littermates in response to a lethal tumor challenge. These findings demonstrate for the first time an in vivo effect of GNLY and suggest that GNLY may prove a useful therapeutic modality for the treatment of cancer.

  View details for Web of Science ID 000243120900009

  View details for PubMedID 17182542

• Cutting edge: Decreased accumulation and regulatory function of CD4(+)CD25(high) T cells in human STAT 5b deficiency JOURNAL OF IMMUNOLOGY Cohen, A. C., Nadeau, K. C., Tu, W., Hwa, V., Dionis, K., Bezrodnik, L., Teper, A., Gaillard, M., Heinrich, J., Krensky, A. M., Rosenfeld, R. G., Lewis, D. B. 2006; 177 (5): 2770-2774

  Abstract

  We show that STAT5b is important for the in vivo accumulation of CD4+ CD25(high) T cells with regulatory cell function. A patient homozygous for a missense A630P STAT5b mutation displayed immune dysregulation and decreased numbers of CD4+ CD25(high) T cells. STAT5b(A630P/A630P) CD4+ CD25(high) T cells had low expression of forkhead box P3 and an impaired ability to suppress the proliferation of or to kill CD4+ CD25- T cells. Expression of CD25, a component of the high-affinity IL-2R, was also reduced in response to IL-2 or after in vitro propagation. The impact of the STAT5b mutation was selective in that IL-2-mediated up-regulation of the common gamma-chain cytokine receptor and perforin, and activation-induced expressions of CD154 and IFN-gamma were normal. These results indicate that STAT5b propagates an important IL-2-mediated signal for the in vivo accumulation of functional regulatory T cells.

  View details for Web of Science ID 000240002800007

  View details for PubMedID 16920911

• Phytoncides (wood essential oils) induce human natural killer cell activity IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY Li, Q., Nakadai, A., Matsushima, H., Miyazaki, Y., Krensky, A. M., Kawada, T., Morimoto, K. 2006; 28 (2): 319-333

  Abstract

  To explore the effect of forest bathing on the human immune system, we investigated the effect of phytoncides (wood essential oils) on natural killer (NK) activity and the expression of perforin, granzyme A and granulysin in human NK cells. We used NK-92MI cell, an interleukin-2 independent human NK cell line derived from the NK-92 cell, in the present study. NK-92MI cells express the CD56 surface marker, perforin, granzyme A, and granulysin by flow cytometry and are highly cytotoxic to K562 cells in chromium release assay. Phytoncides significantly increase cytolytic activity of NK-92MI cells in a dose-dependent manner and significantly increase the expression of perforin, granzyme A, and granulysin in the NK-92MI cells. Phytoncides also partially, but significantly, restore the decreased human NK activity and the decreased perforin, granzyme A, and granulysin expression in NK-92MI cells induced by dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide. Pretreatment with phytoncides partially prevents DDVP-induced inhibition of NK activity. Taken together, these data indicate that phytoncides significantly enhance human NK activity and this effect is at least partially mediated by induction of intracellular perforin, granzyme A, and granulysin.

  View details for DOI 10.1080/08923970600809439

  View details for Web of Science ID 000239535300012

  View details for PubMedID 16873099

• Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Ramamoorthy, A., Thennarasu, S., Tan, A., Lee, D., Clayberger, C., Krensky, A. M. 2006; 1758 (2): 154-163

  Abstract

  A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486-1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and 31P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. 31P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.

  View details for DOI 10.1016/j.bbamem.2006.02.014

  View details for Web of Science ID 000237759200003

  View details for PubMedID 16579960

• Coordinate expression of CC chemokine ligand 5, granulysin, and perforin in CD8(+) T cells provides a host defense mechanism against Mycobacterium tuberculosis JOURNAL OF IMMUNOLOGY Stegelmann, F., Bastian, M., Swoboda, K., Bhat, R., Kiessler, V., KRENSKY, A. M., Roellinghoff, M., Modlin, R. L., Stenger, S. 2005; 175 (11): 7474-7483

  Abstract

  The ability of CD8+ T cells to kill intracellular pathogens depends upon their capacity to attract infected cells as well as their secretion of cytolytic and antimicrobial effector molecules. We examined the Ag-induced expression of three immune effector molecules contained within cytoplasmic granules of human CD8+ T cells: the chemokine CCL5, the cytolytic molecule perforin, and the antimicrobial protein granulysin. Macrophages infected with virulent Mycobacterium tuberculosis triggered the expression of CCL5 in CD8+ T cells only in donors with previous exposure to the tuberculosis bacteria, not in naive donors. Functionally, CCL5 efficiently attracted M. tuberculosis-infected macrophages, but failed to exert direct antibacterial activity. Infected macrophages also triggered the expression of granulysin in CD8+ T cells, and granulysin was found to be highly active against drug-susceptible and drug-resistant M. tuberculosis clinical isolates. The vast majority of CCL5-positive cells coexpressed granulysin and perforin. Taken together, this report provides evidence that a subset of CD8+ T cells coordinately expresses CCL5, perforin and granulysin, thereby providing a host mechanism to attract M. tuberculosis-infected macrophages and kill the intracellular pathogen.

  View details for Web of Science ID 000233544200049

  View details for PubMedID 16301655

• Dimethyl 2,2-dichlorovinyl phosphate (DDVP) markedly decreases the expression of perforin, granzyme A and granulysin in human NK-92CI cell line TOXICOLOGY Li, Q., Nakadai, A., Ishizaki, M., Morimoto, K., Ueda, A., KRENSKY, A. M., Kawada, T. 2005; 213 (1-2): 107-116

  Abstract

  Natural killer (NK), lymphokine-activated killer (LAK) and cytotoxic T lymphocyte (CTL) cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes and granulysin. We previously have found that dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide significantly inhibited NK, LAK and CTL activities via the inhibition of granzyme activity. To further explore the mechanism of organophosphorus pesticide-induced inhibition of cell-mediated cytolysis, we asked here whether organophosphorus pesticides affect the expression of perforin, granzyme and granulysin in NK cells. We used NK-92CI cell, an interleukin-2 (IL-2) independent human NK cell line. We confirmed that NK-92CI cells express CD56 surface marker, perforin, granzyme A and granulysin by flow cytometry and immunofluorescence microscope, and that it is highly cytotoxic to K562 cells in chromium release assay. We found that DDVP significantly decreases the expression of perforin, granzyme A and granulysin in NK-92CI cells in a dose-dependent manner. Immunocytochemical results showed that DDVP significantly decreases perforin, granzyme A and granulysin positive granules in NK-92CI cell, which may be due to the degranulation. We also found that DDVP have a modest, but a significant inhibitory effect on the transcription of mRNA of perforin, granzyme A and granulysin.

  View details for DOI 10.1016/j.tox.2005.05.018

  View details for Web of Science ID 000232251700011

  View details for PubMedID 16002202

• Granulysin: A novel host defense molecule AMERICAN JOURNAL OF TRANSPLANTATION Krensky, A. M., CLAYBERGER, C. 2005; 5 (8): 1789-1792

  Abstract

  Granulysin is a novel cationic molecule present in the granules of cytotoxic T lymphocytes and natural killer cells. Cytotoxic T lymphocytes have long been associated with graft destruction in transplant rejection. Recent studies implicate granulysin in cell-mediated cytotoxicity, chemoattraction, immune activation and as a potential diagnostic biomarker for transplant rejection.

  View details for DOI 10.1111/j.1600-6143.2005.00970.x

  View details for Web of Science ID 000230291500003

  View details for PubMedID 15996224

• Glycosylated recombinant human XCL1/lymphotactin exhibits enhanced biologic activity JOURNAL OF IMMUNOLOGICAL METHODS Dong, C., Chua, A., Ganguly, S., KRENSKY, A. M., Clayberger, C. 2005; 302 (1-2): 136-144

  Abstract

  Chemokines are a family of small, secreted chemoattractant cytokines that regulate distribution and function of leukocytes during immune responses. While most chemokines are members of the CC or CXC subgroups, XCL1, also known as lymphotactin, is the sole member of the C subgroup. XCL1 is produced by activated CD8(+) T cells, NK cells, gammadelta T cells, and mast cells. XCL1 differs from other chemokines in that it contains only a single disulfide bond and a mucin-like domain at its carboxy terminus that is glycosylated. Understanding the biologic functions of chemokines has largely depended upon expression of these recombinant molecules in E. coli. To examine the effects of glycosylation on the biologic activity of XCL1, we designed constructs for expression of human XCL1 in insect S2 cells. Comparison of this material with that expressed in E. coli reveals that glycosylation significantly increases the biologic activity of XCL1.

  View details for DOI 10.1016/j.jim.2005.05.008

  View details for Web of Science ID 000231490600013

  View details for PubMedID 15992811

• Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator JOURNAL OF IMMUNOLOGY Deng, A. M., Chen, S. X., Li, Q., Lyu, S. C., CLAYBERGER, C., Krensky, A. M. 2005; 174 (9): 5243-5248

  Abstract

  Granulysin, a cationic protein produced by activated human CTL and NK cells, is cytolytic against microbial and tumor targets. In this study we show that granulysin also functions as a chemoattractant and activates monocytes to produce cytokines/chemokines. Although granulysin-mediated cytotoxicity occurs at micromolar concentrations, chemoattraction occurs in the nanomolar range, and immune activation occurs over a wide range of concentrations (nanomolar to micromolar). Granulysin causes a 2- to 7-fold increase in chemotaxis of monocytes, CD4(+), and CD8(+) memory (CD45RO) but not naive (CD45RA) T cells, NK cells, and mature, but not immature, monocyte-derived dendritic cells. Pertussis toxin treatment abrogates chemoattraction by granulysin, indicating involvement of G-protein-coupled receptor(s). At low concentrations (10 nM), granulysin promotes a 3- to 10-fold increase in MCP-1 and RANTES produced by monocytes and U937 cells, while a 2-fold increase in TNF-alpha production by LPS-stimulated monocytes requires higher concentrations of granulysin (micromolar). Taken together, these data indicate that the local concentration of granulysin is critical for the biologic activity, with high concentrations resulting in cytotoxicity while lower concentrations, presumably further from the site of granulysin release, actively recruit immune cells to sites of inflammation.

  View details for Web of Science ID 000228630800011

  View details for PubMedID 15843520

• Transactivation of the CCL5/RANTES gene by Epstein-Barr virus latent membrane protein (Retracted article. See vol. 129, pg. 2762, 2011) INTERNATIONAL JOURNAL OF CANCER Uchihara, J. N., KRENSKY, A. M., Matsuda, T., Kawakami, H., Okudaira, T., Masuda, M., Ohta, T., Takasu, N., Mori, N. 2005; 114 (5): 747-755

  Abstract

  Chemokines and chemokine receptors mediate lymphocyte migration and tissue localization. To analyze CCL5 (RANTES) expression by EBV-infected cells, we examined the expression of CCL5 in BL cell lines. Among 4 BL cell lines, those infected with EBV selectively expressed the CCL5 gene and secreted CCL5. Four cell lines also expressed CCR5, a receptor for CCL5. EBV-encoded LMP-1, a pleiotropic protein that effects gene expression, cell transformation, growth and death, induces expression of CCL5 mRNA and secretion of CCL5 in the EBV-negative BL cell line BJAB and the embryonic kidney cell line 293T. HDACI-stimulated endogenous LMP-1 also induced CCL5 expression in an EBV-positive BL cell line. Analysis of the CCL5 promoter revealed that it is activated by both LMP-1 C-terminal activation domains, CTAR-1 and CTAR-2, which can activate NF-kappaB signaling. Coexpression of IkappaBalpha, IkappaBbeta, IKKalpha, IKKbeta, NIK and TRAF2 dominant-negative constructs, with LMP-1 inhibited the activation of the CCL5 promoter by LMP-1, suggesting that LMP-1 induces CCL5 via NF-kappaB signaling. The NF-kappaB binding sites, R(A/B), located at positions -71 to -43 relative to the putative transcription start site in the CCL5 promoter, were essential for the activation of CCL5 gene expression by LMP-1. These results indicate that the activation of the NF-kappaB pathway by LMP-1 is required for the activation of CCL5 expression.

  View details for DOI 10.1002/ijc.20784

  View details for Web of Science ID 000227535600009

  View details for PubMedID 15609310

• A novel apoptosis pathway activated by the carboxyl terminus of p21 BLOOD Dong, C., Li, Q., Lyu, S. C., KRENSKY, A. M., Clayberger, C. 2005; 105 (3): 1187-1194

  Abstract

  Delivery of biologically active peptides into cells may help elucidate intracellular signal transduction pathways, identify additional in vivo functions, and develop new therapeutics. Although p21 was first identified as a major regulator of cell cycle progression, it is now clear that p21 subserves multiple functions. The amino terminus of p21 interacts with cyclins and cyclin-dependent kinases, while the carboxyl terminus interacts with proliferating cell nuclear antigen (PCNA), growth arrest and DNA damage-inducible gene 45 (GADD45), calmodulin, SET, and CCAAT/enhancer binding protein-alpha (C/EBP-alpha). A chimeric peptide, p21-IRS, consisting of the carboxyl terminal domain of p21 conjugated to a pentapeptide (RYIRS) rapidly enters lymphoid cells and activates apoptosis. In the present study, we investigate the molecular events involved in p21-activated apoptosis. Comparison of p21-IRS with other known proapoptotic agents demonstrates that p21-IRS activates a novel apoptotic pathway: mitochondria are central to the process, but caspases and a decrease in Deltapsi(m) are not involved. Targeting the p21 peptide to specific cell populations may allow development of novel therapies to eliminate aberrant cells in human diseases.

  View details for DOI 10.1182/blood-2004-06-2188

  View details for Web of Science ID 000226596700048

  View details for PubMedID 15466931

• Hemolysis of erythrocytes by granulysin-derived peptides but not by granulysin ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Li, Q., Dong, C., Deng, A. M., Katsumata, M., Nakadai, A., Kawada, T., Okada, S., CLAYBERGER, C., Krensky, A. M. 2005; 49 (1): 388-397

  Abstract

  Granulysin, a 9-kDa protein localized in human cytolytic T lymphocytes and natural killer cell granules, is cytolytic against tumors and microbes but not against red blood cells. Synthetic peptides corresponding to the central region of granulysin recapitulate the lytic activity of the intact molecule, and some peptides cause hemolysis of red blood cells. Peptides in which cysteine residues were replaced by serine maintain their activity against microbes but lose activity against human cells, suggesting their potential as antibiotics. Studies were undertaken to determine the mechanism of resistance of red blood cells to granulysin and sensitivity to a subset of granulysin-derived peptides. Granulysin lyses immature reticulocytes, which have mitochondria, but not red blood cells. Granulysin lyses U937 cells but not U937 cells lacking mitochondrial DNA and a functional respiratory chain (U937rho(o) degrees cells), further demonstrating the requirement of intact mitochondria for granulysin-mediated death. Peptide G8, which corresponds to helix 2/loop 2/helix 3, lyses red blood cells, while peptide G9, which is identical except that the cysteine residues were replaced by serine, does not lyse red blood cells. Granulysin peptide-induced hemolysis is markedly inhibited by an anion transporter inhibitor and by Na(+), K(+), and Ca(2+) channel blockers but not by Na(+)/K(+) pump, cotransport, or Cl(-) channel blockers. Although recombinant granulysin and G9 peptide do not induce hemolysis, they both competitively inhibit G8-induced hemolysis. The finding that some derivatives of granulysin are hemolytic may have important implications for the design of granulysin-based antimicrobial therapeutics.

  View details for DOI 10.1128/AAC.49.1.388-397.2005

  View details for Web of Science ID 000226035300050

  View details for PubMedID 15616319

  View details for PubMedCentralID PMC538851

• Lesional T cells and dermal dendrocytes in psoriasis plaque express increased levels of granulysin JOURNAL OF THE AMERICAN ACADEMY OF DERMATOLOGY Raychaudhuri, S. P., Jiang, W. Y., Raychaudhuri, S. K., Krens, A. M. 2004; 51 (6): 1006-1008

  Abstract

  Granulysin is a broad-spectrum potent antimicrobial peptide produced by the immunocytes. We determined granulysin levels in certain cutaneous inflammatory diseases and correlated expression of granulysin with the relative risks of secondary infections in these conditions. In immunohistochemistry stains a monoclonal antigranulysin antibody was used at 1:150 dilutions. Compared with atopic dermatitis and nummular eczema lesions where secondary infection with Staphylococcus aureus is very common, we found that a significantly increased number of granulysin-positive T cells (P < .01) were present in psoriatic plaques. Psoriasis plaques are heavily colonized with S aureus . It is a well-known observation that despite open cracks and fissures these plaques do not get infected. Increased levels of granulysin provide an explanation for relative immunity of psoriatic plaques against both gram-positive and gram-negative bacterial infections.

  View details for DOI 10.1016/j.jaad.2003.10.679

  View details for Web of Science ID 000225745300027

  View details for PubMedID 15583601

• Elevated expression of CCL5/RANTES in adult T-cell leukemia cells: Possible transactivation of the CCL5 gene by human T-cell leukemia virus type I tax INTERNATIONAL JOURNAL OF CANCER Mori, N., KRENSKY, A. M., Ohshima, K., Tomita, M., Matsuda, T., Ohta, T., Yamada, Y., Tomonaga, M., Ikeda, S., Yamamoto, N. 2004; 111 (4): 548-557

  Abstract

  HTLV-I is the etiologic agent of ATL and of tropical spastic paraparesis/HTLV-I-associated myelopathy. Infiltration of various tissues by circulating leukemic cells and HTLV-I-infected T cells is a characteristic of ATL and HTLV-I-associated inflammatory diseases. Chemokines play important roles in migration and tissue localization of various lymphocyte subsets. Here, we report the highly frequent expression of CCL5 (RANTES) in ATL and HTLV-I-infected T-cell lines. Among various human T-cell lines, those infected with HTLV-I selectively expressed the CCL5 gene and secreted CCL5. Furthermore, CCL5 was expressed by leukemic cells in peripheral blood and lymph nodes from patients with ATL. Inducible expression of HTLV-I transcriptional activator Tax in a human T-cell line Jurkat, up-regulated CCL5 mRNA and induced CCL5 secretion. Analysis of the CCL5 promoter revealed that this gene is activated by Tax, via the activation of NF-kappaB, whose responsive element, R(A/B), is located at positions -71 to -43 relative to the putative transcription start site. Aberrant expression of CCL5 by HTLV-I-infected T cells may impact on the pathophysiology of HTLV-I-associated diseases.

  View details for DOI 10.1002/ijc.20266

  View details for Web of Science ID 000223186000012

  View details for PubMedID 15239133

• NK cells use perforin rather than granulysin for anticryptococcal activity JOURNAL OF IMMUNOLOGY Ma, L. L., Wang, C. L., Neely, G. G., Epelman, S., KRENSKY, A. M., Mody, C. H. 2004; 173 (5): 3357-3365

  Abstract

  Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood. Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing. By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown. Primary human NK cells and a human NK cell line YT were found to constitutively express granulysin and perforin, and possessed anticryptococcal activity, in contrast to CD8 T lymphocytes, which required stimulation. When granulysin protein and mRNA were blocked by granulysin small interfering RNA, the NK cell-mediated antifungal effect was not affected in contrast to the abrogated activity observed in CD8 T lymphocytes. However, when perforin was inhibited by concanamycin A, and silenced using hairpin small interfering RNA, the anticryptococcal activities of NK cells were abrogated. Furthermore, when granulysin and perforin were both inhibited, the anticryptococcal activities of the NK cells were not reduced further than by silencing perforin alone. These results indicate that the antifungal activity is constitutively expressed in NK cells in contrast to CD8 T lymphocytes, in which it requires prior activation, and perforin, but not granulysin, plays the dominant role in NK cell anticryptococcal activity, in contrast to CD8 T lymphocytes, in which granulysin, but not perforin, plays the dominant role in anticryptococcal activity.

  View details for Web of Science ID 000223529800061

  View details for PubMedID 15322199

• gamma delta T cells inhibit in vitro growth of the asexual blood stages of Plasmodium falciparum by a granule exocytosis-dependent cytotoxic pathway that requires granulysin EUROPEAN JOURNAL OF IMMUNOLOGY Farouk, S. E., Mincheva-Nilsson, L., KRENSKY, A. M., Dieli, F., Troye-Blomberg, M. 2004; 34 (8): 2248-2256

  Abstract

  Several reports have stated the ability of gamma delta T cells to inhibit the growth of the asexual blood stages of Plasmodium falciparum in vitro. However, little information is available about the mechanisms involved. In this study, in vitro systems were used to study the role of the granule exocytosis-dependent cytotoxic pathway in the growth inhibition/killing of P. falciparum by human gamma delta T cells. Our results show that the inhibition requires cell-to-cell contact and that gamma delta T cells kill the asexual blood stages of P. falciparum through a granule exocytosis-dependent cytotoxic pathway after recognition of certain ligands or molecules expressed on the surface of infected erythrocytes or merozoites. The in vitro inhibitory capacity of gamma delta T cells was strongly correlated with the expression of granulysin in the cytotoxic granules, while non-inhibitory CD4+ and CD8+ T cells expressed very little, implicating a role for granulysin in parasite inhibition. This was further suggested by the addition of neutralizing anti-granulysin antibodies, which abrogated the parasite inhibitory capacity of the gamma delta T cells. Taken together, our results suggest that the capacity of gamma delta T cells for inhibition/killing of P. falciparum is based on the granule exocytosis-dependent cytotoxic pathway and that the presence of granulysin is essential to maintain efficient killing.

  View details for DOI 10.1002/eji.200424861

  View details for Web of Science ID 000223177800020

  View details for PubMedID 15259022

• DQ 65-79, a peptide derived from HLA class II, mimics p21 to block T cell proliferation JOURNAL OF IMMUNOLOGY Dong, C., Lyu, S. C., KRENSKY, A. M., CLAYBERGER, C. 2003; 171 (10): 5064-5070

  Abstract

  DQ 65-79, a peptide derived from residues 65-79 of the alpha-chain HLA class II molecule DQA03011, blocks T cell proliferation and induces T cell apoptosis. Using a yeast two-hybrid assay, we previously identified proliferating cell nuclear Ag (PCNA) as an intracellular ligand for DQ 65-79. In this study, we show that three regions of PCNA, residues 81-100, 121-140, and 241-261, interact with DQ 65-79. Residues 241-261 of PCNA also interact with the C terminus (residues 139-160) of the cell cycle regulator, p21, suggesting that DQ 65-79 and p21 might function similarly. We show here that DQ 65-79 competitively inhibits binding of p21 to PCNA and that both DQ 65-79 and p21 139-160 induce T cell apoptosis, suggesting that DQ 65-79 and p21 act similarly to inhibit cell growth.

  View details for Web of Science ID 000186643300017

  View details for PubMedID 14607903

• Granulysin CURRENT OPINION IN IMMUNOLOGY CLAYBERGER, C., KRENSKY, A. M. 2003; 15 (5): 560-565

  Abstract

  Granulysin, a molecule expressed by human natural killer cells and activated T lymphocytes, exhibits cytolytic activity against a variety of microbes and tumors. Progress in understanding the structure, function and clinical relevance of granulysin over the past year encompasses three main areas: first, the solution of its crystal structure, providing new insights into its potential mechanism of target cell damage; second, inhibition of its function with small interfering RNA, indicating its relevance in microbial immunity; and third, association of granulysin expression in natural killer cells with good outcomes in cancer, indicating its potential utility as a diagnostic and suggesting its relevance to human disease.

  View details for DOI 10.1016/S0952-7915(03)00097-9

  View details for Web of Science ID 000185731800014

  View details for PubMedID 14499265

• Intracellular mediators of granulysin-induced cell death JOURNAL OF IMMUNOLOGY Okada, S., Li, Q., Whitin, J. C., CLAYBERGER, C., Krensky, A. M. 2003; 171 (5): 2556-2562

  Abstract

  Granulysin, a molecule present in the granules of CTL and NK cells, is cytolytic against microbes and tumors. Granulysin induces apoptosis of mammalian cells by damaging mitochondria and causing the release of cytochrome c and apoptosis-inducing factor, resulting in DNA fragmentation. Here we show that Ca2+ and K+ channels as well as reactive oxygen species are involved in granulysin-mediated Jurkat cell death. The Ca2+ channel blockers, nickel and econazole, and the K+ channel blockers, tetraethylammonium chloride, apamin, and charybdotoxin, inhibit the granulysin-induced increase in intracellular Ca2+ ([Ca2+](i)), the decrease in intracellular K+, and apoptosis. Thapsigargin, which releases Ca2+ from the endoplasmic reticulum, prevents a subsequent granulysin-induced increase in [Ca2+](i) in Jurkat cells, indicating that the initial increase in [Ca2+](i) is from intracellular stores. The rise in [Ca2+](i) precedes a decrease in intracellular K+, and elevated extracellular K+ prevents granulysin-mediated cell death. In granulysin-treated cells, electron transport is uncoupled, and reactive oxygen species are generated. Finally, an increase in intracellular glutathione protects target cells from granulysin-induced lysis, indicating the importance of the redox state in granulysin-mediated cell death.

  View details for Web of Science ID 000184970900047

  View details for PubMedID 12928406

• Helicobacter pylori induces RANTES through activation of NF-kappa B. Infection and immunity Mori, N., Krensky, A. M., Geleziunas, R., Wada, A., Hirayama, T., Sasakawa, C., Yamamoto, N. 2003; 71 (7): 3748-3756

  Abstract

  Helicobacter pylori-infected gastric mucosa displays a conspicuous infiltration of mononuclear cells and neutrophils. RANTES (short for "regulated upon activation, normal T cell expressed and secreted") is a chemoattractant cytokine (chemokine) important in the infiltration of T lymphocytes and monocytes. RANTES may therefore contribute to the cellular infiltrate in the H. pylori-infected gastric mucosa. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated RANTES expression. We observed that gastric epithelial cells produced RANTES upon coculture with H. pylori. In addition, H. pylori induced RANTES mRNA expression and an increase in luciferase activity in cells which were transfected with a luciferase reporter construct derived from the RANTES promoter, in gastric epithelial cells, indicating that the induction of RANTES production occurred at the transcriptional level. Induction of RANTES was dependent on an intact cag pathogenicity island. Activation of the RANTES promoter by H. pylori occurred through the action of NF-kappa B. Transfection of kinase-deficient mutants of I kappa B kinase (IKK) and NF-kappa B-inducing kinase (NIK) inhibited H. pylori-mediated RANTES activation. In contrast, tumor necrosis factor alpha- or interleukin-1/Toll-like receptor signaling molecules-such as mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1, MyD88, and interleukin-1 receptor-associated kinase-did not play a role in RANTES activation by H. pylori. Collectively, H. pylori induced NF-kappa B activation through an intracellular signaling pathway that involved IKK and NIK, leading to RANTES gene transcription. RANTES induction by H. pylori may play an important role in gastric inflammation.

  View details for PubMedID 12819056

  View details for PubMedCentralID PMC162018

• CD40 ligand trimer enhances the response of CD8(+) T cells to Mycobacterium tuberculosis JOURNAL OF IMMUNOLOGY Samten, B., Wizel, B., Shams, H., Weis, S. E., Klucar, P., Wu, S. P., Vankayalapati, R., Thomas, E. K., Okada, S., KRENSKY, A. M., Barnes, P. F. 2003; 170 (6): 3180-3186

  Abstract

  We investigated the effect of recombinant CD40 ligand trimer (CD40LT) on the functional capacity of peripheral blood CD8(+) T cells from healthy tuberculin reactors that were cultured with Mycobacterium tuberculosis-infected autologous monocytes. CD40LT enhanced the capacity of M. tuberculosis-responsive CD8(+) T cells to produce IFN-gamma by increasing the number of IFN-gamma-producing CD8(+) T cells and the amount of IFN-gamma produced per cell. CD40LT-induced IFN-gamma production was dependent on production of IL-12 and IL-18, but did not require IL-15. CD40LT up-regulated expression of the transcription factors phosphorylated CREB and c-Jun, both of which have been previously shown to stimulate IFN-gamma mRNA transcription by binding to the IFN-gamma promoter. CD40LT also enhanced the capacity of CD8(+) T cells to lyse M. tuberculosis-infected monocytes, and increased CTL activity was associated with higher expression of perforin and granulysin, but not of Fas ligand. We conclude that CD40LT can enhance CD8(+) T cell effector function in response to M. tuberculosis.

  View details for Web of Science ID 000181412500051

  View details for PubMedID 12626576

• Human NKT cells express granulysin and exhibit antimycobacterial activity JOURNAL OF IMMUNOLOGY Gansert, J. L., Kiessler, V., Engele, M., Wittke, F., Rollinghoff, M., KRENSKY, A. M., Porcelli, S. A., Modlin, R. L., Stenger, S. 2003; 170 (6): 3154-3161

  Abstract

  Human NKT cells are a unique subset of T cells that express an invariant V alpha 24 TCR that recognizes the nonclassical Ag-presenting molecule CD1d. Activation of NKT cells is greatly augmented by the marine sponge-derived glycolipid alpha-galactosylceramide (alpha GalCer). Because human monocyte-derived cells express CD1d and can harbor the intracellular pathogen Mycobacterium tuberculosis, we asked whether the addition of alpha GalCer could be used to induce effector functions of NKT cells against infected monocytes, macrophages, and monocyte-derived dendritic cells. NKT cells secreted IFN-gamma, proliferated, and exerted lytic activity in response to alpha GalCer-pulsed monocyte-derived cells. Importantly, alpha GalCer-activated NKT cells restricted the growth of intracellular M. tuberculosis in a CD1d-dependent manner. NKT cells that exhibited antimycobacterial activity also expressed granulysin, an antimicrobial peptide shown to mediate an antimycobacterial activity through perturbation of the mycobacterial surface. Degranulation of NKT cells resulted in depletion of granulysin and abrogation of antimycobacterial activity. The detection of CD1d in granulomas of tuberculosis patients supports the potential interaction of NKT cells with CD1d-expressing cells at the site of disease activity. These studies provide evidence that alpha Gal Cer-activated CD1d-restricted T cells can participate in human host defense against M. tuberculosis infection.

  View details for Web of Science ID 000181412500048

  View details for PubMedID 12626573

• Society for Pediatric Research Presidential Address 2002: First principles PEDIATRIC RESEARCH KRENSKY, A. M. 2003; 53 (3): 513-515

  View details for DOI 10.1203/01.PDR.0000052077.86447.56

  View details for Web of Science ID 000181143200023

  View details for PubMedID 12595603

• Granulysin crystal structure and a structure-derived lytic mechanism JOURNAL OF MOLECULAR BIOLOGY Anderson, D. H., Sawaya, M. R., Cascio, D., Ernst, W., Modlin, R., Krensky, A., Eisenberg, D. 2003; 325 (2): 355-365

  Abstract

  Our crystal structure of granulysin suggests a mechanism for lysis of bacterial membranes by granulysin, a 74-residue basic protein from human cytolytic T lymphocyte and natural killer cells. We determined the initial crystal structure of selenomethionyl granulysin by MAD phasing at 2A resolution. We present the structure model refined using native diffraction data to 0.96A resolution. The five-helical bundle of granulysin resembles other "saposin folds" (such as NK-lysin). Positive charges distribute in a ring around the granulysin molecule, and one face has net positive charge. Sulfate ions bind near the segment of the molecule identified as most membrane-lytic and of highest hydrophobic moment. The ion locations may indicate granulysin's orientation of initial approach towards the membrane. The crystal packing reveals one way to pack a sheet of granulysin molecules at the cell surface for a concerted lysis effort. The energy of binding granulysin charges to the bacterial membrane could drive the subsequent lytic processes. The loosely packed core facilitates a hinge or scissors motion towards exposure of hydrophobic surface that we propose tunnels the granulysin into the fracturing target membrane.

  View details for DOI 10.1016/S0022-2836(02)01234-2

  View details for Web of Science ID 000180514700009

  View details for PubMedID 12488100

• Selective depression of interferon-gamma and granulysin production with increase of proliferative response by V gamma 9/V delta T cells in children with tuberculosis JOURNAL OF INFECTIOUS DISEASES Dieli, F., Sireci, G., Caccamo, N., Di Sano, C., Titone, L., Romano, A., Di Carlo, P., Barera, A., Accardo-Palumbo, A., KRENSKY, A. M., Salerno, A. 2002; 186 (12): 1835-1839

  Abstract

  Vgamma9/Vdelta2 T cells can contribute to protective immune response against Mycobacterium tuberculosis, although the extent to which and mechanisms by which they could actually protect against human tuberculosis remain unclear. We have previously reported that Vgamma9/Vdelta2 T cells from tuberculin purified protein derivative (PPD)-positive children, either healthy or affected by different clinical forms of tuberculosis, strongly proliferate to different phosphoantigens in vitro, whereas Vgamma9/Vdelta2 T cells from PPD-negative healthy subjects proliferate very poorly. We report here that Vgamma9/Vdelta2 T cells from tuberculous children have an increased proliferative activity, but decreased interferon (IFN)-gamma production and granulysin expression. After successful chemotherapy, the Vgamma9/Vdelta2 T cell proliferative response strongly decreased, whereas IFN-gamma and granulysin production consistently increased. Disease-associated changes in Vgamma9/Vdelta2 T cell effector functions in patients with tuberculosis are consistent with the possibility that these T cells may play a protective role in immune response against M. tuberculosis infection.

  View details for Web of Science ID 000179392800018

  View details for PubMedID 12447771

• CD8 T cell-mediated killing of Cryptococcus neoformans requires granulysin and is dependent on CD4 T cells and IL-15 JOURNAL OF IMMUNOLOGY Ma, L. L., Spurrell, J. C., Wang, J. F., Neely, G. G., Epelman, S., KRENSKY, A. M., Mody, C. H. 2002; 169 (10): 5787-5795

  Abstract

  Granulysin is located in the acidic granules of cytotoxic T cells. Although the purified protein has antimicrobial activity against a broad spectrum of microbial pathogens, direct evidence for granulysin-mediated cytotoxicity has heretofore been lacking. Studies were performed to examine the regulation and activity of granulysin expressed by CD8 T cells using Cryptococcus neoformans, which is one of the most common opportunistic pathogens of AIDS patients. IL-15-activated CD8 T cells acquired anticryptococcal activity, which correlated with the up-regulation of granulysin. When granules containing granulysin were depleted using SrCl(2,) or when the gene was silenced using 21-nt small interfering RNA duplexes, the antifungal effect of CD8 T cells was abrogated. Concanamycin A and EGTA did not affect the antifungal effect, suggesting that the activity of granulysin was perforin independent. Following stimulation by the C. neoformans mitogen, CD8 T cells expressed granulysin and acquired antifungal activity. This activity required CD4 T cells and was dependent upon accessory cells. Furthermore, IL-15 was both necessary and sufficient for granulysin up-regulation in CD8 T cells. These observations are most consistent with a mechanism whereby C. neoformans mitogen is presented to CD4 T cells, which in turn activate accessory cells. The resultant IL-15 activates CD8 T cells to express granulysin, which is responsible for antifungal activity.

  View details for Web of Science ID 000179170300052

  View details for PubMedID 12421959

• Identification of epitopes of Mycobacterium tuberculosis 16-kDa protein recognized by human leukocyte antigen-A*0201 CD8(+) T lymphocytes JOURNAL OF INFECTIOUS DISEASES Caccamo, N., Milano, S., Di Sano, C., Cigna, D., Ivanyi, J., KRENSKY, A. M., Dieli, F., Salerno, A. 2002; 186 (7): 991-998

  Abstract

  CD8(+) T cells could make an important contribution to protection against tuberculosis (TB), but the antigenic determinants recognized in the context of major histocompatibility complex class I molecules remain ill defined. Our aim was to identify nonamer peptides derived from the acr/16-kDa antigen. Two immunogenic peptides (p21-29 and p120-128) were identified by their ability to elicit cytotoxic CD8(+) T cells from juvenile patients recovering from TB. Epitope-specific recognition was demonstrated by the lysis of both Mycobacterium tuberculosis-infected and peptide-pulsed macrophages, the release of cytotoxic granules, and interferon-gamma and tumor necrosis factor-alpha production. CD8(+) T cell responses to p21-29 and p120-128 were detected ex vivo in freshly isolated peripheral blood mononuclear cells from patients with TB but not in those from healthy control subjects. Our data suggest that these antigenic peptides can play a critical role in effective immunity against mycobacterial infection and TB.

  View details for Web of Science ID 000177991100014

  View details for PubMedID 12232840

• Functional domains and DNA-binding sequences of RFLAT-1/KLF13, a Kruppel-like transcription factor of activated T lymphocytes JOURNAL OF BIOLOGICAL CHEMISTRY Song, A., Patel, A., Thamatrakoln, K., Liu, C., Feng, D. D., CLAYBERGER, C., KRENSKY, A. M. 2002; 277 (33): 30055-30065

  Abstract

  RFLAT-1/KLF13, a member of the Krüppel-like family of transcription factors, was identified as a transcription factor expressed 3-5 days after T lymphocyte activation. It binds to the promoter of the chemokine gene RANTES (regulated on activation normal T cell expressed and secreted) and regulates its "late" expression in activated T-cells. In this study, a series of experiments to define the functional domains of RFLAT-1/KLF13 were undertaken to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this factor. Using the GAL4 fusion system, distinct transcriptional activation and repression domains were identified. The RFLAT-1 minimum activation domain is localized to amino acids 1-35, whereas the repression domain resides in amino acids 67-168. Deletion analysis on the RFLAT-1 protein further supports these domain functions. The RFLAT-1 activation domain is similar to that of its closest family member, basic transcription element-binding protein 1. This domain is highly hydrophobic, and site-directed mutagenesis demonstrated that both negatively charged and hydrophobic residues are important for transactivation. The nuclear localization signal of RFLAT-1 was also identified using the RFLAT-1/green fluorescence protein fusion approach. RFLAT-1 contains two potent, independent nuclear localization signals; one is immediately upstream of the zinc finger DNA-binding domain, and the other is within the zinc fingers. Using mutational analysis, we also determined that the critical binding sequence of RFLAT-1 is CTCCC. The intact CTCCC box on the RANTES promoter is necessary for RFLAT-1-mediated RANTES transcription and is also required for the synergy between RFLAT-1 and NF-kappaB proteins.

  View details for DOI 10.1074/jbc.M204278200

  View details for Web of Science ID 000177509300088

  View details for PubMedID 12050170

• A translational rheostat for RFLAT-1 regulates RANTES expression in T lymphocytes JOURNAL OF CLINICAL INVESTIGATION Nikolcheva, T., Pyronnet, S., Chou, S. Y., Sonenberg, N., Song, A., CLAYBERGER, C., Krensky, A. M. 2002; 110 (1): 119-126

  Abstract

  Activation of T lymphocytes by specific antigen triggers a 3- to 7-day maturation process. Terminal differentiation begins late after T cell activation and involves expression of effector genes, including the chemokine RANTES and its major transcriptional regulator, RANTES factor of late-activated T lymphocytes-1 (RFLAT-1). In this article we demonstrate that RFLAT-1 expression is translationally regulated through its 5'-UTR and in a cell type-specific manner. Overexpression of the translation initiation factor eIF4E increases RFLAT-1 protein, while inhibition of Mnk1, which phosphorylates eIF4E, reduces RFLAT-1 production, indicating cap-dependent translational regulation. These events are regulated by ERK-1/2 and p38 MAP kinases and allow T cells to rapidly adjust RANTES expression in response to changes in the cellular environment, such as stress and/or growth factors. These findings provide a molecular mechanism for a rheostat effect of increasing or decreasing RANTES expression at sites of inflammation. Memory T cells, already poised to make RANTES, are finely regulated by translational control of the major transcription factor regulating RANTES expression. This is the first example of such a mechanism regulating a chemokine, but it seems likely that this will prove to be a general way for cells to rapidly respond to stress, cytokines, and other proinflammatory factors in their local environment.

  View details for DOI 10.1172/JCI200215336

  View details for Web of Science ID 000176665100016

  View details for PubMedID 12093895

  View details for PubMedCentralID PMC151028

• DQ 65-79, a peptide derived from HLA class II, induces I kappa B expression JOURNAL OF IMMUNOLOGY Jiang, Y., Chen, D., Lyu, S. C., Ling, X. F., KRENSKY, A. M., Clayberger, C. 2002; 168 (7): 3323-3328

  Abstract

  A synthetic peptide corresponding to residues 65-79 of the alpha helix of the alpha-chain of the class II HLA molecule DQA03011 (DQ 65-79) inhibits the proliferation of human T lymphocytes in an allele nonrestricted manner. By using microarray technology, we found that expression of 29 genes was increased or decreased in a human CTL cell line after treatment with DQ 65-79. This study focuses on one of these genes, IkappaB-alpha, whose expression is increased by DQ 65-79. IkappaB proteins, including IkappaB-alpha and IkappaB-beta, are increased in T cells treated with DQ 65-79. Nuclear translocation of the NF-kappaB subunits p65 and p50 is decreased in T cells after treatment with DQ 65-79, while elevated levels of p65 and p50 are present in cytosol. DQ 65-79 inhibits the degradation of IkappaB-alpha mRNA and inhibits the activity of IkappaB kinase. These findings indicate that the DQ 65-79 peptide increases the level of IkappaB proteins, thereby preventing nuclear translocation of the transcription factor, NF-kappaB, and inhibiting T cell proliferation.

  View details for Web of Science ID 000174566400029

  View details for PubMedID 11907089

• A T cell-specific enhancer of the human CD40 ligand gene JOURNAL OF BIOLOGICAL CHEMISTRY Schubert, L. A., Cron, R. Q., Cleary, A. M., Brunner, M., Song, A., Lu, L. S., JULLIEN, P., KRENSKY, A. M., Lewis, D. B. 2002; 277 (9): 7386-7395

  Abstract

  We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-kappaB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3'-flanking NF-kappaB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-kappaB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-kappaB p50-specific antiserum. Together, these results suggest that the CD40L gene 3'-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.

  View details for DOI 10.1074/jbc.M110350200

  View details for Web of Science ID 000174104300083

  View details for PubMedID 11751888

• Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by V gamma 9/V delta 2 T lymphocytes 11th International Congress of Immunology Dieli, F., Troye-Blomberg, M., Ivanyi, J., Fournie, J. J., KRENSKY, A. M., Bonneville, M., Peyrat, M. A., Caccamo, N., Sireci, G., Salerno, A. UNIV CHICAGO PRESS. 2001: 1082–85

  Abstract

  Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

  View details for Web of Science ID 000171233200020

  View details for PubMedID 11574927

• Chemokines and allograft rejection: Narrowing the list of suspects TRANSPLANTATION Nelson, P. J., KRENSKY, A. M. 2001; 72 (7): 1195-1197

  View details for Web of Science ID 000171740100002

  View details for PubMedID 11602841

• Immunologic tolerance PEDIATRIC NEPHROLOGY KRENSKY, A. M. 2001; 16 (8): 675-679

  Abstract

  Immunologic tolerance, an active state of antigen specific non-responsiveness, is important in understanding autoimmune diseases and the potential for transplant acceptance. Recent progress in basic studies and preclinical models involving mixed chimerism, costimulatory blockade, immune deviation, and HLA derived peptides suggest that clinical applications are now possible. The National Institutes of Health and Juvenile Diabetes Research Foundation recently launched an Immune Tolerance Network made up of investigators from around the world to develop clinical trials in immune tolerance in autoimmune disease, transplantation and allergy. This review highlights historical perspectives and recent progress in induction and maintenance of immunologic tolerance relevant to pediatric nephrology.

  View details for Web of Science ID 000170342100014

  View details for PubMedID 11519901

• A role of the mitochondrial apoptosis-inducing factor in granulysin-induced apoptosis JOURNAL OF IMMUNOLOGY PARDO, J., Perez-Galan, P., Gamen, S., Marzo, I., Monleon, O., Kaspar, A. A., Susin, S. A., Kroemer, G., KRENSKY, A. M., Naval, J., Anel, A. 2001; 167 (3): 1222-1229

  Abstract

  Granulysin is a cytolytic molecule released by CTL via granule-mediated exocytosis. In a previous study we showed that granulysin induced apoptosis using both caspase- and ceramide-dependent and -independent pathways. In the present study we further characterize the biochemical mechanism for granulysin-induced apoptosis of tumor cells. Granulysin-induced death is significantly inhibited by Bcl-2 overexpression and is associated with a rapid (1-5 h) loss of mitochondrial membrane potential, which is not mediated by ceramide generation and is not inhibited by the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide generation induced by granulysin is a slow event, only observable at longer incubation times (12 h). Apoptosis induced by exogenous natural (C(18)) ceramide is truly associated with mitochondrial membrane potential loss, but contrary to granulysin, this event is inhibited by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Ceramide-induced apoptosis is also completely prevented by Bcl-2 overexpression. The nuclear morphology of cells dying after granulysin treatment in the presence of caspase inhibitors suggested the involvement of mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced cell death. We demonstrate using confocal microscopy that AIF is translocated from mitochondria to the nucleus during granulysin-induced apoptosis. The majority of Bcl-2 transfectants are protected from granulysin-induced cell death, mitochondrial membrane potential loss, and AIF translocation, while a small percentage are not protected. In this small percentage the typical nuclear apoptotic morphology is delayed, being of the AIF type at 5 h time, while at longer times (12 h) the normal apoptotic morphology is predominant. These and previous results support a key role for the mitochondrial pathway of apoptosis, and especially for AIF, during granulysin-induced tumoral cell death.

  View details for Web of Science ID 000170949400012

  View details for PubMedID 11466337

• A distinct pathway of cell-mediated apoptosis initiated by granulysin JOURNAL OF IMMUNOLOGY Kaspar, A. A., Okada, S., Kumar, J., Poulain, F. R., Drouvalakis, K. A., Kelekar, A., Hanson, D. A., Kluck, R. M., Hitoshi, Y., Johnson, D. E., Froelich, C. J., Thompson, C. B., Newmeyer, D. D., Anel, A., CLAYBERGER, C., KRENSKY, A. M. 2001; 167 (1): 350-356

  Abstract

  Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

  View details for Web of Science ID 000170949100048

  View details for PubMedID 11418670

• Chemokines, chemokine receptors, and allograft rejection IMMUNITY Nelson, P. J., KRENSKY, A. M. 2001; 14 (4): 377-386

  View details for Web of Science ID 000168246700005

  View details for PubMedID 11336683

• A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection JOURNAL OF BIOLOGICAL CHEMISTRY Horuk, R., CLAYBERGER, C., KRENSKY, A. M., Wang, Z. H., Grone, H. J., Weber, C., Weber, K. S., Nelson, P. J., May, K., Rosser, M., Dunning, L., Liang, M., Buckmann, B., GHANNAM, A., Ng, H. P., Islam, I., Bauman, J. G., Wei, G. P., Monahan, S., Xu, W., Snider, R. M., Morrissey, M. M., Hesselgesser, J., Perez, H. D. 2001; 276 (6): 4199-4204

  Abstract

  Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

  View details for Web of Science ID 000166921200063

  View details for PubMedID 11054419

• Granulysin: a novel antimicrobial EXPERT OPINION ON INVESTIGATIONAL DRUGS Kumar, J., Okada, S., CLAYBERGER, C., KRENSKY, A. M. 2001; 10 (2): 321-329

  Abstract

  Granulysin is a novel lytic molecule produced by human cytolytic T-lymphocytes (CTLs) and natural killer (NK) cells. It is active against a broad range of microbes, including Gram-positive and -negative bacteria, parasites and Mycobacterium tuberculosis. It is functionally related to other antibacterial peptides, like defensins and magainins, but is structurally distinct. It has structural similarity to porcine NK-lysin and to amoebapores made by Entamoeba histolytica. Synthetic peptides derived from granulysin have differential activity against eukaryotic cells and bacteria. Selective bactericidal peptides may have therapeutic roles as novel antibiotics.

  View details for Web of Science ID 000166783200010

  View details for PubMedID 11178344

• T-cell release of granulysin contributes to host defense in leprosy NATURE MEDICINE OCHOA, M. T., Stenger, S., Sieling, P. A., Thoma-Uszynski, S., Sabet, S., Cho, S. G., KRENSKY, A. M., Rollinghoff, M., Sarno, E. N., Burdick, A. E., Rea, T. H., Modlin, R. L. 2001; 7 (2): 174-179

  Abstract

  A novel mechanism by which T cells contribute to host defense against microbial pathogens is release of the antimicrobial protein granulysin. We investigated the role of granulysin in human infectious disease using leprosy as a model. Granulysin-expressing T cells were detected in cutaneous leprosy lesions at a six-fold greater frequency in patients with the localized tuberculoid as compared with the disseminated lepromatous form of the disease. In contrast, perforin, a cytolytic molecule that colocalizes with granulysin in cytotoxic granules, was expressed at similar levels across the spectrum of disease. Within leprosy lesions, granulysin colocalized in CD4+ T cells and was expressed in CD4+ T-cell lines derived from skin lesions. These CD4+ T-cell lines lysed targets by the granule exocytosis pathway and reduced the viability of mycobacteria in infected targets. Given the broad antimicrobial spectrum of granulysin, these data provide evidence that T-cell release of granulysin contributes to host defense in human infectious disease.

  View details for Web of Science ID 000166756300033

  View details for PubMedID 11175847

• Granulysin expression is a marker for acute rejection and steroid resistance in human renal transplantation HUMAN IMMUNOLOGY Sarwal, M. M., Jani, A., Chang, S., Huie, P., Wang, Z., Salvatierra, O., CLAYBERGER, C., Sibley, R., KRENSKY, A. M., Pavlakis, M. 2001; 62 (1): 21-31

  Abstract

  Differentiating etiologies of transplant dysfunction without biopsy and optimizing therapy for acute rejection by predicting steroid resistance will reduce patient morbidity. Granulysin is a cytolytic molecule released by CTL and NK cells and coexpressed with effectors of acute allograft rejection, like perforin and granzymes. Granulysin mRNA and protein expression were studied in peripheral blood lymphocytes (PBL; n = 61 total, n = 10 with intercurrent infections) and biopsy tissue from adult and children renal transplant recipients (n = 97) by competitive quantitative-reverse transcriptase-PCR (QC-RT-PCR) and immunohistochemistry. Differences in cell phenotypes were studied in steroid sensitive and resistant acute rejection biopsies. Granulysin was studied in phytohemagglutinin (PHA) stimulated cell lines (donor PBL and CD45RO(+) T cells) by FACS, Western blotting, and RT-PCR after pretreating with cyclosporine A (CSA), azathioprine, mycophenolic acid, and steroids. Granulysin mRNA was significantly increased in patient PBL and transplant biopsies during acute rejection (p < 0.0001) and infection (p < 0.001). Rejecting biopsies alone (n = 53) had mononuclear cell granulysin staining. Steroid resistant biopsies (n = 25) had denser granulysin staining (>2 cells/high power field) and CD45RO(+) lymphocytes, when compared with steroid sensitive (n = 28) rejecting tissue. Granulysin levels were unchanged after azathioprine and mycophenolic acid treatment, decreased after treating activated PBL with steroids and cyclosporine A (CSA), and paradoxically, increased (p < 0.05) after treating CD45RO(+) CTL with CSA. Elevated PBL granulysin is a peripheral marker for acute rejection and infection and dense granulysin staining a tissue marker for steroid resistance. Memory CTL abound in steroid resistant grafts and may have a markedly different response to CSA immunotherapy, suggesting a possible mechanism for steroid resistance.

  View details for Web of Science ID 000167037400003

  View details for PubMedID 11165712

• Granulysin blocks replication of varicella-zoster virus and triggers apoptosis of infected cells VIRAL IMMUNOLOGY Hata, A., Zerboni, L., Sommer, M., Kaspar, A. A., CLAYBERGER, C., KRENSKY, A. M., Arvin, A. M. 2001; 14 (2): 125-133

  Abstract

  Granulysin, a lytic protein present in cytolytic granules of human natural killer and cytotoxic T cells, entered cells infected with varicella-zoster virus (VZV). Exposure to granulysin accelerated death of infected cells as assessed by apoptosis markers. The functional domain of granulysin that mediated its antiviral effects was amino acid 23-51; this domain also mediates the additional antitumor cell effects of granulysin. Because granulysin is a product of natural killer cells and T lymphocytes, it is possible that its antiviral activity may act as a mediator of innate and adaptive immune mechanisms.

  View details for Web of Science ID 000168993100005

  View details for PubMedID 11398808

• Granulysin, a T cell product, kills bacteria by altering membrane permeability JOURNAL OF IMMUNOLOGY Ernst, W. A., Thoma-Uszynski, S., Teitelbaum, P., Ko, C., Hanson, D. A., CLAYBERGER, C., KRENSKY, A. M., Leippe, M., Bloom, B. R., Ganz, T., Modlin, R. L. 2000; 165 (12): 7102-7108

  Abstract

  Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

  View details for Web of Science ID 000165790500058

  View details for PubMedID 11120840

• Role of granulysin in immunity to leprosy Workshop on Leprosy Research at the New Millennium OCHOA, M. T., Thoma-Uszynski, S., Sieling, P. A., Sabet, S., Cho, S., KRENSKY, A. M., Burdick, A., Rea, T. H., Modlin, R. L. LEPRA. 2000: S115–S115

  View details for Web of Science ID 000166958500040

  View details for PubMedID 11201866

• Molecular biology of transplantation NEPHRON KRENSKY, A. M. 2000; 86 (3): 260-265

  Abstract

  The past decade has witnessed an explosion in our understanding of the molecules involved in transplant rejection and acceptance. This review highlights our current understanding of antigen recognition, signal transduction, and effector mechanisms of the immune system. Such information has proven important in understanding the action of immunosuppressive drugs and will prove useful in the design of new immunotherapies.

  View details for Web of Science ID 000165335000002

  View details for PubMedID 11096281

• The Immune Tolerance Network: The "Holy Grail" comes to the clinic JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Bluestone, J. A., Matthews, J. B., KRENSKY, A. M. 2000; 11 (11): 2141-2146

  View details for Web of Science ID 000090138500024

  View details for PubMedID 11053493

• Transcriptional regulation of RANTES expression in T lymphocytes IMMUNOLOGICAL REVIEWS Song, A., Nikolcheva, T., KRENSKY, A. M. 2000; 177: 236-245

  Abstract

  We first identified the RANTES chemokine as part of a search for genes expressed by T lymphocytes "late", 3-5 days, after T-cell activation. The kinetics of expression of RANTES and a small number of other genes are unusual and the mechanism of such delayed expression is unknown. In order to uncover a mechanism for such "late" expression, we identified and characterized the RANTES promoter and a novel transcription factor regulating RANTES expression in T lymphocytes. RANTES factor of late activated T lymphocytes (RFLAT)-1 is a member of the Krüppel-like family of transcription factors. Like RANTES, RFLAT-1 expression is "late" after T-cell activation. But, unlike RANTES, regulation of RFLAT-1 expression appears to be translational rather than transcriptional.

  View details for Web of Science ID 000165685700021

  View details for PubMedID 11138780

• An immunosuppressive and anti-inflammatory HLA class I-derived peptide binds vascular cell adhesion molecule-1 TRANSPLANTATION Ling, X. F., Tamaki, T., Xiao, Y., Kamangar, S., CLAYBERGER, C., Lewis, D. B., KRENSKY, A. M. 2000; 70 (4): 662-667

  Abstract

  A synthetic peptide corresponding to residues 75-84 of HLA-B2702 modulates immune responses in rodents and humans both in vitro and in vivo.We used a yeast two-hybrid screening, an in vitro biochemical method, and an in vivo animal model.Two cellular receptors for this novel immunomodulatory peptide were identified using a yeast two-hybrid screen: immunoglobulin binding protein (BiP), a member of the heat shock protein 70 family, and vascular cell adhesion molecule (VCAM)-1. Identification of BiP as a ligand for this peptide confirms earlier biochemical findings, while the interaction with VCAM-1 suggests an alternative mechanism of action. Binding to the B2702 peptide but not to closely related variants was confirmed by ligand Western blot analysis and correlated with immunomodulatory activity of each peptide. In mice, an ovalbumin-induced allergic pulmonary response was blocked by in vivo administration of either the B2702 peptide or anti-VLA-4 antibody.We propose that the immunomodulatory effect of the B2702 peptide is caused, in part, by binding to VCAM-1, which then prevents the normal interaction of VCAM-1 with VLA-4.

  View details for Web of Science ID 000088986100021

  View details for PubMedID 10972226

• Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin JOURNAL OF IMMUNOLOGY Wang, Z., Choice, E., Kaspar, A., Hanson, D., Okada, S., Lyu, S. C., KRENSKY, A. M., CLAYBERGER, C. 2000; 165 (3): 1486-1490

  Abstract

  Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

  View details for Web of Science ID 000088340600042

  View details for PubMedID 10903754

• Proliferating cell nuclear antigen as the cell cycle sensor for an HLA-derived peptide blocking T cell proliferation JOURNAL OF IMMUNOLOGY Ling, X. F., Kamangar, S., Boytim, M. L., Kelman, Z., Huie, P., Lyu, S. C., Sibley, R. K., Hurwitz, J., CLAYBERGER, C., KRENSKY, A. M. 2000; 164 (12): 6188-6192

  Abstract

  Synthetic peptides corresponding to structural regions of HLA molecules are novel immunosuppressive agents. A peptide corresponding to residues 65-79 of the alpha-chain of HLA-DQA03011 (DQ65-79) blocks cell cycle progression from early G1 to the G1 restriction point, which inhibits cyclin-dependent kinase-2 activity and phosphorylation of the retinoblastoma protein. A yeast two-hybrid screen identified proliferating cell nuclear Ag (PCNA) as a cellular ligand for this peptide, whose interaction with PCNA was further confirmed by in vitro biochemistry. Electron microscopy demonstrates that the DQ65-79 peptide enters the cell and colocalizes with PCNA in the T cell nucleus in vivo. Binding of the DQ65-79 peptide to PCNA did not block polymerase delta (pol delta)-dependent DNA replication in vitro. These findings support a key role for PCNA as a sensor of cell cycle progression and reveal an unanticipated function for conserved regions of HLA molecules.

  View details for Web of Science ID 000087508500014

  View details for PubMedID 10843669

• Interleukin-1 beta induction of the chemokine RANTES promoter in the human astrocytoma line CH235 requires both constitutive and inducible transcription factors JOURNAL OF NEUROIMMUNOLOGY Miyamoto, N. G., Medberry, P. S., Hesselgesser, J., Boehlk, S., Nelson, P. J., KRENSKY, A. M., Perez, H. D. 2000; 105 (1): 78-90

  Abstract

  The chemokine RANTES is an important mediator of inflammatory processes. In this report, we describe the DNA sequence and transcription factor requirements for interleukin-1beta (IL-1beta) induction of the RANTES promoter in the human astrocytoma line CH235. RANTES promoter sequences between -278 and +55 are sufficient for IL-1beta-inducibility. In vitro DNA binding assays demonstrate constitutive binding of Sp1, HMG, Ets domain, and bZIP family members to their cognate sites in the RANTES promoter, whereas NF-kappaB and IRF-1 bind in an IL-1beta-inducible manner. IL-1beta-inducibility of the RANTES promoter requires both constitutive and inducible transcription factors. The formation of a higher order nucleoprotein complex, or 'enhanceosome', may be critical for IL-1beta induction of the RANTES promoter.

  View details for Web of Science ID 000086821200010

  View details for PubMedID 10713367

• A human class II MHC-derived peptide antagonizes phosphatidylinositol 3-kinase to block IL-2 signaling JOURNAL OF CLINICAL INVESTIGATION Boytim, M. L., Lilly, P., Drouvalakis, K., Lyu, S. C., Jung, R., KRENSKY, A. M., Clayberger, C. 2000; 105 (10): 1447-1453

  Abstract

  MHC molecules bind antigenic peptides and present them to T cells. There is a growing body of evidence that MHC molecules also serve other functions. We and others have described synthetic peptides derived from regions of MHC molecules that inhibit T-cell proliferation or cytotoxicity in an allele-nonspecific manner that is independent of interaction with the T-cell receptor. In this report, we describe the mechanism of action of a synthetic MHC class II-derived peptide that blocks T-cell activation induced by IL-2. Both this peptide, corresponding to residues 65-79 of DQA*03011 (DQ 65-79), and rapamycin inhibit p70 S6 kinase activity, but only DQ 65-79 blocks Akt kinase activity, placing the effects of DQ 65-79 upstream of mTOR, a PI kinase family member. DQ 65-79, but not rapamycin, inhibits phosphatidylinositol 3-kinase (PI 3-kinase) activity in vitro. The peptide is taken up by cells, as demonstrated by confocal microscopy. These findings indicate that DQ 65-79 acts as an antagonist with PI 3-kinase, repressing downstream signaling events and inhibiting proliferation. Understanding the mechanism of action of immunomodulatory peptides may provide new insights into T-cell activation and allow the development of novel immunosuppressive agents.

  View details for Web of Science ID 000087075300016

  View details for PubMedID 10811852

  View details for PubMedCentralID PMC315461

• Self-recognition of CD1 by gamma/delta T cells: Implications for innate immunity JOURNAL OF EXPERIMENTAL MEDICINE Spada, F. M., Grant, E. P., Peters, P. J., Sugita, M., Melian, A., Leslie, D. S., Lee, H. K., van Donselaar, E., Hanson, D. A., KRENSKY, A. M., Majdic, O., Porcelli, S. A., Morita, C. T., Brenner, M. B. 2000; 191 (6): 937-948

  Abstract

  The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

  View details for Web of Science ID 000086055400004

  View details for PubMedID 10727456

  View details for PubMedCentralID PMC2193122

• Granulysin - A novel antimicrobial peptide of cytolytic T lymphocytes and natural killer cells BIOCHEMICAL PHARMACOLOGY KRENSKY, A. M. 2000; 59 (4): 317-320

  Abstract

  Granulysin is a novel antimicrobial protein produced by human cytolytic T lymphocytes and natural killer cells. It is active against a broad range of microbes, including Gram-positive and Gram-negative bacteria, fungi, and parasites. The fact that it kills Mycobacterium tuberculosis is particularly important, since the current vaccine (Bacille Calmette-Guerin, BCG) is of limited efficacy and antibiotic resistance is increasing. Although functionally related to other antibacterial peptides, defensins and magainins, granulysin is structurally distinct. Like porcine NK lysin and amoebapores made by Entamoeba histolytica, granulysin is related to saposins, small lipid-associated proteins present in the central nervous system. The identification of this novel molecule indicates a broader and perhaps more significant role for T lymphocytes in both innate and acquired antimicrobial defenses.

  View details for Web of Science ID 000084715000001

  View details for PubMedID 10644038

• The allocation of cadaver kidneys for transplantation in the United States: Consensus and controversy JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Neylan, J. F., Sayegh, M. H., Coffman, T. M., Danovitch, G. M., KRENSKY, A. M., Strom, T. B., Turka, L. A., Harmon, W. E. 1999; 10 (10): 2237-2243

  View details for Web of Science ID 000082747900021

  View details for PubMedID 10505702

• Granulysin: a lethal weapon of cytolytic T cells IMMUNOLOGY TODAY Stenger, S., Rosat, J. P., Bloom, B. R., KRENSKY, A. M., Modlin, R. L. 1999; 20 (9): 390-394

  View details for Web of Science ID 000082280600002

  View details for PubMedID 10462738

• HLA-derived peptides as novel immunomodulatory therapeutics JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY Murphy, B., Krensky, A. M. 1999; 10 (6): 1346-1355

  Abstract

  A growing body of experimental evidence demonstrates that synthetic peptides corresponding to linear sequences of MHC (HLA in humans) proteins have immunomodulatory effects in vitro and in vivo in animal models and in humans. Although the original concept was that these peptides inhibited antigen recognition at the MHC-T cell receptor interface via physical blockade, it is now clear that the mechanisms responsible for the myriad of functional effects are more complex. Recent findings show that some peptides affect signal transduction and cell cycle progression. Fragments of MHC molecules can dampen or downregulate immune responses via a variety of mechanisms. Some soluble MHC molecules or synthetic peptides are capable of inducing and maintaining immunologic tolerance in animals. This information suggests that synthetic peptides themselves or drugs mimicking their effects may represent a new class of immunotherapeutics.

  View details for Web of Science ID 000080487900023

  View details for PubMedID 10361875

• Biosynthesis of granulysin, a novel cytolytic molecule MOLECULAR IMMUNOLOGY Hanson, D. A., Kaspar, A. A., Poulain, F. R., KRENSKY, A. M. 1999; 36 (7): 413-422

  Abstract

  Granulysin is a newly described lytic molecule expressed by CTL and NK cells. Three mRNA (519, 520, and 522) and two protein products of 15 and 9 kDa are encoded by the granulysin gene. Stable transfectants overexpressing the predominate 520 mRNA were generated to determine the protein products originating from the translation of this message. A transfectant of the NK cell tumor YT overexpressed both 15 and 9 kDa proteins while a transfectant of the T cell tumor HuT78 produced mainly 15 kDa granulysin. Thus the 520 mRNA is sufficient for production of both 15 and 9 kDa granulysin. 9 kDa granulysin accumulated via post-translational processing of 15 kDa protein and was present intracellularly but not in the cell culture supernatant, indicating specific retention of the 9 kDa protein. An inhibitor of granule acidification, concanamycin A, blocked the processing of 15 kDa granulysin to the 9 kDa form. A deduced structural difference between the two forms of the protein and a decrease in lytic activity of 9 kDa granulysin at granule pH suggest two mechanisms by which a granulysin expressing cell is protected from autolysis during the biosynthesis of this potentially harmful molecule.

  View details for Web of Science ID 000081669500001

  View details for PubMedID 10449094

• RFLAT-1: A new zinc finger transcription factor that activates RANTES gene expression in T lymphocytes IMMUNITY Song, A., Chen, Y. F., Thamatrakoln, K., Storm, T. A., KRENSKY, A. M. 1999; 10 (1): 93-103

  Abstract

  RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted) is a chemoattractant cytokine (chemokine) important in the generation of inflammatory infiltrate and human immunodeficiency virus entry into immune cells. RANTES is expressed late (3-5 days) after activation in T lymphocytes. Using expression cloning, we identified the first "late" T lymphocyte associated transcription factor and named it "RANTES Factor of Late Activated T Lymphocytes-1" (RFLAT-1). RFLAT-1 is a novel, phosphorylated, zinc finger transcription factor that is expressed in T cells 3 days after activation, coincident with RANTES expression. While Rel proteins play the dominant role in RANTES gene expression in fibroblasts, RFLAT-1 is a strong transactivator for RANTES in T cells.

  View details for Web of Science ID 000078428700010

  View details for PubMedID 10023774

• An antimicrobial activity of cytolytic T cells mediated by granulysin SCIENCE Stenger, S., Hanson, D. A., Teitelbaum, R., Dewan, P., Niazi, K. R., Froelich, C. J., Ganz, T., Thoma-Uszynski, S., Melian, A., Bogdan, C., Porcelli, S. A., Bloom, B. R., KRENSKY, A. M., Modlin, R. L. 1998; 282 (5386): 121-125

  Abstract

  Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

  View details for Web of Science ID 000076294900053

  View details for PubMedID 9756476

• Granulysin-induced apoptosis. I. Involvement of at least two distinct pathways JOURNAL OF IMMUNOLOGY Gamen, S., Hanson, D. A., Kaspar, A., Naval, J., Krensky, A. M., Anel, A. 1998; 161 (4): 1758-1764

  Abstract

  Granulysin is a newly described cytolytic molecule released by CTL and NK cells via granule-mediated exocytosis. It shares homology with saposin-like proteins, including NK-lysin and amoebapores, and has been implicated in the lysis of tumor cells and microbes. In the present study we show that recombinant granulysin alone induces apoptosis of Jurkat cells. This apoptosis is associated with a sixfold increase in the ceramide/sphingomyelin ratio, implicating the activation of sphingomyelinases. Granulysin- and ceramide-induced apoptosis are similar in that they both are only minimally inhibited by the more selective cysteine protease p32 (caspase 3)-like caspase inhibitor N-acetyl-Asp-Glu-Val-Asp aldehyde, while they are significantly inhibited by the more general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Nevertheless, while Z-VAD-fmk almost completely inhibits ceramide-induced apoptosis, a Z-VAD-fmk-resistant component was observed using granulysin. Granulysin also causes apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. These data indicate that granulysin induces target cell death by both ceramide- and caspase-dependent and -independent pathways.

  View details for Web of Science ID 000075345400024

  View details for PubMedID 9712041

• Chemokines, lymphocytes and viruses: what goes around, comes around CURRENT OPINION IN IMMUNOLOGY Nelson, P. J., Krensky, A. M. 1998; 10 (3): 265-270

  Abstract

  Chemokines are believed to be the long-sought soluble mediators of selective lymphocyte recruitment. As most selectin-integrin interactions are nonselective, it is thought that the discrimination seen during lymphocyte infiltration into tissues is brought about by the actions of distinct chemokines. Developments over the past year have demonstrated the expanding roles of these factors in lymphocyte chemoattraction, normal trafficking, and viral immunity.

  View details for Web of Science ID 000074178100005

  View details for PubMedID 9638362

• Pediatric Transplant Grand Rounds. Pediatric kidney transplantation at Stanford. Pediatric transplantation Salvatierra, O., Alexander, S. R., KRENSKY, A. M. 1998; 2 (2): 165-176

  Abstract

  (1) We believe that we have achieved excellent graft survival with pediatric kidney transplantation because: (a) we have had no technical losses; (b) there have been no primary immunologic losses within 4 years following transplantation; (c) we have avoided ATN. (2) Our cyclosporine dosage has been greater than the average dosage reported by the NAPRTCS, and we believe that this has led to: (a) a low incidence of rejection episodes; (b) because of this, good 1 and 2 year average serum creatinine levels. (3) In the management of adult-sized kidneys in infants and small children I have discussed: (a) the rationale and strategy to prevent vascular thrombosis, ATN and primary non-function; (b) the importance of optimizing intravascular volume, as well as renal and aortic blood flow. (4) With regard to the management of congenital urologic abnormalities I have discussed: (a) the strategy to avoid unnecessary surgery and to avoid scar around the aorta and the vena cava, particularly in infants and small children; (b) my philosophy regarding the abnormal bladder and the successful use of the small defunctionalized urinary bladder. We believe that these have been the primary ingredients to the success we have seen. I also harken back and continue to practice the adage advanced by my former mentor Dr. Fred Belzer that 'no kidney is better than a bad kidney!' And this could not be more true than in pediatric kidney transplantation, where graft failure enhanced by suboptimal graft quality may potentially both cripple the child and shorten his/her life.

  View details for PubMedID 10082451

• Inhibition of cell cycle progression by a synthetic peptide corresponding to residues 65-79 of an HLA class II sequence: Functional similarities but mechanistic differences with the immunosuppressive drug rapamycin JOURNAL OF IMMUNOLOGY Boytim, M. L., Lyu, S. C., Jung, R., KRENSKY, A. M., CLAYBERGER, C. 1998; 160 (5): 2215-2222

  Abstract

  A synthetic peptide corresponding to a region of the alpha1 alpha-helix of DQA03011 (DQ 65-79) inhibits the proliferation of human PBL and T cells in an allele-nonspecific manner. It blocks proliferation stimulated by anti-CD3 mAb, PHA-P, and alloantigen, but not by PMA and ionomycin. Substitution of each amino acid with serine shows that residues 66, 68, 69, 71-73, and 75-79 are critical for function. Inhibition of proliferation is long lasting and is not reversible with exogenous IL-2. The peptide can be added 24 to 48 h after stimulation and still block proliferation. The DQ 65-79 peptide does not affect expression of IL-2 or IL-2R; however, IL-2-stimulated proliferation is inhibited. Cell cycle progression is blocked at the G1/S transition, and the activity of cdk2 (cyclin-dependent kinase 2) kinase is impaired by the continued presence of p27. Although these results suggest a mechanism similar to that of rapamycin, the peptide inhibition is not reversed with FK-506, which indicates a distinct mechanism.

  View details for Web of Science ID 000072115700024

  View details for PubMedID 9498760

• Switching gears during T-cell maturation: RANTES and late transcription IMMUNOLOGY TODAY Ortiz, B. D., Nelson, P. J., KRENSKY, A. M. 1997; 18 (10): 468-471

  Abstract

  Although much is understood about the induction of genes expressed early (within 24 h) after T-cell activation, little is known about the regulation of expression of genes expressed 'late' (three or more days) post-stimulation. A better understanding of transcriptional regulation at this important stage of T-cell maturation may yield new insights into T-cell development and new immunotherapeutic targets.

  View details for Web of Science ID A1997YD18000003

  View details for PubMedID 9357137

• SCID mouse models: More than furry flasks NATURE BIOTECHNOLOGY KRENSKY, A. M. 1997; 15 (8): 720-721

  View details for Web of Science ID A1997XM70700016

  View details for PubMedID 9255779

• HLA-derived peptides as novel immunosuppressives NEPHROLOGY DIALYSIS TRANSPLANTATION KRENSKY, A. M., CLAYBERGER, C. 1997; 12 (5): 865-868

  Abstract

  Over the past decade the use of synthetic peptides corresponding to linear sequences of HLA molecules has progressed from a concept to a reality. These peptides are currently being evaluated in clinical trials. In animal models these peptides, given over 1 week with cyclosporin alone, induced long-term immunological tolerance (Figure 3). They may similarly induce tolerance in humans. The next major hurdle for such tolerogenic drugs, however, is to prove efficacy in clinical circumstances. Many of the drugs used to treat transplant patients today (steroids, cyclosporin, azathioprine, mycophenolate mofetil) may actually inhibit the 'active' processes of induction and maintenance of immunological tolerance. Demonstration that new drugs induce tolerance will require efficacy studies in other immune-mediated diseases in which monotherapy is feasible (such as psoriasis), before further advances can be made in the induction of transplant tolerance. In addition, rapid assays of rejection must be developed in order to reverse tolerance induction failures without graft damage or loss. Lastly, it will require heroic physicians, surgeons, and patients to make immunological tolerance a reproducible clinical reality.

  View details for Web of Science ID A1997WZ68100001

  View details for PubMedID 9175033

• New insights into mechanisms of allograft rejection AMERICAN JOURNAL OF THE MEDICAL SCIENCES Pattison, J. M., KRENSKY, A. M. 1997; 313 (5): 257-263

  Abstract

  Understanding of the molecular basis of organ allograft rejection has increased tremendously in the past decade. Insight into the nature of the alloantigen and the mechanisms underlying T cell recognition, activation, and differentiation provide novel targets for immunotherapy. Appreciation of the role that peptides present in the human leukocyte antigen groove play in allorecognition provides a new target for synthetic peptide therapy. Elucidation of signal transduction pathways downstream from the T-cell receptor helps to explain the mechanism of action of immunosuppressive agents and impacts the design of new drugs. An understanding of the role of costimulatory molecules, such as CD28, has given rise to new therapies, such as CTLA4-Ig. Information about the mechanisms of cytotoxicity, chemoattraction, and vascular biology similarly has provided new targets for rational drug design. This article highlights new insights into the mechanism of allograft rejection relevant to the design of new immunotherapies.

  View details for Web of Science ID A1997WY02600002

  View details for PubMedID 9145033

• Granulysin, a new human cytolytic granule-associated protein with possible involvement in cell-mediated cytotoxicity. Seminars in immunology Peña, S. V., KRENSKY, A. M. 1997; 9 (2): 117-125

  Abstract

  A primary process by which cytotoxic T lymphocytes (CTL) and natural killer (NK) cells lyse target cells involves the regulated exocytosis of granules present in the cytoplasm of the effector. These granules contain proteins, such as perforin and the granzymes, that play a direct role in the killing process. The localization of a human T and NK cell-specific protein, granulysin (formerly 519), to cytolytic granules suggests that additional mechanisms may be involved in granule-mediated cytolysis. This protein shares homology with small, granule-associated molecules and is a member of a larger family of proteins known as saposin-like proteins (SAPLIP). SAPLIP share common structural features allowing for association with lipids while retaining the ability to mediate a variety of different functions. Expression of granulysin is induced late after T-cell activation, similar to perforin and the granzymes. Two prominent protein products of 15 and 9kDa were identified in CTL. The 9kDa form localizes to dense, highly cytolytic granules and contains the SAPLIP homology domain. A recombinant granulysin protein, corresponding to the 9kDa form, is cytolytic against tumor cell targets.

  View details for PubMedID 9194222

• Processing, subcellular localization, and function of 519 (granulysin), a human late T cell activation molecule with homology to small, lytic, granule proteins JOURNAL OF IMMUNOLOGY PENA, S. V., Hanson, D. A., Carr, B. A., Goralski, T. J., KRENSKY, A. M. 1997; 158 (6): 2680-2688

  Abstract

  CTL and NK cells share a common cytolytic mechanism that involves regulated exocytosis of lytic molecules stored within cytoplasmic granules. Here we describe the processing, subcellular localization, and function of a T and NK cell-specific granule protein that shares homology with small, lytic granule-associated molecules. The gene coding for this protein, 519, is expressed late after T cell activation. Antisera raised against a 519/glutathione-S-transferase fusion protein and a series of peptides derived from the 519 protein sequence permitted the identification of two small CTL protein products of 15 and 9 kDa that are exocytosed after stimulation through the TCR. The 9-kDa product is a processed form of 519 and differs from the 15-kDa product in both its amino and carboxyl terminus. While both 519 proteins are found in cytoplasmic granules, the 9-kDa form is also present in dense, highly cytolytic granules. Functional studies indicate that this protein is lytic against tumor cell targets. The cell type- and stage-specific expression pattern of 519 along with its subcellular localization are reminiscent of molecules that play a vital role in granule-mediated cytolysis by CTL and NK cells. Its lytic activity suggests the involvement of 519 in CTL effector function.

  View details for Web of Science ID A1997WM43500023

  View details for PubMedID 9058801

• The HLA system, antigen processing and presentation KIDNEY INTERNATIONAL KRENSKY, A. M. 1997: S2-S7

  Abstract

  Human leukocyte antigens (HLA) restrict immune responses by binding antigenic peptides and presenting them in the context of self to T lymphocytes. In transplantation, a vigorous T cell response, termed alloreactivity, is caused by recognition of non-self HLA. The major histocompatibility complex (MHC) encodes two major classes of HLA molecules, designated I and II. In general, HLA class I molecules present peptides derived by proteolysis of intracellular proteins (the endogenous pathway), while HLA class II molecules present peptides sampled from the extracellular body fluids (the exogenous pathway). The dichotomy between class I and class II antigens is reflected in the T cells in that the CD8 cell surface glycoprotein is expressed by cytotoxic T lymphocytes and recognizes HLA class I, while CD4 is expressed on helper T cells and recognizes HLA class II. Although the two classes of HLA molecules are thought to have originated from a common ancestral gene, they have evolved specific structures and intracellular trafficking compartments that account for their functions. The class I proteins bind short peptides of eight to nine amino acids that are tightly anchored at their ends. The class II proteins bind longer peptides that are more heterogeneous in size (12 to 28 amino acids) and have ragged ends. The class I peptides are generated by proteasomes and other mechanisms in the cytosol and translocated from the cytosol into the endoplasmic reticulum (ER). In the ER, functional HLA class I complexes are formed and then transported to the cell surface for presentation. HLA class II molecules are assembled without antigenic peptide. An "invariant chain" promotes the assembly of the class II molecule and protects its groove from binding peptides in the ER. In an endosomal compartment, antigens that have entered the cell via pinocytosis or via receptor-mediated internalization are processed, the invariant chain is degraded, and antigenic peptides bind to the HLA class II molecule. This mature complex is transported to the cell surface for presentation. Alloreactivity is the special case of antigen presentation responsible for transplant rejection.

  View details for Web of Science ID A1997WL56900002

  View details for PubMedID 9067934

• Gene expression of RANTES CHEMOKINES Nelson, P. J., Pattison, J. M., KRENSKY, A. M. 1997; 287: 148-162

  View details for Web of Science ID A1997BJ75A00010

  View details for PubMedID 9330320

• Expression of chemokine RANTES and production of monoclonal antibodies CHEMOKINES KRENSKY, A. M., Nelson, P. J. 1997; 287: 162-174

  Abstract

  RANTES was first identified as a cDNA in a search for genes expressed late (3-5 days) after T-cell activation. Definition of RANTES function depended on the generation of protein. This chapter describes the various techniques used to make recombinant RANTES protein, to test its activity, and to generate monoclonal antibodies to assess RANTES protein cell distribution.

  View details for Web of Science ID A1997BJ75A00011

  View details for PubMedID 9330321

• Immunomodulation by HLA class I-derived peptides 2nd International Conference on New Trends in Clinical and Experimental Immunosuppression KRENSKY, A. M., CLAYBERGER, C. ELSEVIER SCIENCE INC. 1996: 3026–28

  Abstract

  Synthetic peptides corresponding to linear sequences of HLA class I molecules have profound immunomodulatory effects in vitro and in vivo. Recent clinical trials confirm their potential as the therapeutics for transplantation and for a variety of immune-mediated diseases. These peptides also inhibit NK responses in vivo in humans. The importance of the carboxy end of the alpha 1 alpha helix in negative signaling to both T cells and NK cells focuses attention on new targets for immunotherapy.

  View details for Web of Science ID A1996VW99900006

  View details for PubMedID 8962174

• RANTES chemokine expression in transplant-associated accelerated atherosclerosis JOURNAL OF HEART AND LUNG TRANSPLANTATION Pattison, J. M., Nelson, P. J., Huie, P., Sibley, R. K., KRENSKY, A. M. 1996; 15 (12): 1194-1199

  Abstract

  The pathogenesis of transplantation-associated accelerated atherosclerosis is poorly understood, but it is likely to be an alloimmune response involving infiltration of the vessel wall by T lymphocytes and monocytes leading to smooth muscle cell proliferation and extracellular matrix deposition. RANTES is a chemokine that selectively chemoattracts T lymphocytes, NK cells, monocytes, and eosinophils. The expression of RANTES in accelerated atherosclerosis was investigated by in situ hybridization and immunohistochemistry.Coronary arteries from six patients undergoing accelerated atherosclerosis were obtained at the time of retransplantation. Normal coronary arteries from two patients with idiopathic dilated cardiomyopathy were used as controls. Messenger RNA for RANTES was localized with digoxigenin-labeled complementary DNA probes. RANTES protein was detected by use of a monoclonal antibody and a three-step horseradish peroxidase method.RANTES mRNA and protein were detected in the lymphocytes, macrophages, myofibroblasts, and endothelial cells of arteries undergoing accelerated atherosclerosis but not in normal coronary arteries.In view of its in vitro biologic activity and in vivo expression pattern, RANTES may be a pivotal mediator of the cellular infiltrate seen in graft atherosclerosis. This information may help in the design of novel diagnostic and therapeutic approaches to this increasingly important disease process.

  View details for Web of Science ID A1996WA94600004

  View details for PubMedID 8981204

• Immunologic tolerance: Tailored antigen 1st International Cybercongress - Transplantation in the next Millennium: A Landmark in Organ Transplantation Technology KRENSKY, A. M., CLAYBERGER, C. ELSEVIER SCIENCE INC. 1996: 2075–77

  Abstract

  In summary, synthetic peptides corresponding to linear sequences of HLA class I molecules can inhibit T-cell responses in vitro and in vivo. These peptides induce immunologic tolerance by binding to hsp-70 family members, causing an increase in intracellular calcium, and down-regulating the nuclear factor of activated T cells, NF-AT. We suggest that heat shock proteins may function as novel immunophilins (Fig 2). Like cyclophilins and FK 506 binding proteins, heat shock proteins are ubiquitous, are involved in protein folding and trafficking, and bind exogenous drugs. Cyclosporine and FK 506 exert immunosuppressive effects by binding immunophilins, which as a result interrupt the phosphatase activity of calcineurin. Although the precise pathways involved in the synthetic HLA peptide effects are not as well worked out, it seems likely that peptide binding to heat shock protein is disrupting normal events in T-cell activation, giving rise to an apparently permanent state of anergy.

  View details for Web of Science ID A1996VD46300022

  View details for PubMedID 8769160

• Identification of a novel regulatory region critical for expression of the RANTES chemokine in activated T lymphocytes JOURNAL OF IMMUNOLOGY Nelson, P. J., Ortiz, B. D., Pattison, J. M., KRENSKY, A. M. 1996; 157 (3): 1139-1148

  Abstract

  The RANTES chemokine is a T cell-expressed, proinflammatory cytokine recently implicated as a suppressive agent of HIV replication. We have identified tandem kappaB-like sequences within the promoter for RANTES that are critical for RANTES promoter-reporter gene activity in both the T cell tumor line Hut78 and in PHA-activated PBL. This region binds not only Rel family members (including p50-p65 heterodimers and p50-p50 homodimers) but also non-Rel factors up-regulated in PBL 3 to 5 days following activation. The expression of these "late" expressed nuclear factors correlates with an up-regulation of RANTES message found at this point in T cell activation. These factors are also constitutively expressed in functionally mature CD8+ T cells. We hypothesize that these apparently novel proteins are responsible in part for the temporal regulation of RANTES seen in peripheral blood T cells and represent a component of transcriptional regulatory machinery newly expressed at this "late" stage of peripheral T cell development.

  View details for Web of Science ID A1996VG99900022

  View details for PubMedID 8757619

• Tumor-specific, cytotoxic T-lymphocyte response after idiotype vaccination for B-cell, non-Hodgkin's lymphoma BLOOD Nelson, E. L., Li, X. B., Hsu, F. J., Kwak, L. W., Levy, R., CLAYBERGER, C., KRENSKY, A. M. 1996; 88 (2): 580-589

  Abstract

  Patients with non-Hodgkin's B-cell lymphoma who received an antitumor vaccine of idiotypic ig protein showed humoral and proliferative immune responses. Because immunity to some antigens, including tumor antigens and human pathogenic viruses, may be better correlated with the cytolytic cellular immune response, we evaluated 16 non-Hodgkin's lymphoma patients immunized with autologous idiotypic ig molecules for changes in tumor-specific cytotoxic T-lymphocyte precursor (CTLp) frequency using limiting dilution analysis. Eleven patients had a significant increase in tumor-specific CTLp. Eight of these 11 patients remain without evidence of disease or with stable minimal disease. In contrast, all five patients who did not have a significant change in tumor-specific CTLp have developed progressive disease. Patient vaccination with tumor associated protein antigens can increase tumor-specific CTLp frequencies. The correlation of increased tumor specific CTLp with freedom from progression is significant at P = .002. This study indicates that measurement of CTLp frequencies are relevant to the clinical evaluation of human tumor vaccines and suggests that cell-mediated cytolytic immune responses may be an important determinant of vaccine efficacy.

  View details for Web of Science ID A1996UW48800023

  View details for PubMedID 8695806

• HLA-derived peptides which inhibit T cell function bind to members of the heat-shock protein 70 family JOURNAL OF EXPERIMENTAL MEDICINE Nossner, E., Goldberg, J. E., Naftzger, C., Lyu, S. C., CLAYBERGER, C., KRENSKY, A. M. 1996; 183 (2): 339-348

  Abstract

  Synthetic peptides corresponding to sequences of HLA class I molecules have inhibitory effects on T cell function. The peptides investigated in this study have sequences corresponding to the relatively conserved region of the alpha 1 helix of HLA class I molecules that overlaps the "public epitope" Bw4/Bw6. These HLA-derived peptides exhibit inhibitory effects on T lymphocytes and have beneficial effects on the survival of allogenic organ transplants in mice and rats. Peptides corresponding to the Bw4a epitope appear most potent as they inhibit the differentiation of T cell precursors into mature cytotoxic T lymphocytes (CTL) and target cell lysis by established CTL lines and clones. To elucidate the mechanism through which these peptides mediate their inhibitory effect on T lymphocytes, peptide binding proteins were isolated from T cell lysates. We show that the inhibitory Bw4a peptide binds two members of the heat-shock protein (HSP) 70 family, constitutively expressed HSC70 and heat-inducible HSP70. Peptide binding to HSC/HSP70 is sequence specific and follows the rules defined by the HSC70 binding motif. Most intriguing, however, is the strict correlation of peptide binding to HSC/HSP70 and the functional effects such that only inhibitory peptides bind to HSC70 and HSP70 whereas noninhibitory peptides do not bind. This correlation suggests that small molecular weight HLA-derived peptides may modulate T cell responses by directly interacting with HSPs. In contrast to numerous reports of HSP70 expression at the surface of antigen-presenting cells and some tumor cells, we find no evidence that HSC/HSP70 are expressed at the surface of the affected T cells. Therefore, we believe that the peptides' immunodulatory effects are not mediated through a signaling event initiated by interaction of peptide with surface HSP, but favor a model similar to the action of other immunomodulatory compounds, FK506 and cyclosporin A, with a role for HSC/HSP70 similar to that for immunophilins, FKBPs and CyP40.

  View details for Web of Science ID A1996TW10900001

  View details for PubMedID 8627147

  View details for PubMedCentralID PMC2192466

• Stimulus specificity of matrix metalloproteinase dependence of human T cell migration through a model basement membrane JOURNAL OF IMMUNOLOGY Xia, M. H., Leppert, D., Hauser, S. L., Sreedharan, S. P., Nelson, P. J., KRENSKY, A. M., Goetzl, E. J. 1996; 156 (1): 160-167

  Abstract

  Chemotaxis of human T lymphoblastoma cells of the Tsup-1 line, which migrate similarly to blood T cells, through a layer of basement membrane-like Matrigel on a polycarbonate micropore filter was evoked by vasoactive intestinal peptide (VIP; concentration for a maximal response, 10(-7)M), IL-2 (10(-9)M), and the chemokines RANTES (10(-10)M) and macrophage inflammatory protein-1 alpha (10(-10)M). Chemotactic concentrations of each factor increased Tsup-1 cell secretion of matrix metalloproteinase-9 (MMP-9), with significant responses by 4 h for VIP, IL-2, and IL-4, but only after 24 h for macrophage inflammatory protein-1 alpha and RANTES, as quantified by Western blots and zymography. 3H-Labeled type IV human collagen incorporated in the Matrigel layer was degraded by migrating Tsup-1 cells, as assessed by release of radioactive fragments of the collagen. The in situ degradation of type IV collagen in Matrigel by migrating Tsup-1 cells was enhanced most significantly by VIP, IL-2, and IL-4 after 4 h at concentrations that increased the secretion of MMP-9 optimally, but only after 24 h by macrophage inflammatory protein-1 alpha and RANTES. The specific MMP inhibitor GM6001 suppressed Tsup-1 cell MMP activity evoked by all stimuli, as determined by zymography and in situ degradation of 3H-Labeled type IV human collagen. The chemotactic migration of Tsup-1 cells through Matrigel, but not through a filter alone, in response to optimal concentrations of VIP, IL-2, and IL-4, but not the chemokines, was inhibited by GM6001, with a concentration dependence similar to that for suppression of MMP activity. Thus elicitation of T cell chemotactic migration through a model basement membrane by stimuli that increase MMP activity early in the response depends on degradation of matrix proteins by MMP, whereas stimuli that recruit MMP late may rely on early activation of other proteases.

  View details for Web of Science ID A1996TM17100023

  View details for PubMedID 8598457

• Novel immunotherapeutic strategies using MHC derived peptides. Kidney international. Supplement Sayegh, M. H., KRENSKY, A. M. 1996; 53: S13-20

  Abstract

  Organ transplantation is now the treatment of choice for end stage organ failure. The ultimate goal in transplantation remains the development of strategies to induce specific tolerance to the allograft. The major histocompatibility complex (MHC) antigens are the principal targets of the immune response to allografts and T cell recognition of allo-MHC is the initial event which initiates allograft rejection. The availability of sequences of MHC genes in mice, rats and humans has made it possible to prepare synthetic peptides for the study of the role of MHC peptides in allorecognition and tolerance induction. New evidence confirms that there are at least two distinct, but not necessarily mutually exclusive, pathways of allorecognition. In the so-called "direct" pathway T cells recognize intact allo-MHC molecules on the surface of donor cells. These MHC molecules contain an array of endogenous peptides bound in their antigen presentation groove. In the "indirect" pathway, T cells recognize specific processed alloantigen presented as peptides in the context of self MHC by antigen-presenting cells (APCs). In addition, there is ample evidence that synthetic MHC peptides can immunomodulate the alloimmune response both in vitro and vivo, and that potent allo-tolerance can be induced with synthetic MHC peptides. Two types of effects mediated by synthetic MHC peptides have been demonstrated: (1) suppression of the alloimmune response by relatively non-polymorphic peptides and (2) antigen-specific unresponsiveness induced by polymorphic peptides. The mechanisms mediating the immunomodulatory effects of synthetic effects of synthetic class I and class II MHC peptides and the potential for clinical applications are reviewed.

  View details for PubMedID 8770986

• Kinetics of transcription factors regulating the RANTES chemokine gene reveal a developmental switch in nuclear events during T-lymphocyte maturation MOLECULAR AND CELLULAR BIOLOGY Ortiz, B. D., KRENSKY, A. M., Nelson, P. J. 1996; 16 (1): 202-210

  Abstract

  RANTES is a chemoattractant cytokine (chemokine) whose gene is expressed immediately after stimulation of several cell types but upregulated late (3 to 5 days) after activation in normal T lymphocytes. Here we describe two cis-acting elements in the human RANTES promoter that act in T lymphocytes. One site interacts with NFIL6, which is activated within the first 24 h after T-cell activation. The second site binds an apparently novel complex that is upregulated later, between days 3 and 5. These data provide an explanation for the immediate-early expression of RANTES in some cell types and identify apparently novel factors contributing to late RANTES transcription in T cells. The results reveal a developmental switch occurring during normal T-cell maturation coincident with the onset of terminal differentiation and the binding of late-acting factors to sequences of the RANTES promoter.

  View details for Web of Science ID A1996TL34500022

  View details for PubMedID 8524297

• RANTES chemokine expression in diseased and normal human tissues CYTOKINE VONLUETTICHAU, I., Nelson, P. J., Pattison, J. M., VANDERIJN, M., Huie, P., Warnke, R., Wiedermann, C. J., Stahl, R. A., Sibley, R. K., KRENSKY, A. M. 1996; 8 (1): 89-98

  Abstract

  RANTES is a member of a large family of cytokines, called chemokines, which are thought to play a regulatory role in inflammatory processes. We have made recombinant human RANTES protein which was used to generate a panel of anti-RANTES monoclonal antibodies. Following characterization, select anti-RANTES monoclonal antibodies were used for immunohistologic staining of a large panel of normal, diseased and fetal tissue sections. Diseased tissues included eleven lymphomas and eight renal tumors. Most tissues were also tested in parallel for RANTES mRNA by in situ hybridization using RANTES mRNA specific oligomeric probes. As expected, most normal adult tissues contain few, if any, RANTES positive cells. In contrast, RANTES expression dramatically increases in inflammatory sites. In addition, megakaryocytes, some tumours, and select fetal tissues express high levels of RANTES message and protein. These results indicate a wider expression of RANTES than previously appreciated and suggest multiple physiologic roles for this soluble factor.

  View details for PubMedID 8742071

• Cytotoxic T lymphocyte recognition of non-hodgkin's B cell lymphomas IMMUNOLOGIC RESEARCH KRENSKY, A. M. 1996; 15 (2): 91-97

  Abstract

  Human cytolytic T lymphocytes specific for autologous Burkitt's lymphoma express the gamma, delta T cell receptor and recognize immunoglobulin idiotype in an MHC-unrestricted manner. Antibodies against a member of the heat shock protein 70 family inhibit this specific cytotoxicity, implicating these molecules in tumor recognition and antigen presentation. Such data is relevant to the design of novel immunotherapies for cancer and provides new insights into target recognition by gamma, delta T cells.

  View details for Web of Science ID A1996UX44800001

  View details for PubMedID 8839778

• Structure of HLA molecules and immunosuppressive effects of HLA derived peptides. International reviews of immunology KRENSKY, A. M., CLAYBERGER, C. 1996; 13 (3): 173-185

  Abstract

  Elucidation of the structure of MHC molecules has provided profound new insights into their function in antigen presentation. In addition, structural studies have implicated certain regions of MHC molecules in specific functions. Although much of MHC biology has concentrated on the extensive polymorphism among these molecules, there is also evolutionary pressure to maintain the relatively monomorphic portions of these molecules. Drs. Krensky and Clayberger have found that synthetic peptides corresponding to linear sequences of HLA molecules have immunomodulatory effects both in vitro and in vivo. In this paper, they review the structure of HLA molecules and their studies of HLA derived peptides as novel immunotherapeutics. Members of the heat shock protein 70 family are implicated in the HLA derived peptide immunosuppressive pathway.

  View details for PubMedID 8782740

• IMMUNOSUPPRESSIVE PEPTIDES CORRESPONDING TO MHC CLASS-I SEQUENCES CURRENT OPINION IN IMMUNOLOGY CLAYBERGER, C., KRENSKY, A. M. 1995; 7 (5): 644-648

  Abstract

  The induction of tolerance is a long-standing goal in transplantation. Intact MHC molecules, or fragments of them, are being used to render T cells unresponsive both in vitro and in vivo. Elucidation of the mechanisms underlying the effects of these treatments should aid the design of novel therapies for transplantation.

  View details for Web of Science ID A1995TD86600008

  View details for PubMedID 8573307

• HLA-DERIVED PEPTIDES AS NOVEL IMMUNOSUPPRESSIVES PEDIATRIC RESEARCH KRENSKY, A. M. 1995; 38 (3): 275-279

  Abstract

  Peptides corresponding to linear sequences of HLA molecules have been synthesized and tested for immunomodulatory activity in in vitro assays using human T lymphocytes. Sequences from different parts of the HLA molecules have different effects. Peptides corresponding to residues 75-84 of an HLA class I supratypic specificity of limited heterogeneity (HLA-Bw4) had profound inhibitory effects in a variety of in vitro assays of human T lymphocyte function. Furthermore, a 2-wk course of human HLA sequences and cyclosporine therapy induced enduring immunologic tolerance in a rat model of heterotopic heart transplantation. These studies prompted clinical trials which are currently in progress. The peptides appear to induce T cell anergy by causing a prolonged intracellular calcium flux and interrupting normal signal transduction pathways. Furthermore, these peptides bind to members of the heat-shock protein 70 family, implicating these ubiquitous proteins in the immunomodulatory pathway. Such peptides may be normal physiologic mediators. In any case, they represent potential new immunotherapeutics for a variety of immune-mediated diseases.

  View details for Web of Science ID A1995RP60800001

  View details for PubMedID 7494646

• HLA-DERIVED PEPTIDES AS NOVEL IMMUNOTHERAPEUTICS CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY KRENSKY, A. M., CLAYBERGER, C. 1995; 75 (2): 112-116

  Abstract

  Soluble forms of HLA may be natural immunoregulatory molecules and may explain the "transfusion effect" seen in transplant patients in the precyclosporine era. Synthetic peptides corresponding to short linear sequences of HLA molecules have inhibitory effects on human T lymphocytes in vitro and cause tolerance induction in a rat heterotopic heart transplant model. Recent studies indicate that a subset of these peptides causes an increase in intracellular calcium which appears to account for T cell unresponsiveness and tolerance induction. The peptides bind to members of the heat-shock protein 70 family in a specific manner, depending upon peptide sequence. These studies provide a mechanism to explain the biologic activities of these new immunotherapeutic reagents.

  View details for Web of Science ID A1995QT37900002

  View details for PubMedID 7704968

• HUMAN-LEUKOCYTE ANTIGEN-DERIVED PEPTIDES AS NOVEL IMMUNOSUPPRESSIVES PROCEEDINGS OF THE ASSOCIATION OF AMERICAN PHYSICIANS KRENSKY, A. M., CLAYBERGER, C. 1995; 107 (1): 81-85

  Abstract

  Synthetic peptides corresponding to linear sequences of HLA class I and class II molecules can potently inhibit T lymphocytes responses both in vitro and in vivo. The class I and class II peptides studied to date seem to function by different mechanisms. Nevertheless, several different peptides have been shown to potently induce T cell anergy. Opelz and Terasaki first demonstrated that blood transfusions improved graft survival in transplant patients (19). Data from several sources indicate that blood transfusions are immunosuppressive in: 1) increasing infections in trauma patients who have received transfusions (20), 2) increasing metastases and/or relapses in cancer patients who have been transfused (21), and 3) remissions of autoimmune diseases associated with pregnancy and/or transfusion (22). It is likely that the active constituent of blood transfusions is soluble HLA molecule (23,24). Liver transplants, which are profoundly immunosuppressive in themselves, produce large amounts of soluble HLA (25). Both B and T lymphocytes in culture secrete soluble HLA (26). We hypothesize that soluble HLA is a natural immunoregulatory molecule involved in dampening of the immune response, but, even if this is not the case, synthetic peptides corresponding to HLA sequences have profound effects on T lymphocytes and may prove to be effective for the induction of clinical tolerance in transplant patients.

  View details for Web of Science ID A1995TF49700010

  View details for PubMedID 8630749

• GAMMA-DELTA T-CELL RECOGNITION OF TUMOR IG PEPTIDE JOURNAL OF IMMUNOLOGY Kim, H. T., Nelson, E. L., CLAYBERGER, C., SANJANWALA, M., Sklar, J., KRENSKY, A. M. 1995; 154 (4): 1614-1623

  Abstract

  Although gamma delta T cells have been postulated to act as a surveillance mechanism that eliminates transformed or otherwise damaged cells, little is known about tumor recognition by gamma delta T cells, including the Ags that are recognized and the molecules that present them. Previously, we described human gamma delta CTL that recognize the autologous B cell lymphoma. Here we report that these gamma delta CTL lyse heterologous cells transfected with the tumor Ig lambda chain gene. Furthermore, the lambda chain is recognized as processed peptide in an Id-specific manner. T cell recognition does not involve classical MHC molecules, but it could be blocked by Abs directed against the heat shock protein grp75. These findings show a specific gamma delta T cell response to a highly polymorphic Ag such as tumor Id and implicate heat shock protein as a molecule required for recognition.

  View details for PubMedID 7836746

• NOVEL TUMOR-ASSOCIATED ACCESSORY MOLECULES INVOLVED IN THE GAMMA/DELTA CYTOTOXIC T-LYMPHOCYTE BURKITTS-LYMPHOMA INTERACTION CANCER Nelson, E. L., Kim, H. T., MAR, N. D., Goralski, T. J., MCINTYRE, B. W., CLAYBERGER, C., KRENSKY, A. M. 1995; 75 (3): 886-893

  Abstract

  Tumor specific cytotoxic T lymphocytes (CTL) recognize antigen via the T-cell receptor (TCR). In addition, recognition requires accessory molecules involved in adhesion and signal transduction. The authors previously have characterized an autologous, Burkitt's lymphoma specific CTL line that uses the gamma-delta TCR to recognize antigen in a nonclassical context. The current study was undertaken to identify novel accessory molecules involved in this unusual TCR-tumor cell interaction.A panel of monoclonal antibodies was generated against a Burkitt's lymphoma cell line and was screened for inhibition of autologous, tumor specific, cytolysis by a gamma-delta CTL line. Proteins identified by these monoclonal antibodies were further characterized by fluorescent-activated cell sorter analysis, Western blot and immunoprecipitation.Three known (CD5, CD43, and CD11a/CD18) and three novel (BAM-1, BAM-2, and BAM-3) cell surface molecules involved in the gamma-delta CTL-Burkitt's lymphoma interaction were identified and characterized.This study identifies and provides a preliminary characterization of three novel Burkitt's lymphoma-associated molecules involved in the gamma-delta CTL-tumor cell interaction and demonstrates that CD5, CD43, and CD11a/CD18 are involved in this interaction. It is likely that other unidentified accessory molecules are also involved in this and other effector cell-tumor interactions. Identification of such molecules may be useful in the design of new immunotherapeutic approaches.

  View details for PubMedID 7530169

• PROSPECTS FOR INDUCTION OF TOLERANCE IN RENAL-TRANSPLANTATION PEDIATRIC NEPHROLOGY KRENSKY, A. M., CLAYBERGER, C. 1994; 8 (6): 772-779

  Abstract

  Immunological tolerance is the ultimate goal of transplantation immunobiology. Current therapies involve nonspecific immunosuppression with concomitant risks for infection, malignancy, and drug-specific side effects. By inducing specific immune unresponsiveness to the graft it should be possible to maintain transplants without the need for chronic drug administration and without the risk of nonspecific immunosuppression. This review highlights recent progress in the understanding of immunological tolerance, with special attention to the long-term prospects for successful induction of tolerance in renal transplant patients.

  View details for Web of Science ID A1994PW73600030

  View details for PubMedID 7696124

• THE INDUCTION OF TOLERANCE TO ALLOANTIGENS USING HLA-BASED SYNTHETIC PEPTIDES CURRENT OPINION IN IMMUNOLOGY KRENSKY, A. M., CLAYBERGER, C. 1994; 6 (5): 791-796

  Abstract

  The identification of immunomodulatory approaches that allow the induction of antigen-specific unresponsiveness is required for long-term graft survival without the complications of chronic immunosuppression. Recent novel strategies based upon treatment with synthetic peptides corresponding to linear sequences of MHC class I and II molecules reproducibly induce tolerance to alloantigens. Although the mechanisms involved are still not completely understood, the phenomenology reported makes these approaches promising for evaluation in clinical trials.

  View details for Web of Science ID A1994PM73500017

  View details for PubMedID 7826536

• PEPTIDES CORRESPONDING TO THE CD8 AND CD4 BINDING DOMAINS OF HLA MOLECULES BLOCK T-LYMPHOCYTE IMMUNE-RESPONSES IN-VITRO JOURNAL OF IMMUNOLOGY CLAYBERGER, C., Lyu, S. C., DeKruyff, R., Parham, P., KRENSKY, A. M. 1994; 153 (3): 946-951

  Abstract

  CD4 and CD8 are cell surface glycoproteins that serve as co-receptors for Ag with the TCR. Recent studies have shown that both CD4 and CD8 interact with conserved regions of MHC class II and class I, respectively. To investigate further the roles of CD4 and CD8 in the immune response, we prepared synthetic peptides corresponding to the HLA sequences with which CD4 and CD8 are thought to interact. The peptide corresponding to residues 222 to 235 of the HLA class I heavy chain blocked the differentiation of human CTL precursors into active effect cells but affected neither the ability of PBLs to proliferate in response to mitogen nor the cytotoxic activity of established CTLs. In contrast, the peptide corresponding to residues 134 to 152 of the HLA-DR beta-chain inhibited the differentiation of CTL precursors, the proliferative response of freshly isolated PBL, and the proliferation of an established alloreactive CD4+ T cell clone to Ag. The inhibitory effect of the DR.134-152 peptide on CTL differentiation could be overcome by addition of exogenous IL-2 to the limiting dilution cultures, whereas the effect of the HLA-1.222-235 peptide was unaffected by exogenous IL-2. These results directly demonstrate a functional role for these regions of MHC molecules and underscore the central role of both CD4 and CD8 in the effective initiation of a CTL response.

  View details for Web of Science ID A1994NY34200004

  View details for PubMedID 8027565

• TRANSPLANTATION IMMUNOLOGY PEDIATRIC CLINICS OF NORTH AMERICA KRENSKY, A. M., CLAYBERGER, C. 1994; 41 (4): 819-839

  Abstract

  A major function of the immune response is the discrimination of self from nonself. It is this response that must be overcome in transplant rejection. Progress in understanding these basic immune mechanisms has helped to improve clinical outcome and lays the foundation for a new generation of therapies.

  View details for Web of Science ID A1994PA77100012

  View details for PubMedID 8047371

• INDUCTION OF ALLOGRAFT TOLERANCE IN RATS BY AN HLA CLASS-I-DERIVED PEPTIDE AND CYCLOSPORINE-A JOURNAL OF IMMUNOLOGY NISCO, S., Vriens, P., Hoyt, G., Lyu, S., Farfan, F., Pouletty, P., KRENSKY, A. M., CLAYBERGER, C. 1994; 152 (8): 3786-3792

  Abstract

  T cell recognition of MHC molecules initiates a cascade of events resulting in allograft rejection. CTLs damage the graft by targeting nonself-MHC class I molecules. We and others have previously shown that small synthetic peptides corresponding to regions of certain MHC class I molecules can inhibit the CTL response against MHC class I alloantigens in vitro. Here we report that rat heart allografts survived survived indefinitely when transplanted into recipients treated with a synthetic peptide corresponding to residues 75-84 of (B7.75-84) in combination with a subtherapeutic dose of cyclosporine A. Furthermore, this treatment induced long-term donor-specific tolerance that was mediated by anergic cells, indicating that such peptides may have potential as therapeutics for human organ transplantation.

  View details for Web of Science ID A1994NF01800009

  View details for PubMedID 8144948

• RANTES CHEMOKINE EXPRESSION IN CELL-MEDIATED TRANSPLANT REJECTION OF THE KIDNEY LANCET Pattison, J., Nelson, P. J., Huie, P., VONLEUTTICHAU, I., Farshid, G., Sibley, R. K., KRENSKY, A. M. 1994; 343 (8891): 209-211

  Abstract

  RANTES (regulated upon activation, normal T cell expressed and secreted) is a chemotactic cytokine (a chemokine) for memory T lymphocytes, monocytes, and eosinophils. RANTES expression was studied in renal allograft biopsy specimens. Although RANTES was not expressed in samples taken one hour after transplantation, or in native renal biopsy specimens from patients with cyclosporin nephrotoxicity, it was expressed during cell-mediated transplant rejection. RANTES mRNA was detected in infiltrating mononuclear cells and renal tubular epithelium, and RANTES protein was localised to mononuclear cells, tubular epithelium, and vascular endothelium. This suggests RANTES has a role in allograft rejection.

  View details for Web of Science ID A1994MT33200011

  View details for PubMedID 7507196

• T cells in autoimmunity and allograft rejection. Kidney international. Supplement KRENSKY, A. M. 1994; 44: S50-6

  Abstract

  Since T lymphocytes are important regulators and effectors of the immune response, a basic understanding of T lymphocyte activation and differentiation may lay the basis for novel immunotherapies of immune mediated diseases. Potential sites at which to interrupt T lymphocyte mediated diseases include: (1) HLA-peptide interactions, (2) T cell receptor (TCR) recognition of peptides in the context of HLA, (3) biochemical signals mediated via the TCR, (4) numerous other cellular adhesion and signaling pathways, (5) second messengers, (6) transcription factors, and (7) various effector molecules, including granzymes and perforin. We have investigated the effects of peptides corresponding to linear sequences of HLA class I molecules. Previously, we showed that peptides corresponding to regions of the polymorphic alpha 1 and alpha 2 domains could inhibit lysis by cytotoxic T lymphocytes (CTL) specific for those regions. We now show that peptides corresponding to certain conserved regions of the HLA class I molecule can profoundly inhibit CTL. Furthermore, peptides corresponding to a particular conserved region of the HLA class I alpha 1 alpha helix are able to significantly prolong rat heterotopic heart transplants when given in conjunction with a sub-therapeutic course of cyclosporin A. These observations raise the possibility that soluble HLA molecules are natural immunoregulatory factors in vivo and that synthetic peptides may prove useful as novel immunosuppressive drugs.

  View details for PubMedID 8127034

• MONOCYTE HAPTOTAXIS INDUCED BY THE RANTES CHEMOKINE CURRENT BIOLOGY Wiedermann, C. J., Kowald, E., Reinisch, N., Kaehler, C. M., VONLUETTICHAU, I., Pattison, J. M., Huie, P., Sibley, R. K., Nelson, P. J., KRENSKY, A. M. 1993; 3 (11): 735-739

  Abstract

  Soluble mediators and inducible cell-surface molecules coordinate the ordered cascade of events giving rise to inflammation. The specific mechanisms underlying the attraction of antigen-specific cells into a site of inflammation remain sketchy, however. In particular, it is unclear how chemoattractants cause rapidly moving immune cells to adhere to the blood vessel wall and to enter inflamed tissues.Here we show that RANTES, a potent chemo-attractant for monocytes and T lymphocytes, is inducibly expressed within an inflamed organ, binds to endothelial cells, and promotes haptotaxis, the migration of cells induced by surface-bound gradients.These findings lead us to propose a model for the role of RANTES in the migration of antigen-specific immune cells into an inflammatory site.

  View details for Web of Science ID A1993MR82300002

  View details for PubMedID 15335836

• The nature of allorecognition. Current opinion in nephrology and hypertension KRENSKY, A. M., CLAYBERGER, C. 1993; 2 (6): 898-903

  Abstract

  Understanding the nature of allorecognition is fundamental to the design of antigen-specific therapies for transplantation. As recently as 10 years ago it was generally believed that recognition of major histocompatibility complex (MHC) molecules by T lymphocytes was direct and represented the "simplest" kind of T-lymphocyte interaction. It is now clear that the nature of allorecognition is complex, involving a variety of different forms of MHC antigens with or without peptides contained in their antigen-binding groove. In addition, there is renewed interest in alternative forms of allorecognition, including so-called indirect allorecognition, in which donor alloantigens are recognized as peptides in the context of recipient self. Lastly, it appears that cells other than T lymphocytes, eg, natural killer cells, are capable of antigen-specific recognition and may be responsible for heretofore underappreciated mechanisms of transplant rejection.

  View details for PubMedID 7922230

• TNF-ALPHA INDUCES EXPRESSION OF THE CHEMOATTRACTANT CYTOKINE RANTES IN CULTURED MOUSE MESANGIAL CELLS KIDNEY INTERNATIONAL Wolf, G., Aberle, S., Thaiss, F., Nelson, P. J., KRENSKY, A. M., NEILSON, E. G., Stahl, R. A. 1993; 44 (4): 795-804

  Abstract

  We investigated the effect of several immune-relevant cytokines on expression of the chemoattractant intercrine/chemokine RANTES in a mouse mesangial cell line (MMC). Fifty ng/ml recombinant tumor necrosis factor alpha (TNF alpha) induced a marked increase in RANTES transcripts after two hours. RANTES mRNA remained elevated for 24 to 48 hours after stimulation, and could be abolished by co-incubation with 30 micrograms/ml of a neutralizing rabbit anti-TNF alpha antibody. Protein expression of RANTES, as assessed by indirect immunofluorescence and Western blotting, increased in MMCs 24 hours after TNF alpha stimulation. Interleukin-1 beta, tumor necrosis factor beta (TNF beta), and lipopolysaccharide (LPS) also increased expression of RANTES mRNA. In addition, RANTES mRNA expression was stimulated in glomeruli harvested from rats following renal in vivo perfusion with TNF alpha. Our results indicate that mesangial cells produce the small cytokine RANTES. This factor, in concert with other chemoattractants, may play a role in the glomerular recruitment of inflammatory cells like macrophages/monocytes.

  View details for Web of Science ID A1993LX88600016

  View details for PubMedID 7505037

• GENOMIC ORGANIZATION AND TRANSCRIPTIONAL REGULATION OF THE RANTES CHEMOKINE GENE JOURNAL OF IMMUNOLOGY Nelson, P. J., Kim, H. T., Manning, W. C., Goralski, T. J., KRENSKY, A. M. 1993; 151 (5): 2601-2612

  Abstract

  RANTES is a member of a large supergene family of pro-inflammatory cytokines called CC chemokines that appear to play a fundamental role in inflammatory processes. The RANTES protein causes release of histamine from basophils and is a chemoattractant for CD45RO/CD4+ "memory" T lymphocytes, monocytes, and eosinophils. Although expression of RANTES was first thought to be limited to activated T cells, recent data have shown that it is produced by a variety of tissue types in response to specific stimuli. RANTES mRNA is expressed late (3 to 5 days) after activation of resting T cells whereas in fibroblasts, renal epithelial and mesangial cells, RANTES mRNA is quickly up-regulated by TNF-alpha stimulation. In order to gain a better understanding of the molecular mechanisms that regulate expression of the RANTES locus, we have characterized the RANTES gene and determined a putative promoter region. The RANTES gene spans approximately 7.1 kb and is composed of three exons of 133, 112 and 1075 bases and two introns of approximately 1.4 and 4.4 kb with the position of intron/exon boundaries conserved relative to the other CC chemokine family members. Approximately 1 kb of DNA from the immediate 5' upstream region of RANTES was sequenced and found to contain a large number of potential consensus elements for specific T cell/hemopoietic, myeloid, muscle, and ubiquitously expressed DNA-binding factors. RANTES-promoter-luciferase gene fusion assays demonstrate high levels of reporter gene activity in a "mature" T cell line Hut78, the erythroleukemic cell line HEL, and the rhabdomyosarcoma cell line RD, with little or no activity in the "early" T cell line Jurkat, the gamma delta T cell line PEER, the thymic tumor Molt4, or the pre-erythroid cell line K562. Deletion analysis of the promoter region indicates that different transcriptional mechanisms control expression of RANTES in the various tissues studied.

  View details for PubMedID 7689610

• INSULIN-LIKE GROWTH-FACTOR BINDING PROTEIN-3 PROTEASE ACTIVITY IN THE URINE OF CHILDREN WITH CHRONIC-RENAL-FAILURE PEDIATRIC NEPHROLOGY Lee, D. Y., Cohen, P., KRENSKY, A. M., Rosenfeld, R. G., Yorgin, P. D. 1993; 7 (4): 416-423

  Abstract

  The insulin-like growth factors, IGF-1 and IGF-II, are polypeptides that potentiate cellular growth. In addition to binding to specific cell surface receptors, the IGFs bind with high affinity to a family of proteins, the insulin-like growth factor binding proteins (IGFBPs). Serum and urine IGFBP patterns are altered in individuals with chronic renal failure (CRF). We recently reported that the urinary IGFBP pattern of CRF patients is unique for increased insulin-like growth factor binding protein-1 (U-IGFBP-1) levels. In this study, we used western ligand blotting (WLB), western immunoblotting (WIB), and radioimmunoassay (RIA) to further evaluate serum and urine IGFBP profiles of children with CRF (n = 14). Five patients with CRF displayed decreased serum IGFBP-3 profiles by WLB. Serum IGFBP-3 WIB profiles were remarkable for 30- and 20-kDa fragments of IGFBP-3 not seen in control serum. Serum IGFBP-3 levels, as determined by RIA, were slightly elevated. Serum levels of IGFBP-2 also were increased, although not at a level reaching statistical significance. WLB of CRF urine revealed a large increase in U-IGFBP-1 and a complete absence of urinary IGFBP-3. Recent studies of serum from pregnant women and seminal plasma have demonstrated a similar absence of intact IGFBP-3, due to the presence of a specific IGFBP-3 protease. To evaluate whether an IGFBP-3 protease accounts for the absence of intact U-IGFBP-3 in children with CRF, urine and serum samples from individuals with CRF and controls were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1993LM62900019

  View details for PubMedID 7691141

• THE CD8 CORECEPTOR INTERACTION WITH THE ALPHA-3-DOMAIN OF HLA CLASS-I IS CRITICAL TO THE DIFFERENTIATION OF HUMAN CYTOTOXIC T-LYMPHOCYTES SPECIFIC FOR HLA-A2 AND HLA-CW4 HUMAN IMMUNOLOGY WESLEY, P. K., CLAYBERGER, C., Lyu, S. C., KRENSKY, A. M. 1993; 36 (3): 149-155

  Abstract

  The CD8 coreceptor interacts with MHC class I molecules through an acidic loop in the MHC alpha 3 domain. Mutations in this region reduced binding between cells expressing mutant HLA molecules and CHO cells transfected with CD8 alpha chain, with mutations at residue 227 having the greatest effects. This study was undertaken to examine the role of the CD8-HLA interaction in the generation of primary and long-term CTLs. HLA-A*0201 genes (wild type or mutated at residue 227) were transfected into a cell line that lacked expression of HLA-A or B molecules but expressed HLA-Cw4. These cells were used as stimulators for PBLs from a normal donor. Cultures were tested for cytotoxicity at various times thereafter. Transfectants expressing the HLA-A*0201 mutant gene were poor stimulators of primary HLA-A2-specific CTLs. In long-term culture, HLA-Cw4-specific CTLs predominated, indicating that continuous expansion of allogeneic CTLs depends upon an efficient CD8-MHC class I interaction.

  View details for Web of Science ID A1993LC17900003

  View details for PubMedID 8320133

• PEPTIDES CORRESPONDING TO T-CELL RECEPTOR HLA CONTACT REGIONS INHIBIT CLASS-I RESTRICTED IMMUNE-RESPONSES JEAN HAMBURGER MEMORIAL CONGRESS / 14TH INTERNATIONAL CONGRESS OF THE TRANSPLANTATION SOC CLAYBERGER, C., Lyu, S. C., Pouletty, P., KRENSKY, A. M. ELSEVIER SCIENCE INC. 1993: 477–78

  View details for Web of Science ID A1993KN62100184

  View details for PubMedID 8438385

• PEPTIDES CORRESPONDING TO THE CD8 BINDING REGION OF HLA CLASS-I BLOCK THE DIFFERENTIATION OF CYTOTOXIC LYMPHOCYTE-T PRECURSORS JEAN HAMBURGER MEMORIAL CONGRESS / 14TH INTERNATIONAL CONGRESS OF THE TRANSPLANTATION SOC KRENSKY, A. M., Lyu, S. C., Pouletty, P., Benjamin, R., Parham, P., CLAYBERGER, C. ELSEVIER SCIENCE INC. 1993: 483–84

  View details for Web of Science ID A1993KN62100187

  View details for PubMedID 8438388

• EFFECT OF THE SUBSTITUTION OF CRITICAL RESIDUES ON THE ALLORECOGNITION OF HLA-B27 CLINICAL AND EXPERIMENTAL RHEUMATOLOGY Ozen, S., CLAYBERGER, C., KRENSKY, A. M., Benjamin, R. 1992; 10 (6): 583-587

  Abstract

  The three-dimensional structure of the HLA class I molecules has highlighted the importance of the "groove" formed by the helices. We used site-directed mutagenesis to construct a series of HLA-B27 mutants with different substitutions at the sites of the conserved amino acid residues of HLA-B27 subtypes, specifically residue 77 which is thought to be critical to the binding site of the molecule, and a residue at the CD8 binding site. We formed an anti-B27 CTL line and derived six anti-B27 clones. Each of the six clones showed a different pattern of reaction, reflecting the diversity of the epitopes recognized. All nine mutants were effective in altering allorecognition by HLA-B27 specific CTL, although positions 45 and 77 caused the most drastic effect. The residue in position 77 is also the last amino acid of the peptide sequence shared with Klebsiella. Our results highlight the importance of certain epitopes in allorecognition that may have important implications for the immunotherapy of autoimmune diseases.

  View details for Web of Science ID A1992KD27100006

  View details for PubMedID 1282853

• HUMAN CYTOTOXIC LYMPHOCYTES-T SPECIFIC FOR AUTOLOGOUS FOLLICULAR LYMPHOMA RECOGNIZE IMMUNOGLOBULIN IN A MAJOR HISTOCOMPATIBILITY COMPLEX RESTRICTED FASHION CANCER LUNAFINEMAN, S., Lee, J. E., WESLEY, P. K., CLAYBERGER, C., KRENSKY, A. M. 1992; 70 (8): 2181-2186

  Abstract

  Previously, autologous Burkitt lymphoma-specific cytotoxic T-lymphocytes (CTL) were found to express the gamma and delta T-cell receptor and recognize tumor idiotype in a major histocompatibility complex (MHC) unrestricted fashion.In this study, the authors established autologous CTL lines and clones specific for a B-cell follicular lymphoma.These CTL are tumor specific and inhibited by antiimmunoglobulin monoclonal antibodies, but unlike the Burkitt lymphoma-specific CTL, they are MHC restricted and express the alpha and beta T-cell receptor.These studies suggest that different B-cell lymphomas can induce CTL of different phenotypes and MHC restriction.

  View details for Web of Science ID A1992JT85900027

  View details for PubMedID 1394049

• GENOMIC STRUCTURE AND ALTERNATIVE SPLICING OF 519, A GENE EXPRESSED LATE AFTER T-CELL ACTIVATION JOURNAL OF IMMUNOLOGY Manning, W. C., OFARRELL, S., Goralski, T. J., KRENSKY, A. M. 1992; 148 (12): 4036-4042

  Abstract

  Relatively little is known about the transcriptional control of genes expressed late after T cell activation. We have identified four genes expressed 3 to 5 days after T cell activation by alloantigen or mitogen. Here we report the genomic organization of 519, one of these late T cell activation Ag. Analysis of the genomic clone revealed that 519 consists of six exons. Ribonuclease protection experiments indicated that the most abundant transcript arising from this region is an alternatively spliced form of 519, referred to as 520, which lacks exon 2 and is similar in sequence to NKG5, a cDNA identified in NK cells. These experiments also revealed the existence of two other alternatively spliced RNA transcripts, with heterogeneity in exon 2. Primer extension analysis and ribonuclease protection assays demonstrated that there are two prominent start sites for transcription; however, there is no evidence for the NKG5 transcript in T cells, indicating that NKG5 may represent a NK cell-specific form of 520. The 5' flanking region of this gene contains several previously identified sequences involved in transcriptional regulation, as well as some potentially interesting novel conserved motifs.

  View details for Web of Science ID A1992HY05600047

  View details for PubMedID 1318339

• IDENTIFICATION AND MOLECULAR-CLONING OF TACTILE - A NOVEL HUMAN T-CELL ACTIVATION ANTIGEN THAT IS A MEMBER OF THE IG GENE SUPERFAMILY JOURNAL OF IMMUNOLOGY Wang, P. L., OFARRELL, S., CLAYBERGER, C., KRENSKY, A. M. 1992; 148 (8): 2600-2608

  Abstract

  We have identified and cloned cDNA for a novel cell-surface protein that we have named Tactile for T cell activation, increased late expression. It is expressed on normal T cell lines and clones, and some transformed T cells, but no other cultured cell lines tested. It is expressed at low levels on peripheral T cells and is strongly up-regulated after activation, peaking 6 to 9 days after the activating stimulus. It is also up-regulated on NK cells activated in allogeneic cultures. It is not found on peripheral B cells but is expressed at very low levels on activated B cells. Tactile-specific mAb immunoprecipitates a band of 160 kDa when reduced and bands of 240, 180, and 160 kDa nonreduced. Using an antiserum produced with affinity-purified Tactile protein to screen a lambda gt11 library, we have identified Tactile cDNA. Northern blot analysis shows an expression pattern similar to that of the protein and transfection of COS cells with the full-length 5.2-kb cDNA results in cell-surface expression. Comparison with the sequence databanks show that Tactile is a member of the immunoglobulin gene superfamily, with similarity to Drosophila amalgam, the melanoma Ag MUC-18, members of the carcinoembryonic Ag family, the poliovirus receptor, and the neural cell adhesion molecule. The deduced primary sequence encodes a protein with three Ig domains, a long serine/threonine/proline-rich region typical of an extensively O-glycosylated domain, a transmembrane domain, and a 45 residue cytoplasmic domain. These data suggest that Tactile may be involved in adhesive interactions of activated T and NK cells during the late phase of the immune response.

  View details for Web of Science ID A1992HN74000041

  View details for PubMedID 1313846

• ANCHORING POCKETS IN HUMAN HISTOCOMPATIBILITY COMPLEX LEUKOCYTE ANTIGEN (HLA) CLASS-I MOLECULES - ANALYSIS OF THE CONSERVED B-(45)-POCKET OF HLA-B27 JOURNAL OF EXPERIMENTAL MEDICINE BUXTON, S. E., Benjamin, R. J., CLAYBERGER, C., Parham, P., KRENSKY, A. M. 1992; 175 (3): 809-820

  Abstract

  Dissection of the peptide binding grooves of seven subtypes of human histocompatibility leukocyte antigen (HLA)-B27 into the six specificity pockets defined by the 2.6-A structure of HLA-A*0201 revealed just one pocket, the B ("45") pocket, that is conserved among all the HLA-B27 subtypes. Functional studies of mutant HLA-B*2705 molecules with point substitutions in residues of the B pocket show that this structure, and the glutamine residue at position 45 in particular, plays a critical role in cell surface expression, peptide binding, and in the presentation of both exogenous and endogenous peptides by HLA-B*2705. We predict that the B pocket of HLA-B*2705 interacts with an amino acid side chain that anchors peptides in the binding groove, and that this peptide motif is present in most endogenously processed peptides that bind to all seven subtypes of HLA-B27.

  View details for Web of Science ID A1992HF64000019

  View details for PubMedID 1371304

• INTERLEUKIN-3 IS A GROWTH-FACTOR FOR HUMAN FOLLICULAR B-CELL LYMPHOMA JOURNAL OF EXPERIMENTAL MEDICINE CLAYBERGER, C., LUNAFINEMAN, S., Lee, J. E., Pillai, A., Campbell, M., Levy, R., KRENSKY, A. M. 1992; 175 (2): 371-376

  Abstract

  More than one-half of adults with non-Hodgkin's B cell lymphomas present with low-grade follicular lymphomas. These tumor cells are found in close association with follicular T lymphocytes and dendritic cells, suggesting that the surrounding cells may play a role in the support of follicular tumors. Supernatants from activated human peripheral blood lymphocytes were found to promote the in vitro proliferation of follicular tumor cells. This effect was entirely due to interleukin 3 (IL-3), a factor generally thought to cause the growth and differentiation of immature hematopoietic cells. IL-3 receptors were detected on fresh isolates of all primary follicular cell tumors examined. These findings suggest that follicular cell tumors may be dependent in vivo on IL-3 and that therapies directed against IL-3, its receptor, or the T cells that produce it may be effective treatment for follicular lymphoma.

  View details for Web of Science ID A1992HB06100007

  View details for PubMedID 1732410

• ISOLATION AND CHARACTERIZATION OF CDNA FROM RENAL TUBULAR EPITHELIUM ENCODING MURINE RANTES KIDNEY INTERNATIONAL Heeger, P., Wolf, G., Meyers, C., Sun, M. J., OFARRELL, S. C., KRENSKY, A. M., NEILSON, E. G. 1992; 41 (1): 220-225

  Abstract

  We have been interested in identifying proinflammatory molecules which might play a role in attracting monocytes and T cells to the kidney. Some of the new intercrines are potential candidates. In this report we have isolated cDNA encoding murine Rantes (MuRantes) from renal tubular epithelium (MCT cells). MuRantes is a 91 amino acid member of the -C-C- or intercrine beta subgroup of the Scy superfamily. The amino acid sequence for mature MuRantes was deduced from its coding cDNA and was found to be 90% homologous to its mature human counterpart (HuRantes). MCT epithelium expresses a single mRNA transcript for MuRantes of approximately 1100 bp. The MuRantes protein could be detected in cell lysates of MCT epithelium by western blotting and in the cytoplasm of MCT cells by immunofluorescence using a polyclonal antibody generated against HuRantes fusion protein. A search protocol using MuRantes-specific primers and cDNA amplification revealed that mRNAs for MuRantes are expressed additionally in syngeneic mesangial cells (MMC cells), whole kidney, liver, and spleen, as well as in nephritogenic antigen-specific CD4+ helper and CD8+ effector T cells. cDNA amplification studies also demonstrated a significant elevation in mRNA transcripts encoding MuRantes in response to the stimulation of MCT epithelium with TNF alpha and IL-1 alpha in culture, but not with TGF beta, gamma IFN, or IL-6. Our findings indicate that proximal tubular epithelium is an authentic source of MuRantes, and that transcripts encoding MuRantes are responsive to the modulating influence of paracrine factors having a known role in the development of parenchymal injury.

  View details for Web of Science ID A1992GX91800031

  View details for PubMedID 1375672

• GENERATION OF MONOCLONAL-ANTIBODIES AGAINST SOLUBLE HUMAN T-CELL RECEPTOR POLYPEPTIDES EUROPEAN JOURNAL OF IMMUNOLOGY DEVAUX, B., BJORKMAN, P. J., Stevenson, C., GREIF, W., ELLIOTT, J. F., Sagerstrom, C., CLAYBERGER, C., KRENSKY, A. M., DAVIS, M. M. 1991; 21 (9): 2111-2119

  Abstract

  One approach to the diagnosis and therapy of T cell-mediated diseases is to develop reagents specific for T cell receptor (TcR) variable (V) regions. To date, however, TcR expressed on the surface of antigen-specific T lymphocytes have proven to be poorly immunogenic. As a result, few monoclonal antibodies (mAb) recognizing human variable regions are available. In this report, we have used the "phosphatidylinositol linkage" strategy to generate soluble forms of two human allogeneic TcR derived from human cytotoxic T lymphocytes (CTL) known to be specific for HLA-A2 and HLA-Aw68/HLA-Aw69, respectively. Monomeric TcR alpha and beta chains from the HLA-A2-specific CTL were purified in large quantities from CHO cells and each was used to immunize mice to generate mAb. In particular, the anti-beta chain mAb, denoted anti-V beta 13, stain a significant (approximately 5%) fraction of human peripheral blood alpha/beta T lymphocytes, immunoprecipitate native anti-A2 TcR molecules, and activate T cells transfected with the relevant alpha and beta chain cDNA. Anti-alpha chain mAb were also obtained against a constant region determinant which can immunoprecipitate detergent-solubilized polypeptides. In general, we find that immunizations with soluble protein are far superior to those with cells bearing TcR chimeras or in combination with the purified protein.

  View details for Web of Science ID A1991GF65500019

  View details for PubMedID 1832385

• LYMPHOCYTE ADHESION MOLECULES IN T-CELL-MEDIATED LYSIS OF HUMAN KIDNEY-CELLS KIDNEY INTERNATIONAL Suranyi, M. G., Bishop, G. A., CLAYBERGER, C., KRENSKY, A. M., Leenaerts, P., Aversa, G., Hall, B. M. 1991; 39 (2): 312-319

  Abstract

  The complementary adhesion molecules LFA-1 (CD11a, 18)/ICAM-1 (CD54) and LFA-2 (CD2)/LFA-3 (CD58) have been shown to be important in T cell interaction with lymphoid target cells. The role of these ligand pairs in cytotoxicity against somatic cells is less well established. While LFA-3 is expressed by all cells in the kidney, ICAM-1 expression is low in normal kidneys but is increased in allograft rejection. An in vitro cytotoxicity assay was used to examine the relative importance of the two adhesion ligands in immune damage against kidney cells in rejection. HLA-A2 specific cytotoxic T lymphocyte (CTL) recognition of cultured human kidney cells (HKC), of predominantly renal tubular cell origin, was studied. Immunofluorescence studies showed that both induced and uninduced HKC target cells expressed ICAM-1, MHC class I and LFA-3, but only MHC class I and class II antigens and ICAM-1 were significantly upregulated by cytokine induction. Effector cells expressed LFA-1 and LFA-2 but little or no ICAM-1 and LFA-3. Cytokine induction of ICAM-1 expression on HKC target cells increased their susceptibility to lysis. Monoclonal antibody against ICAM-1 or LFA-1 produced the greatest inhibition of HKC lysis, and their effects were not additive. Antibody against LFA-2 (CD2) or LFA-3 also produced significant inhibition, but to a lesser degree, and no additive effect was found.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1991FA89000012

  View details for PubMedID 1706002

• MOLECULAR-BASIS OF TRANSPLANT REJECTION AND ACCEPTANCE PEDIATRIC NEPHROLOGY KRENSKY, A. M. 1991; 5 (4): 422-427

  Abstract

  The field of kidney transplantation is advancing so rapidly that it is difficult to keep up with the various therapeutic protocols, let alone our current understanding of transplantation immunobiology. This basic science review summarizes our current understanding of aspects of the cellular and molecular basis of renal transplant rejection and then details studies from the author's laboratory designed to specifically interrupt transplant rejection. The interaction of HLA with T cell receptors and the cascade of events following T cell activation are highlighted.

  View details for Web of Science ID A1991FW26400011

  View details for PubMedID 1911115

• CONTINUOUS VENOVENOUS HEMOFILTRATION PEDIATRIC NEPHROLOGY Yorgin, P. D., KRENSKY, A. M., Tune, B. M. 1990; 4 (6): 640-642

  Abstract

  Continuous venovenous hemofiltration (CVVH) is a technique characterized by a venovenous circuit and a pump to perfuse the hemofilter. CVVH is suited to individualization of ultrafiltration and solute clearance in patients with acute renal failure and volume overload, specifically when there is impaired cardiovascular function or where arterial access is problematic. Examples, indications and relative advantages of this and other dialytic modalities are discussed.

  View details for Web of Science ID A1990EP15800015

  View details for PubMedID 2088467

• MOLECULAR-CLONING AND CHARACTERIZATION OF WP34, A PHOSPHORYLATED HUMAN LYMPHOCYTE DIFFERENTIATION AND ACTIVATION ANTIGEN EUROPEAN JOURNAL OF IMMUNOLOGY Kadiyala, R. K., MCINTYRE, B. W., KRENSKY, A. M. 1990; 20 (11): 2417-2423

  Abstract

  An absorbed antiserum was made by incubating a cytotoxic T lymphocyte immune antiserum with the T cell leukemia HPB-ALL, thus removing reactivity to known lymphocyte function-associated antigens. This antiserum was used to screen a cDNA expression library and isolate a novel human lymphocyte cDNA clone designated WP34, WP34 transcript is expressed in functional T cells and a variety of hematopoietic cell lines and tissues, including fetal liver and thymus, but not in HPB-ALL or any non-hematopoietic cell lines or tissues tested. The WP34 protein is an acidic, phosphorylated molecule with a pI of 4.5 and molecular mass of 50 kDa. WP34 protein expression is absent in resting peripheral blood lymphocytes but can be induced with antigen stimulation, while the transcript is constitutively expressed. Sequence analysis indicates that WP34 is the human homologue of the recently described murine molecule LSP1.

  View details for Web of Science ID A1990EM97200008

  View details for PubMedID 2174784

• THE IMPORTANCE OF THE ICAM-1 LFA-1-DEPENDENT PATHWAY IN INVITRO CYTOTOXICITY AGAINST CULTURED HUMAN KIDNEY-CELLS 7TH SCIENTIFIC CONGRESS OF THE TRANSPLANTATION SOC OF AUSTRALIA AND NEW ZEALAND Suranyi, M. G., Bishop, G. A., CLAYBERGER, C., KRENSKY, A. M., Leenaerts, P., Hall, B. M. ELSEVIER SCIENCE INC. 1990: 2101–2

  View details for Web of Science ID A1990ED69000006

  View details for PubMedID 2219308

• TREATMENT OF STEROID-RESISTANT FOCAL SEGMENTAL GLOMERULOSCLEROSIS WITH PULSE METHYLPREDNISOLONE AND ALKYLATING-AGENTS PEDIATRIC NEPHROLOGY Mendoza, S. A., Reznik, V. M., Griswold, W. R., KRENSKY, A. M., Yorgin, P. D., Tune, B. M. 1990; 4 (4): 303-307

  Abstract

  In children, steroid-resistant nephrotic syndrome due to focal segmental glomerulosclerosis (FSGS) is frequently a progressive condition resulting in end-stage renal disease. There have been no reports of effective treatment for this condition. For the past several years, the Pediatric Nephrology services at the University of California, San Diego and Stanford University Schools of Medicine have treated these patients with a protocol involving infusions of high doses of methylprednisolone, often in combination with oral alkylating agents. Twenty-three children have been treated in this manner with a follow-up of 46 +/- 5 months. Twelve of these children are in complete remission. Six have minimal to moderate proteinuria. Four children remain nephrotic. Each of these children has a normal glomerular filtration rate. One child developed chronic renal failure and subsequently died while on dialysis. These results appear significantly better than previous series of children with FSGS. A controlled, multi-center trial of this protocol has been proposed.

  View details for Web of Science ID A1990DR21100001

  View details for PubMedID 2206894

• RECOGNITION OF AN HLA PUBLIC DETERMINANT (BW4) BY HUMAN ALLOGENEIC CYTOTOXIC LYMPHOCYTES-T JOURNAL OF IMMUNOLOGY CLAYBERGER, C., Rosen, M., Parham, P., KRENSKY, A. M. 1990; 144 (11): 4172-4176

  Abstract

  The human class I HLA molecules are composed of both polymorphic, or private, and conserved, or public, regions. The private regions are recognized by alloreactive T cells and also serve as restriction elements for peptide presentation to autologous cells. Although the ability of public determinants to elicit antibody responses is well documented, little is known of their role in T cell function. In this study we examine the ability of one of these public HLA determinants, designated Bw4, to serve as a target Ag for CTL. We show that for some individuals the HLA-Bw4 epitope can function as a restriction element for CTL. This finding has important implications for organ transplantation.

  View details for Web of Science ID A1990DF84400013

  View details for PubMedID 2341716

• A BINDING-SITE FOR THE T-CELL CO-RECEPTOR CD8 ON THE ALPHA-3 DOMAIN OF HLA-A2 NATURE Salter, R. D., Benjamin, R. J., WESLEY, P. K., BUXTON, S. E., Garrett, T. P., CLAYBERGER, C., KRENSKY, A. M., Norment, A. M., Littman, D. R., Parham, P. 1990; 345 (6270): 41-46

  Abstract

  Adhesion measurements between CD8 and 48 point mutants of HLA-A2.1 show that the CD8 alpha-chain binds to the alpha 3 domain of HLA-A2.1. Three clusters of alpha 3 residues contribute to the binding, with an exposed, negatively charged loop (residues 223-229) playing a dominant role. CD8 binding correlates with cytotoxic T-cell recognition and sensitivity to inhibition by anti-CD8 antibodies. Impaired alloreactive T-cell recognition of an HLA-A2.1 mutant with reduced affinity for CD8 is not restored by functional CD8 binding sites on an antigenically irrelevant class I molecule. Therefore, complexes of CD8 and the T-cell receptor bound to the same class I major histocompatibility complex molecule seem to be necessary for T-cell activation.

  View details for Web of Science ID A1990DB95700049

  View details for PubMedID 2109837

• LOCALIZATION OF A HUMAN T-CELL-SPECIFIC GENE, RANTES (D17S136E), TO CHROMOSOME 17Q11.2-Q12 GENOMICS Donlon, T. A., KRENSKY, A. M., Wallace, M. R., Collins, F. S., LOVETT, M., CLAYBERGER, C. 1990; 6 (3): 548-553

  Abstract

  We report here the localization of the gene for a human T-cell-specific molecule, designated RANTES, to human chromosome region 17q11.2-q12 by in situ hybridization and analysis of somatic cell hybrids using a cDNA probe to the gene. We have recently shown that this gene, which encodes a small, secreted, putative lymphokine, is a member of a larger gene family some of whose members reside on chromosome 4 but most of whose members have not to date been mapped. A secondary hybridization peak was noted on the region of human chromosome 5q31-q34, which may represent the location of other members of the gene family. Interestingly, this latter region overlaps with the location of an extended linked cluster of growth factor and receptor genes, some of which may be coregulated with members of the RANTES gene family.

  View details for Web of Science ID A1990CQ97700017

  View details for PubMedID 1691736

• LYMPHOCYTE-T ANTIGEN INTERACTIONS IN TRANSPLANT REJECTION NEW ENGLAND JOURNAL OF MEDICINE KRENSKY, A. M., WEISS, A., Crabtree, G., DAVIS, M. M., Parham, P. 1990; 322 (8): 510-517

  View details for Web of Science ID A1990CP69600005

  View details for PubMedID 2405272

• LOCALIZATION OF THE HUMAN LYMPHOCYTE-T ACTIVATION GENE-519 (D2S69E) TO CHROMOSOME 2P12-]Q11 CYTOGENETICS AND CELL GENETICS Donlon, T. A., KRENSKY, A. M., CLAYBERGER, C. 1990; 53 (4): 230-231

  Abstract

  In situ hybridization was used to localize the lymphocyte activation gene 519 (D2S69E) on human metaphase chromosomes. A significant proportion of the grains were situated over chromosome 2p12----q11, a region which contains other genes involved in immunologic functions.

  View details for Web of Science ID A1990ED25500013

  View details for PubMedID 2209093

• MICROVASCULAR DESTRUCTION IN RENAL-TRANSPLANT REJECTION TRANSPLANTATION Bishop, G. A., WAUGH, J. A., Landers, D. V., KRENSKY, A. M., Hall, B. M. 1989; 48 (3): 408-414

  Abstract

  In normal kidneys, peritubular and glomerular capillaries can be readily identified by their intense expression of HLA class I and class II compared to other cells within the graft. This high density of expression of MHC, plus their exposure to activated circulating lymphocytes, makes these cells the likely early and primary target of rejection responses. The fate of these capillaries during renal allograft rejection was examined using an indirect immunoperoxidase staining technique and monoclonal antibodies to class I and class II MHC antigens as well as other antigens on capillary endothelium including ICAM-1, LFA-3, and a novel antigen identified by E1.5. Expression of HLA-DR by peritubular capillaries was decreased during rejection, and this disappearance of peritubular capillaries with severe rejection was confirmed by loss of other markers of microvascular endothelium. These studies suggest peritubular capillaries may be the major target of the acute rejection response, and the techniques described allow assessment of degree of damage to these structures in renal allograft biopsies.

  View details for Web of Science ID A1989AQ44300011

  View details for PubMedID 2476878

• HISTAMINE REGULATES THE GENERATION OF HUMAN CYTOLYTIC LYMPHOCYTES-T CELLULAR IMMUNOLOGY Khan, M. M., KEANEY, K. M., Melmon, K. L., CLAYBERGER, C., KRENSKY, A. M. 1989; 121 (1): 60-73

  Abstract

  Human cytolytic T lymphocytes (CTL) were generated in the presence and absence of histamine in order to define the role of this autacoid in immune regulation. Histamine (10(-8)-10(-4) M) suppressed the generation of class I specific CTL but, at 10(-4) M, actually increased class II specific cytolysis. Histamine acted at the level of CTL generation; histamine was not present in the cytolytic assay. When histamine was added to the cytolytic assay with CTL grown without histamine, the lytic ability of the effector cells was similar to that of controls. Histamine-induced suppression of class I specific cytolysis was blocked by continuous culture with the H2 antagonist ranitidine but not with the H1 antagonist pyrilamine. These data suggest that suppression was mediated by the H2 receptor. Continuous culture with histamine had no effect on T cell proliferation or the expression of cell surface molecules. Histamine-induced suppression of class I specific cytolysis was reversed by the addition of PHA to the cytotoxicity assay, showing that the cytolytic machinery was intact. These data provide evidence that histamine is involved in regulation of cytolytic T cells.

  View details for Web of Science ID A1989U778900005

  View details for PubMedID 2541932

• CYTO-TOXIC LYMPHOCYTE-T SPECIFIC FOR SELF TUMOR IMMUNOGLOBULIN EXPRESS T-CELL RECEPTOR DELTA-CHAIN JOURNAL OF EXPERIMENTAL MEDICINE Wright, A., Lee, J. E., Link, M. P., Smith, S. D., Carroll, W., Levy, R., CLAYBERGER, C., KRENSKY, A. M. 1989; 169 (5): 1557-1564

  Abstract

  CTL are thought to play a role in the elimination of transformed cells in vivo. The effectiveness of such CTL is in part dependent on recognition of tumor specific antigens. Among the best characterized tumor-specific antigens are the unique or idiotypic determinants on the Ig of B cell lymphomas. Here we describe the generation and properties of human CTL specific for the idiotype on autologous B cell tumors. These cells are CD3+,CD4-,CD8- and express the delta chain of the TCR. Such cells may prove useful in tumor-specific adoptive therapy.

  View details for Web of Science ID A1989U366600004

  View details for PubMedID 2541219

• POLYMORPHISM IN THE ALPHA-3 DOMAIN OF HLA-A MOLECULES AFFECTS BINDING TO CD8 NATURE Salter, R. D., Norment, A. M., Chen, B. P., CLAYBERGER, C., KRENSKY, A. M., Littman, D. R., Parham, P. 1989; 338 (6213): 345-347

  Abstract

  Cytotoxic T lymphocytes (CTL) expressing the CD8 glycoprotein recognize peptide antigens presented by class I major histocompatibility complex (MHC) molecules. This correlation and the absence of CD8 polymorphism led to the hypothesis that CD8 binds to a conserved site of class I MHC molecules. Using a cell-cell binding assay we previously demonstrated specific interaction between human class I MHC (HLA-A,B,C) molecules and CD8. Subsequent analysis of the products of 17 HLA-A,B alleles revealed a natural polymorphism for CD8 binding in the human population. Two molecules, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A,B,C molecules have alanine. Site-directed mutagenesis shows that this single substitution in the alpha 3 domain is responsible for the CD8 binding phenotype and also affects recognition by alloreactive and influenza-specific CTL. Our results indicate that CD8 binds to the alpha 3 domain of class I MHC molecules.

  View details for Web of Science ID A1989T800800061

  View details for PubMedID 2784196

• A PANEL OF UNIQUE HLA-A2 MUTANT MOLECULES DEFINE EPITOPES RECOGNIZED BY HLA-A2-SPECIFIC ANTIBODIES AND CYTO-TOXIC LYMPHOCYTES-T JOURNAL OF IMMUNOLOGY HOGAN, K. T., CLAYBERGER, C., Bernhard, E. J., Walk, S. F., Ridge, J. P., Parham, P., KRENSKY, A. M., Engelhard, V. H. 1989; 142 (6): 2097-2104

  Abstract

  HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156.

  View details for Web of Science ID A1989T588500048

  View details for PubMedID 2466083

• THE MOLECULAR-BASIS OF RENAL-TRANSPLANT REJECTION SEMINARS IN NEPHROLOGY KRENSKY, A. M., CLAYBERGER, C. 1989; 9 (1): 116-119

  View details for Web of Science ID A1989AA33400025

  View details for PubMedID 2662297

• EXPRESSION OF LFA-1 IN NON-HODGKINS LYMPHOMA CANCER Medeiros, L. J., Weiss, L. M., Picker, L. J., CLAYBERGER, C., Horning, S. J., KRENSKY, A. M., Warnke, R. A. 1989; 63 (2): 255-259

  Abstract

  Lymphocyte function-associated antigen 1 (LFA-1) is a glycoprotein involved in virtually all aspects of the immune response requiring direct cell to cell contact. It has been suggested that lack of LFA-1 expression in lymphomas may represent a mechanism of escape from immunologic surveillance. We investigated the expression of LFA-1 in a series of more than 250 lymphoid neoplasms and reactive lymphoid proliferations using a frozen section immunoperoxidase technique. LFA-1 was expressed by all lymphoid populations in the reactive cases. In contrast, absence of LFA-1 alpha or beta chains was found in 44% of non-Hodgkin's lymphomas, including 50% of B-cell lymphomas. These findings suggest that loss of LFA-1 expression may be of great use in the differential diagnosis of benign versus malignant lymphoproliferations. Eighty percent of initial biopsy specimens of low-grade lymphoma exhibited LFA-1 expression, whereas only 8% of recurrent specimens retained expression of both LFA-1 subunits. However, we found no correlation between LFA-1 expression and clinical course in a series of 64 patients with diffuse large cell lymphomas.

  View details for Web of Science ID A1989R775500008

  View details for PubMedID 2642732

• DIVERSITY OF CLASS-I HLA MOLECULES - FUNCTIONAL AND EVOLUTIONARY INTERACTIONS WITH T-CELLS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Parham, P., Benjamin, R. J., Chen, B. P., CLAYBERGER, C., Ennis, P. D., KRENSKY, A. M., Lawlor, D. A., Littman, D. R., Norment, A. M., Orr, H. T., Salter, R. D., ZEMMOUR, J. 1989; 54: 529-543

  View details for Web of Science ID A1989JX74500061

  View details for PubMedID 2700944

• CYTO-TOXIC LYMPHOCYTE-T-DEFINED EPITOPE DIFFERENCES BETWEEN HLA-A2.1 AND HLA-A2.2 MAP TO 2 DISTINCT REGIONS OF THE MOLECULE JOURNAL OF IMMUNOLOGY HOGAN, K. T., CLAYBERGER, C., Le, A. T., Walk, S. F., Ridge, J. P., Parham, P., KRENSKY, A. M., Engelhard, V. H. 1988; 141 (11): 4005-4011

  Abstract

  Hemi-exon shuffling and site-directed mutagenesis have been used to determine which amino acid differences between HLA-A2.1 and HLA-A2.2 alter the CTL-defined epitopes on these two molecules. Two genes were constructed that encode novel molecules in which the effect of amino acid differences at residues 9, 43, and 95, or at residue 156 could be separately evaluated. Using both human and murine CTL that were specific for either HLA-A2.1 or HLA-A2.2, four types of epitopes were identified: 1) epitopes that were insensitive to substitutions at either residues 9, 43, and 95, or residue 156 but were lost when all four positions were changed; 2) epitopes that were dependent on the residues 9, 43, 95, but not residue 156; 3) epitopes that were dependent on residue 156, but not amino acid residues 9, 43, and 95; and 4) epitopes that were dependent on residues 9, 43, and 95, as well as amino acid residue 156. Overall, there was a roughly equal distribution of clones recognizing each of these types of epitopes. Additional molecules were constructed by hemi-exon shuffling between the HLA-A2.2 and HLA-A2.3 genes, and by site-directed mutagenesis, to analyze the epitopes recognized by two HLA-A2.2/A2.1 cross-reactive murine CTL that do not recognize HLA-A2.3. Although the epitopes recognized by these CTL were unaffected by changes occurring at residues 9, 43, and 95, or at residues 149, 152, and 156 alone, simultaneous changes in both of these regions acted in concert to destroy the epitopes. Both of the CTL recognized epitopes that were lost when substitutions were made at residues 9, 43, 95, 149, and 152. The epitope recognized by one of the CTL was also destroyed by the substitution of residues 9, 43, 95, 152, and 156. Overall, these results indicate that residues 9, 43, and 95, as well as residues in the alpha-helical region of the molecule, are all capable of contributing to the definition of the epitopes recognized by HLA-A2.1- and HLA-A2.2-specific CTL. They further indicate that some epitopes can be mapped to a particular region of the molecule, whereas other epitopes are formed through a complex interaction of residues in distant regions of the molecule.

  View details for Web of Science ID A1988R004700046

  View details for PubMedID 2460555

• IDENTIFICATION BY SITE-DIRECTED MUTAGENESIS OF AMINO-ACID RESIDUES CONTRIBUTING TO SEROLOGIC AND CTL-DEFINED EPITOPE DIFFERENCES BETWEEN HLA-A2.1 AND HLA-A2.3 JOURNAL OF IMMUNOLOGY HOGAN, K. T., CLAYBERGER, C., Bernhard, E. J., Walk, S. F., Ridge, J. P., Parham, P., KRENSKY, A. M., Engelhard, V. H. 1988; 141 (7): 2519-2525

  Abstract

  Site-directed mutagenesis of HLA-A2.1 has been used to identify the amino acid substitutions in HLA-A2.3 that are responsible for the lack of recognition of the latter molecule by the HLA-A2/A28 specific antibody, CR11-351, and by HLA-A2.1 specific CTL. Three genes were constructed that encoded HLA-A2 derivatives containing one of the amino acids known to occur in HLA-A2.3: Thr for Ala149, Glu for Val152, and Trp for Leu156. Three additional genes were constructed that encoded the different possible combinations of two amino acid substitutions at these residues. Finally, a gene encoding all three substitutions and equivalent to HLA-A2.3 was constructed. These genes were transfected into the class I negative, human cell line Hmy2.C1R. Analysis of this panel of cells revealed that recognition by the antibody CR11-351 was completely lost when Thr was substituted for Ala149, whereas substitutions at amino acids 152 and 156, either singly or in combination, had no effect on the binding of this antibody. The epitopes recognized by the allogeneic and xenogeneic HLA-A2.1 specific CTL clones used in this study were all affected by either one or two amino acid substitutions. Of those epitopes sensitive to single amino acid changes, none were affected by the substitution of Thr for Ala149, whereas all of them were affected by at least one of the substitutions of Glu for Val 152 or Trp for Leu156. Overall, amino acid residue 152 exerted a stronger effect on the epitopes recognized by HLA-A2.1 specific CTL than did residue 156. Of those epitopes affected only by multiple amino acid substitutions, double substitutions at residues 149 and 152 or at 152 and 156 resulted in a loss of recognition, whereas a mutant with substitutions at residues 149 and 156 was recognized normally. This reemphasizes the importance of residue 152 and indicates that residue 149 can affect epitope formation in conjunction with another amino acid substitution. These results are discussed in the context of current models for the recognition of alloantigens and in light of the recently published three-dimensional structure of the HLA-A2.1 molecule.

  View details for Web of Science ID A1988Q240000052

  View details for PubMedID 2459215

• A HUMAN T-CELL-SPECIFIC MOLECULE IS A MEMBER OF A NEW GENE FAMILY JOURNAL OF IMMUNOLOGY Schall, T. J., Jongstra, J., Dyer, B. J., Jorgensen, J., CLAYBERGER, C., DAVIS, M. M., KRENSKY, A. M. 1988; 141 (3): 1018-1025

  Abstract

  We have used a cDNA library enriched for T cell-specific sequences to isolate genes expressed by T cells but not by other cell types. We report here one such gene, designated RANTES, which encodes a novel T cell-specific molecule. The RANTES gene product is predicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines, and there are no sites for N-linked glycosylation. RANTES is expressed by cultured T cell lines that are Ag specific and growth factor dependent. RANTES expression is inducible in PBL by Ag or mitogen. In CTL, expression of RANTES decreases after stimulation with Ag and growth factors. Interestingly, RANTES was not expressed by any T cell tumor line tested. There is significant homology between the RANTES sequence and several other T cell genes, suggesting that they comprise a previously undescribed family of small T cell molecules.

  View details for Web of Science ID A1988P361900046

  View details for PubMedID 2456327

• RESCUE OF DAUDI CELL HLA EXPRESSION BY TRANSFECTION OF THE MOUSE BETA-2-MICROGLOBULIN GENE JOURNAL OF EXPERIMENTAL MEDICINE Seong, R. H., CLAYBERGER, C. A., KRENSKY, A. M., Parnes, J. R. 1988; 167 (2): 288-299

  Abstract

  The Daudi cell line is a B-lymphoblastoid line derived from a Burkitt lymphoma. Daudi cells lack cell surface expression of class I HLA molecules despite the presence of intracellular class I heavy chains. They have a defect in the gene encoding beta 2-microglobulin (beta 2m), resulting in lack of translatable mRNA for this protein. It has been thought that this deficiency is responsible for the lack of cell surface class I expression. However, data have recently been presented demonstrating that at least one mouse class I heavy chain can be expressed on the cell surface in the absence of beta 2m. These results raised the questions of whether the lack of beta 2m is the only defect in Daudi and whether transfer of this single gene could restore surface class I expression. We found that transfection of the mouse beta 2m gene into Daudi indeed rescued cell surface expression of class I HLA molecules, and that these molecules could be recognized both by monomorphic and allospecific mAbs. CTL clones specific for HLA-B17 or a determinant shared by HLA-B17 and HLA-A2 killed the Daudi cells transfected with the beta 2m gene, but not untransfected Daudi or Daudi transfected with vector alone. Mouse beta 2m on the transfected Daudi cells could exchange with human beta 2m when the cells were incubated in human serum. This exchange did not alter the ability of the cells to be killed by the specific CTLs. These results demonstrate that the lack of beta 2m is the sole reason for lack of surface class I molecules in Daudi cells, and that beta 2m is required for cell surface expression of the specific class I heavy chains of Daudi.

  View details for Web of Science ID A1988M473200004

  View details for PubMedID 3279151

• RECOMBINANT GAMMA-INTERFERON INCREASES THE BINDING OF PERIPHERAL-BLOOD MONONUCLEAR LEUKOCYTES AND A LEU-3+ LYMPHOCYTE-T CLONE TO CULTURED KERATINOCYTES AND TO A MALIGNANT CUTANEOUS SQUAMOUS CARCINOMA CELL-LINE THAT IS BLOCKED BY ANTIBODY AGAINST THE LFA-1 MOLECULE JOURNAL OF INVESTIGATIVE DERMATOLOGY Nickoloff, B. J., LEWINSOHN, D. M., BUTCHER, E. C., KRENSKY, A. M., CLAYBERGER, C. 1988; 90 (1): 17-22

  Abstract

  Because keratinocytes (KCs) express HLA-DR in a wide variety of skin diseases in which mononuclear leukocytes are observed in close apposition to KCs (i.e., graft-versus-host disease), and since gamma interferon (IFN-gamma) induces HLA-DR expression on KCs, we asked whether IFN-gamma treatment of KCs would influence the adherence of mononuclear leukocytes. When allogeneic peripheral blood mononuclear leukocytes (PBML) and a Leu-3+ T cell clone were coincubated with IFN-gamma-treated KCs (300 U/ml, 3 days), there was a marked increase in binding compared with nontreated KCs. Similar binding results were obtained using a cutaneous squamous carcinoma cell line (SCL-1) after IFN-gamma treatment. The IFN effect was relatively specific for IFN-gamma, as neither IFN-alpha nor -beta had any effect. Tumor necrosis factor exposure (500 U/ml, 3 days) increased the binding of the Leu-3+ T cell clone to both KCs and SCL-1 cells. Neutrophils displayed a less marked (but statistically significant) increase in binding to IFN-gamma-treated KCs. Using the Leu-3+ cell clone and SCL-1 cells, detailed kinetic analysis of the effect of IFN-gamma on binding was performed. The increased adherence between the cells began to appear after only 7 hours of treatment with r-IFN-gamma (300 U/ml) and reached a plateau at 48 hours, with significantly enhanced binding continuing for at least 48 hours after removal of IFN-gamma. The mechanism of binding was explored by preincubation of the PBML/Leu-3+ T cells with anti-LFA-1 (lymphocyte function-associated antigen) antibody (0.6-6.0 micrograms/ml), which totally inhibited the binding with no effect by anti-LFA-2 or -3 or class I or II antibodies despite documented binding of these antibodies to the cells. These results suggest that, after exposure to IFN-gamma, the ability of KCs to bind mononuclear leukocytes is strongly enhanced, and this adherence may be important in leukocyte trafficking in the skin as well as contributing to altered KC-leukocyte interaction, which may be of fundamental importance in a variety of skin disease.

  View details for Web of Science ID A1988L481700004

  View details for PubMedID 2447190

• HLA-A2 PEPTIDES CAN REGULATE CYTOLYSIS BY HUMAN ALLOGENEIC LYMPHOCYTES-T NATURE CLAYBERGER, C., Parham, P., Rothbard, J., Ludwig, D. S., SCHOOLNIK, G. K., KRENSKY, A. M. 1987; 330 (6150): 763-765

  Abstract

  The class-I and class-II molecules encoded by the major histocompatibility complex (MHC) are homologous proteins which allow cytotoxic and helper T cells to recognize foreign antigens. Recent studies have shown that the form of the antigen recognized by T cells is generally not a native protein but rather a short peptide fragment and that class-II molecules specifically bind antigenic peptides. Furthermore, the three-dimensional structure of the human MHC class-I molecule, HLA-A2, is consistent with a peptide-binding function for MHC class-I molecules. An outstanding question concerns the molecular nature and involvement of MHC-bound peptides in antigens recognized by alloreactive T cells. In this study the effects of peptides derived from HLA-A2 on cytolysis of alloreactive cytotoxic T cells (TC) cells are presented. Peptides can inhibit lysis by binding to the T cell or sensitize to lysis by binding an HLA-A2-related class-I molecule (HLA-Aw69) on the target cell. Thus, allospecific TC cells can recognize HLA-derived peptides in the context of the MHC.

  View details for Web of Science ID A1987L431600073

  View details for PubMedID 3501071

• RHABDOMYOLYSIS COMPLICATING DOXYLAMINE OVERDOSE CLINICAL PEDIATRICS Mendoza, F. S., ATIBA, J. O., KRENSKY, A. M., SCANNELL, L. M. 1987; 26 (11): 595-597

  Abstract

  A 16-year-old male presenting with anticholinergic symptoms was found to have hematuria and oliguria. Evaluation of the patient revealed a serum creatinine of 2.2 mg/dl, myoglobinuria, and a creatine phosphokinase (CPK) level of 78, 750 IU/l with 99 percent fraction 3 isoenzyme. A toxic screen showed the presence of doxylamine, an antihistamine of the ethanolamine class, at a level of 75 times therapeutic. The patient did not have a history of trauma or seizures. The extremely high CPK level with the doxylamine overdose suggests that doxylamine may be associated with nontraumatic rhabdomyolysis. This is the first case report of rhabdomyolysis being associated with an antihistamine overdose.

  View details for Web of Science ID A1987K862000009

  View details for PubMedID 3665331

• ABSENCE OF CELL-SURFACE LFA-1 AS A MECHANISM OF ESCAPE FROM IMMUNOSURVEILLANCE LANCET CLAYBERGER, C., Medeiros, L. J., Link, M. P., Warnke, R. A., Wright, A., KOLLER, T. D., Smith, S. D., KRENSKY, A. M. 1987; 2 (8558): 533-536

  Abstract

  During studies of T-cell recognition of autologous tumour cells, a number of tumour cell lines derived from patients with lymphoma proved to be poor stimulators of both autologous and allogeneic T-cell responses. Analysis of the tumour cell surface molecules indicated that expression of the lymphocyte-function-associated antigen, LFA-1, was lacking, whereas normal leucocytes from these patients expressed normal levels of LFA-1. Examination of other lymphoid tumours revealed that most high grade lymphomas, but not most low or intermediate grade lymphomas, do not express the LFA-1 molecule. Furthermore, in an initial survey, the tumours from 5 of 7 patients with non-relapsing large cell lymphomas expressed LFA-1 whereas only 3 of 18 patients with relapsing lymphomas had tumours that did so. These findings suggest that tumour cells lacking surface LFA-1 cannot initiate an effective immune response in vivo. This lack of immunogenicity might contribute to escape from immunosurveillance.

  View details for Web of Science ID A1987J846800004

  View details for PubMedID 2887833

• INVITRO MUTAGENESIS AT A SINGLE RESIDUE INTRODUCES B-CELL AND T-CELL EPITOPES INTO A CLASS-I HLA MOLECULE JOURNAL OF EXPERIMENTAL MEDICINE Salter, R. D., CLAYBERGER, C., LOMEN, C. E., KRENSKY, A. M., Parham, P. 1987; 166 (1): 283-288

  Abstract

  We have studied the interaction of HLA class I antigens with alloreactive cytotoxic T lymphocytes and monoclonal antibodies using site-directed mutagenesis and expression of an HLA-Aw68.1 gene. Two mutants containing distinct substitutions at polymorphic residues near the NH2-terminal end of the alpha 2 domain were made. One mutant with substitutions at positions 95 and 97 corresponding to residues found in HLA-A2.1 showed no alterations in binding of HLA-Aw68- or HLA-A2-specific monoclonal antibodies, but was reactive with some HLA-A2-specific CTL clones. A second mutant, in which glycine at position 107 was replaced with tryptophan found at that position in HLA-A2.1, was recognized by HLA-A2-specific CTL clones and HLA-A2, Aw69-specific monoclonal antibodies. Thus, substitution of a single amino acid residue at position 107 of the HLA-Aw68.1 molecule generates an allospecific determinant shared with HLA-A2.1 and recognized by both B and T lymphocytes.

  View details for Web of Science ID A1987H960000025

  View details for PubMedID 2439636

• HUMAN ALLOSPECIFIC CYTOLYTIC LYMPHOCYTE-T LYSIS OF A MURINE CELL TRANSFECTED WITH HLA-A2 JOURNAL OF IMMUNOLOGY KOLLER, T. D., CLAYBERGER, C., Maryanski, J. L., KRENSKY, A. M. 1987; 138 (7): 2044-2049

  Abstract

  A variety of molecules are involved in the interaction of human allospecific cytolytic T lymphocytes (CTL) with target cells. Monoclonal antibodies specific for these molecules inhibit CTL-target conjugate formation and/or lysis. To further study recognition and lysis of targets by human CTL, we used a murine mastocytoma cell line transfected with the histocompatibility leukocyte antigen (HLA)-A2 gene (P815-A2+) as a target for human HLA-A2-specific CTL. We find that only a subset of human HLA-A2-specific CTL can lyse murine P815-A2+ cells, suggesting that the murine cells may lack one or more accessory molecules needed for CTL recognition and lysis.

  View details for Web of Science ID A1987G571600005

  View details for PubMedID 3549894

• EXPRESSION OF LEU-19 (NKH-1) ANTIGEN ON IL 2-DEPENDENT CYTOTOXIC AND NONCYTOTOXIC T-CELL LINES JOURNAL OF IMMUNOLOGY Lanier, L. L., LE, A. M., Ding, A., Evans, E. L., KRENSKY, A. M., CLAYBERGER, C., Phillips, J. H. 1987; 138 (7): 2019-2023

  Abstract

  The Leu-19 (NKH-1) antigen is expressed on human peripheral blood NK cells and a subset of peripheral blood cytotoxic T lymphocytes that kill "NK-sensitive" tumor cell targets without major histocompatibility complex restriction. In the present study, we demonstrate that the Leu-19 (NKH-1) antigen is also expressed on most interleukin 2 (IL 2) dependent T cell lines and clones that have been maintained in long term culture. The Leu-19 (NKH-1) antigen expressed on an antigen-specific, class I directed cytotoxic T lymphocyte cell line was an approximately 200,000 to 220,000 dalton protein, similar to Leu-19 (NKH-1) protein expressed on natural killer cells and KG1a, an immature stem cell leukemia cell line. Furthermore, Leu-19 (NKH-1) was expressed on both CD4+ and CD8+ IL 2 dependent T cell clones, and was present on both cytotoxic and non-cytotoxic T cell clones. Thus expression of Leu-19 (NKH-1) antigen on cultured cell lines does not directly correlate with cytotoxic function, antigenic specificity, or cell lineage.

  View details for Web of Science ID A1987G571600002

  View details for PubMedID 2951430

• THE ISOLATION AND SEQUENCE OF A NOVEL GENE FROM A HUMAN FUNCTIONAL T-CELL LINE JOURNAL OF EXPERIMENTAL MEDICINE Jongstra, J., Schall, T. J., Dyer, B. J., CLAYBERGER, C., Jorgensen, J., DAVIS, M. M., KRENSKY, A. M. 1987; 165 (3): 601-614

  Abstract

  Using a subtractive hybridization procedure we have constructed a cDNA library enriched for sequences present in functional human T cell lines, but not in human EBV-transformed B cell lines. We have isolated a cDNA clone, AH2-519, representing a novel gene, designated 519. This novel gene is expressed in functional human cytolytic and Th cell lines but not in a variety of other cell lines, including several long-term human T cell tumor lines. The expression of gene 519 is inducible in cultures of normal human PBL using antigenic or mitogenic stimulation. Neither the DNA sequence determined from a full-length cDNA clone overlapping with clone AH2-519 nor the amino acid sequence of its predicted protein product has significant homology to published sequences in the GenBank or NBRF databases. The restricted expression of gene 519 suggests that its gene product is involved in the growth and/or differentiation of normal T cells. The data also show that normal, nontransformed, functional T cells express gene products that can not be readily identified in long-term tumor lines of the same cell lineage.

  View details for Web of Science ID A1987G292200002

  View details for PubMedID 2434598

  View details for PubMedCentralID PMC2188281

• IDENTIFICATION AND CHARACTERIZATION OF 2 NOVEL LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN-L24 AND ANTIGEN-L25 JOURNAL OF IMMUNOLOGY CLAYBERGER, C., KRENSKY, A. M., MCINTYRE, B. W., KOLLER, T. D., Parham, P., Brodsky, F., LINN, D. J., Evans, E. L. 1987; 138 (5): 1510-1514

  Abstract

  We describe the function and cell distribution of two novel cell surface antigens, L24 and L25. These antigens are broadly distributed on human lymphocytes. Monoclonal antibodies specific for these molecules block lysis by Class I- and II-specific cytotoxic T lymphocytes, but do not affect any other T cell functions tested. Anti-L24 antibody immunoprecipitates a molecule composed of two disulfide-linked monomers of 140 kd each. Anti-L25 antibody immunoprecipitates three proteins of 150, 85, and 75 kd. The study of these and other function associated molecules may provide insight into mechanisms of cytotoxic T lymphocyte recognition and/or function.

  View details for Web of Science ID A1987G268700031

  View details for PubMedID 3492556

• INHIBITION OF ALLOREACTIVE CYTOTOXIC LYMPHOCYTES-T BY PEPTIDES FROM THE ALPHA-2 DOMAIN OF HLA-A2 NATURE Parham, P., CLAYBERGER, C., ZORN, S. L., Ludwig, D. S., SCHOOLNIK, G. K., KRENSKY, A. M. 1987; 325 (6105): 625-628

  Abstract

  Class I major histocompatibility complex (MHC) molecules function in the recognition of antigens by cytotoxic T lymphocytes (CTL). Although this biological role is firmly established and much has been learnt about their structure and polymorphic variation, little is known of the regions of class I molecules that are involved in functional interactions with components of the T-cell surface. Here we show that peptides derived from residues 98-113 of the alpha 2 domain of HLA-A2 specifically inhibit the recognition of target cells by many HLA-A2-specific CTL. In addition to identifying a region that is probably involved in binding the T-cell receptor these results raise the possibility that alloreactive CTL may recognize degraded fragments of class I histocompatibility antigens.

  View details for Web of Science ID A1987F973500058

  View details for PubMedID 2433598

• MAPPING FUNCTIONAL EPITOPES OF THE HUMAN LFA-1 GLYCOPROTEIN - MONOCLONAL-ANTIBODY INHIBITION OF NK AND CTL EFFECTORS HUMAN IMMUNOLOGY Mentzer, S. J., KRENSKY, A. M., Burakoff, S. J. 1986; 17 (3): 288-296

  Abstract

  Anti-LFA-1 monoclonal antibody (MoAb) was originally identified by screening antibodies for their ability to inhibit cytolysis in the absence of complement. Anti-LFA-1 MoAb has been shown to inhibit both natural killer (NK) and cytolytic T lymphocyte (CTL) mediated cytolysis. To further define the utilization of this molecule in cell-mediated cytolysis, we used a panel of MoAb to functional epitopes on both the alpha and beta chains of the LFA-1 heterodimer. The panel was used to compare OKT3- NK effectors and OKT3+ CTL clones. As expected, function-associated MoAb to CTL antigens (T3, T8, LFA-2) and target cell antigens (HLA, LFA-3) blocked only CTL clones and not NK effectors. In contrast, anti-LFA-1 MoAb blocked both NK effectors and CTL clones. In addition, the panel of anti-LFA-1 MoAb demonstrated an identical hierarchy of functionally relevant LFA-1 epitopes. Given the similar utilization of LFA-1 in NK and CTL mediated cytotoxicity assays, we explored the ability of MoAb to different epitopes on LFA-1 to inhibit conjugate formation. Anti-LFA-1 MoAb inhibition of NK-target binding paralleled the inhibition of CTL-target binding. Thus, functional epitopes on the LFA-1 molecule have been defined for NK and CTL effectors. The identical hierarchy of functional epitopes indicates that the LFA-1 molecule is similarly utilized in NK and CTL mediated cytotoxicity and that the relevant epitopes are involved in effector-target conjugate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

  View details for Web of Science ID A1986E622200008

  View details for PubMedID 2432044

• FUNCTION AND CELL DISTRIBUTION OF KC-1, A NOVEL NATURAL-KILLER CELL-ASSOCIATED ANTIGEN JOURNAL OF IMMUNOLOGY CLAYBERGER, C., DYER, B., McIntyre, B., KOLLER, T. D., Hardy, B., Parham, P., Lanier, L. L., KRENSKY, A. M. 1986; 136 (5): 1537-1541

  Abstract

  Natural killer (NK) cell have been implicated in immune responses to tumor and viral antigens. We describe here a monoclonal antibody, anti-KC-1, that blocks lysis of NK targets by fresh but not activated NK cells. Anti-KC-1 has no effect on cytotoxic T lymphocyte activity or on antibody-dependent cellular cytotoxicity. This antibody may be useful in the analysis of NK cell activation and the mechanism of lysis.

  View details for Web of Science ID A1986A163400002

  View details for PubMedID 3081628

• The modulation of generation/function of human cytotoxic lymphocytes by autacoids: unusually compartmentalized cAMP? Proceedings of the Western Pharmacology Society Khan, M. M., KEANEY, K. M., KRENSKY, A. M., Melmon, K. L. 1986; 29: 31-34

  View details for PubMedID 2876437

• DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS OF T-CELL SURFACE-ANTIGENS TRANSPLANTATION KRENSKY, A. M., CLAYBERGER, C. 1985; 39 (4): 339-348

  View details for Web of Science ID A1985AFP8600001

  View details for PubMedID 3920793

• PHYTOHEMAGGLUTININ ACTIVATION OF T-CELLS THROUGH THE SHEEP RED BLOOD-CELL RECEPTOR NATURE OFLYNN, K., KRENSKY, A. M., Beverley, P. C., Burakoff, S. J., Linch, D. C. 1985; 313 (6004): 686-687

  Abstract

  Expression of receptors for sheep red blood cells and the ability to proliferate in response to phytohaemagglutinin (PHA) are the traditional properties of human T cells, but the function of the sheep red cell receptor (the T11 antigen) is controversial and the mechanism of PHA-induced mitogenesis unclear. Mitogenesis involves a complex series of cell-mediated and factor-dependent interactions, but a rise in intracellular free calcium concentration, [Ca2+]i, seems to be an important primary event in T-cell activation. We have now investigated the effects of three monoclonal antibodies, previously shown to inhibit mitogen-induced proliferation, on T-cell [Ca2+]i. We find that anti-LFA-2 and OKT11, which react with the sheep red cell receptor, have no effect on [Ca2+]i, nor do they inhibit the rise in [Ca2+]i induced by concanavalin A (Con A) or the mitogenic anti-T3 monoclonal antibody UCHT1 (ref. 11). They do, however, block PHA-induced Ca2+ mobilization. Anti-LFA-1, which reacts with the lymphocyte function-associated antigen, has no effect on intracellular Ca2+. These studies suggest that the sheep red blood cell receptor is an activation pathway for T cells and that the effects of PHA are mediated through this pathway.

  View details for Web of Science ID A1985ACA2300054

  View details for PubMedID 3871914

• THE HUMAN CYTOLYTIC LYMPHOCYTE-T RESPONSE TO TRANSPLANTATION ANTIGENS PEDIATRIC RESEARCH KRENSKY, A. M. 1985; 19 (12): 1231-1234

  Abstract

  Cytolytic T lymphocytes are important effectors in the immune response to allografts, viral infections, and some tumors. Cytolytic T lymphocyte lines and clones have been generated and used to define functionally relevant effector and target cell surface molecules. The major target antigens of the human allogeneic response are the major histocompatibility complex antigens. Functionally relevant effector antigens include LFA-1, LFA-2, LFA-3, OKT4, OKT8, OKT3, and Ti, the T cell receptor. The diagnostic and therapeutic implications of T cell surface molecules are discussed.

  View details for Web of Science ID A1985AVG1100001

  View details for PubMedID 3878492

• Lymphocyte subsets and surface molecules in man. Clinical immunology reviews KRENSKY, A. M., Lanier, L. L., Engleman, E. G. 1985; 4 (1): 95-138

  View details for PubMedID 2992859

• FUNCTIONAL DISTINCTIONS BETWEEN THE LFA-1, LFA-2, AND LFA-3 MEMBRANE-PROTEINS ON HUMAN CTL ARE REVEALED WITH TRYPSIN-PRETREATED TARGET-CELLS JOURNAL OF IMMUNOLOGY GROMKOWSKI, S. H., KRENSKY, A. M., Martz, E., Burakoff, S. J. 1985; 134 (1): 244-249

  Abstract

  We asked whether we could distinguish the roles of the human lymphocyte membrane proteins LFA-1, LFA-2, and LFA-3 in the function of CTL-mediated killing. Little is known about the functions of these molecularly distinct proteins beyond the facts that i) binding of a monoclonal antibody (MAb) to any one of them is sufficient to inhibit killing, ii) that in each case inhibition involves prevention of CTL-target cell conjugate formation, and iii) that MAb to LFA-1 and LFA-2 inhibit best when bound to the CTL, whereas anti-LFA-3 inhibits only when bound to the target cell. This latter is despite the fact that (in our test system) LFA-1 and LFA-3 are expressed both on the CTL and on the target. When the target cells were pretreated with trypsin, the sensitivity of CTL-mediated killing was affected in a different way for each site. Inhibition of anti-LFA-1 was increased by approximately 20-fold. Inhibition by anti-LFA-2 was unaffected. Inhibition by anti-LFA-3 was abolished. Trypsin did not remove the specific antigens recognized by the various CTL, HLA-A,B,C or HLA-DR. Nor did it remove LFA-1 from the target cell. It did, however, selectively remove LFA-3 from the target cell. These results indicate, for the first time, that LFA-1 and LFA-2 have functionally distinct roles. They suggest that an unidentified trypsin-sensitive target cell molecule, operationally designated the "trypsin-sensitive counter blocker" (TSCB), plays an important role in the function of LFA-1, possibly by providing a target cell binding site for LFA-1 on the CTL. The hypothesis that this TSCB is identical to LFA-3 (and the related possibility that LFA-1 and LFA-3 are mutual ligands) is not favored by our data, but is not excluded. Finally, the data indicate that the mechanisms by which MAb inhibit killing differ at the LFA-1 and LFA-3 sites. They are consistent with LFA-1 providing adhesion strengthening by binding to another site (the TSCB?) and with LFA-3 delivering an inhibitory signal when provoked with MAb.

  View details for Web of Science ID A1985TX32500039

  View details for PubMedID 3871102

• THE FUNCTION OF LFA-1 IN CELL-MEDIATED KILLING AND ADHESION - STUDIES ON HERITABLE LFA-1, MAC-1 DEFICIENCY AND ON LYMPHOID-CELL SELF AGGREGATION ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY Springer, T. A., Rothlein, R., Anderson, D. C., Burakoff, S. J., KRENSKY, A. M. 1985; 184: 311-322

  View details for Web of Science ID A1985AMW7000021

  View details for PubMedID 3898754

• LFA-1 MEMBRANE MOLECULE IN THE REGULATION OF HOMOTYPIC ADHESIONS OF HUMAN LYMPHOCYTES-B JOURNAL OF IMMUNOLOGY Mentzer, S. J., GROMKOWSKI, S. H., KRENSKY, A. M., Burakoff, S. J., Martz, E. 1985; 135 (1): 9-11

  Abstract

  We report here that MAb to human LFA-1 inhibit spontaneous homotypic adhesions of human B lymphocytes. This is, to our knowledge, the first report of a MAb that inhibits human homotypic intercellular adhesions for any cell type. LFA-1 has previously been recognized as a molecule capable of regulating specific immunologic adhesions between T lymphocytes and antigen-bearing target cells. The present findings show that the role of LFA-1 is not limited to adhesions initiated by specific immunologic recognition. The results indicate that the LFA-1 molecule is capable of regulating lymphocyte adhesions, possibly because it is a direct participant in adhesion formation.

  View details for Web of Science ID A1985AKS7900004

  View details for PubMedID 3889159

• DETERMINANTS RECOGNIZED BY HUMAN CYTO-TOXIC T-CELLS ON A NATURAL HYBRID CLASS-I HLA MOLECULE JOURNAL OF EXPERIMENTAL MEDICINE CLAYBERGER, C., Holmes, N., Wang, P. L., KOLLER, T. D., Parham, P., KRENSKY, A. M. 1985; 162 (5): 1709-1714

  Abstract

  The major histocompatibility complex class I HLA molecules are the primary determinants recognized by allogeneic cytotoxic T lymphocytes (CTL), and serve as restricting elements for CTL recognition of viral, chemical, or minor histocompatibility antigens. HLA-Aw69 is a naturally occurring hybrid class I molecule that we have used to investigate the regions of class I antigens involved in human CTL recognition. HLA-Aw69 appears to have resulted from an exon shuffle between two closely related class I genes: the alpha 1 domain of HLA-Aw69 is identical to that of HLA-Aw68, while the alpha 2 and alpha 3 domains are identical to HLA-A2. The determinants recognized by human allogeneic CTL clones specific for HLA-A2, -Aw68, and/or -Aw69 fall into three patterns: (a) CTL determinants are located on both the alpha 1 and alpha 2 domains; (b) interaction of the alpha 1 and alpha 2 domains results in new combinatorial determinants; (c) interaction of the alpha 1 and alpha 2 domains in the hybrid molecule results in the loss of CTL determinants that are present on both parental molecules. Thus, using human CTL clones, target cells, and HLA molecules, we show that the interaction of the alpha 1 and alpha 2 domains alters CTL determinants in ways not directly predictable from primary structure.

  View details for Web of Science ID A1985ATL1600021

  View details for PubMedID 2414389

• TARGET SPECIFICITY AND CELL-SURFACE STRUCTURES INVOLVED IN THE HUMAN CYTOLYTIC LYMPHOCYTE-T RESPONSE TO ENDOTHELIAL-CELLS JOURNAL OF IMMUNOLOGY CLAYBERGER, C., Uyehara, T., Hardy, B., Eaton, J., Karasek, M., KRENSKY, A. M. 1985; 135 (1): 12-18

  Abstract

  Two long-term cytolytic T lymphocyte (CTL) lines derived from the peripheral blood lymphocytes (PBL) of a single donor were analyzed for target specificity and involvement of cell surface molecules in CTL-target interactions. One line, AH2, was generated after stimulation with B lymphoblastoid cells. Cytolysis by these cells was restricted to targets expressing the appropriate HLA-A2 specificity and was blocked by mAb recognizing CD2, CD3, CD8, LFA-1, and LFA-3. The second line, AE1, was generated after stimulation with cultured endothelial cells derived from human newborn preputial microvessels. These CTL lysed all human target cells tested, except autologous cells and the Class I negative cell line Daudi. In addition, mAb specific for CD2, CD3, and CD8 did not affect cytolysis. Anti-LFA-1 and -LFA-3 mAb blocked cytolysis of B lymphoblastoid targets but not endothelial targets. These results indicate that some CTL utilize as yet uncharacterized cell surface structures for CTL-target interactions.

  View details for Web of Science ID A1985AKS7900005

  View details for PubMedID 2582029

• HUMAN T-CELL CLONES EXPRESS FUNCTIONAL HOMING RECEPTORS REQUIRED FOR NORMAL LYMPHOCYTE TRAFFICKING JOURNAL OF EXPERIMENTAL MEDICINE NAVARRO, R. F., JALKANEN, S. T., HSU, M., SOENDERSTRUPHANSEN, G., GORONZY, J., WEYAND, C., Fathman, C. G., CLAYBERGER, C., KRENSKY, A. M., BUTCHER, E. C. 1985; 162 (3): 1075-1080

  Abstract

  To function efficiently in vivo, lymphocytes must circulate from the blood into lymphoid tissues and other sites of immune reaction. Herein, we show that human cytotoxic and helper T cell clones and lines, maintained in vitro with IL-2, express the functional capacity to recognize and bind to high endothelial venules (HEV), a capacity essential for lymphocyte exit from the blood, and hence for normal lymphocyte trafficking. The expression of functional homing receptors distinguishes human T cell clones from their murine counterparts, which uniformly lack receptors for HEV and are unable to migrate normally from the blood in vivo. The results raise the possibility that human T cell clones may be more effective in mediating in vivo immune responses than is suggested by murine models.

  View details for Web of Science ID A1985AQH0300021

  View details for PubMedID 3875680

  View details for PubMedCentralID PMC2187804

• HERITABLE LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN-1 DEFICIENCY - ABNORMALITIES OF CYTO-TOXICITY AND PROLIFERATION ASSOCIATED WITH ABNORMAL EXPRESSION OF LFA-1 JOURNAL OF IMMUNOLOGY KRENSKY, A. M., Mentzer, S. J., CLAYBERGER, C., Anderson, D. C., Schmalstieg, F. C., Burakoff, S. J., Springer, T. A. 1985; 135 (5): 3102-3108

  Abstract

  The effect of heritable LFA-1 deficiency on T lymphocyte function was measured. After primary mixed lymphocyte stimulation, all six patients studied showed diminished allospecific T lymphocyte cytolytic and NK activity as compared with kindred and normal controls. MLR and mitogen-induced proliferative responses were consistently depressed. LFA-1-deficient, EBV-transformed B cell lines were poor stimulators of T cell responses. Primary cytolytic responses by lymphocytes from severely LFA-1-deficient patients (less than 0.2% of normal surface expression) were consistently more profoundly depressed than those by lymphocytes from moderately deficient patients (about 5% of normal surface expression). These results demonstrate the importance of LFA-1 in lymphocyte function. After repeated MLR restimulation, proliferative and cytolytic capacity improved and CTL lines could be established from all patients. Cytolysis by lines from one but not a second severe patient, and by four of four moderate patients, was inhibited by anti-LFA-1 MAb, and at 10-fold lower concentrations than required for inhibition of killing by control CTL lines. The locus of inhibition was on the target cell for the severely deficient CTL line, and on both the target and effector cells for moderately deficient CTL lines. In contrast, the locus of inhibition for normal CTL is on the effector cell. These findings show that LFA-1 can participate bidirectionally in cell interactions. The in vitro results are discussed in terms of the clinical findings in patients.

  View details for Web of Science ID A1985ASW4100028

  View details for PubMedID 3900204

• MHC-UNRESTRICTED CYTOLYTIC LYMPHOCYTES-T SURVEY OF IMMUNOLOGIC RESEARCH KRENSKY, A. M., CLAYBERGER, C. 1985; 4 (4): 297-302

  View details for Web of Science ID A1985C494200005

  View details for PubMedID 3939157

• HUMAN CYTOLYTIC LYMPHOCYTE-T INTERACTIONS WITH VASCULAR ENDOTHELIUM AND FIBROBLASTS - ROLE OF EFFECTOR AND TARGET-CELL MOLECULES JOURNAL OF IMMUNOLOGY Collins, T., KRENSKY, A. M., CLAYBERGER, C., Fiers, W., Gimbrone, M. A., Burakoff, S. J., Pober, J. S. 1984; 133 (4): 1878-1884

  Abstract

  Monoclonal antibodies (mAb) against cell surface structures have been used to identify several molecules involved in the interaction of human cytolytic T lymphocytes (CTL) with lymphoid and other bone marrow-derived targets. In allograft rejection or in graft-vs-host disease, however, major cellular targets are vascular and stromal cells, especially endothelium. Yet little is known about whether the same cell surface molecules are involved in the interactions of CTL with these cell types. We assessed the ability of mAb against effector or target cell structures to inhibit cytolysis of susceptible, cultured human vascular endothelium or dermal fibroblasts by a cloned human CTL line. Using mAb reactive with T3, T4, LFA-1, LFA-2, LFA-3, and HLA-DR, we found a qualitatively similar but quantitatively different pattern of inhibition of cytolysis as previously established for lymphoid targets by using the same CTL clone. These results have two implications: 1) the target cell structures recognized by CTL molecules such as T4, LFA-1 and LFA-2 are present on diverse cell types; and 2) the relative importance of such interactions may vary with target cell type. Furthermore, our studies provide several insights into the mechanisms of the interacting molecules. Our model system, and the use of pathophysiologically important target cells, may be useful for further analysis of CTL-mediated immune injury.

  View details for Web of Science ID A1984TJ51800031

  View details for PubMedID 6432903

• GLYCOPROTEINS OF 210,000 AND 130,000 MW ON ACTIVATED T-CELLS - CELL DISTRIBUTION AND ANTIGENIC RELATION TO COMPONENTS ON RESTING CELLS AND T-CELL LINES JOURNAL OF IMMUNOLOGY Hemler, M. E., SANCHEZMADRID, F., Flotte, T. J., KRENSKY, A. M., Burakoff, S. J., Bhan, A. K., Springer, T. A., STROMINGER, J. L. 1984; 132 (6): 3011-3018

  Abstract

  A glycoprotein complex of 210,000 and 130,000 m.w., found on mitogen or alloantigen-stimulated human T cells and not on other hematopoietic cells, has been defined by a monoclonal antibody (Mab). The components of this complex are a subset of a larger family of proteins (210,000, 165,000 and 130,000 m.w.) defined by a second Mab. In a panel of hematopoietic cell lines and cell types, only activated T cells (including the cell line HUT-102) express the 210,000/130,000 complex and these cells also express the IL 2 receptor, a characteristic marker for activated T cells. The 210,000/130,000 m.w. complex (reactive with the Mab TS2/7) is present on all long-term activated T cells, including both the OKT4 and OKT8 subsets. The 210,000 m.w. subunit is expressed only on activated T cells. Other lymphoid cells express either the 130,000 m.w. subunit alone (unactivated lymphocytes, thymocytes, HUT-78) or the 130,000 subunit together with a 165,000 subunit (MOLT-4, HSB, and other leukemic T cell lines). The 210,000/130,000 m.w., 165,000/130,000 m.w. and 130,000 m.w. complexes are antigenically related in that all share reactivity with the Mab A- 1A5 . Among non-lymphoid hematopoietic cells and cell lines, none express the 210,000 m.w. chain; adherent cells (monocytes) and myeloid cell lines each express single proteins of 130,000 to 155,000 m.w. Granulocytes and red blood cells are negative and platelets express multiple bands (165,000 and 140,000 m.w.). Immunoperoxidase staining of tissue sections showed that a broad range of tissues and cell types had material cross-reactive with the lymphoid 130,000 m.w. protein. However, only a discrete subset of those tissues and cells including blood vessel walls, connective tissue, smooth muscle, kidney mesangial cells, and some non-cellular matrix tissue, had material cross-reactive with the 210,000 m.w. protein on activated T lymphocytes.

  View details for Web of Science ID A1984SS42400052

  View details for PubMedID 6327814

• LFA-1, LFA-2, AND LFA-3 ANTIGENS ARE INVOLVED IN CTL-TARGET CONJUGATION JOURNAL OF IMMUNOLOGY KRENSKY, A. M., Robbins, E., Springer, T. A., Burakoff, S. J. 1984; 132 (5): 2180-2182

  Abstract

  Three cell surface antigens associated with the CTL-target cell interaction were previously identified by generation of mAb against OKT4+, HLA-DR-specific CTL, and selection for inhibition of cytolysis in a 51Cr-release assay. In this report, we showed that these mAb inhibit cytolysis by blocking CTL-target cell conjugate formation. It appears that LFA-1, LFA-2, and LFA-3 are cell surface structures involved in strengthening effector-target adhesion that accompanies antigen-specific recognition.

  View details for Web of Science ID A1984SN37200006

  View details for PubMedID 6201533

• HUMAN-LYMPHOCYTE FUNCTION ASSOCIATED ANTIGENS SURVEY OF IMMUNOLOGIC RESEARCH KRENSKY, A. M., SANCHEZMADRID, F., Springer, T. A., Burakoff, S. J. 1984; 3 (1): 39-44

  Abstract

  Three cell surface molecules, designated LFA-1, LFA-2, and LFA-3 were identified by mAbs selected for their ability to block cytolysis by an OKT4+, HLA-DR-specific CTL line. The LFA mAbs block all CTL and proliferative functions studied. In addition, anti-LFA-1 mAbs inhibit NK-mediated cytolysis. By analogy with murine LFA-1, human LFA-1 may be involved in the adhesion stage of cellular interactions. LFA-2, the SRBC receptor molecule, appears to be a T cell function-specific molecule. We have not yet established whether LFA-2 participates in antigen recognition or whether it is involved in antigen-non-specific interactions. The anti-LFA-3 mAb specifically blocks function by binding to the target cells, implying that LFA-3 may be a target ligand for an effector-specific receptor. The CTL-target interaction involves a number of steps, including antigen recognition, cell adhesion, and delivery of the lethal hit [22]. The LFA antigens show the complexity of this process at the molecular level. The anti-LFA monoclonal antibodies will be useful probes into the T cell immune response and may prove clinically relevant, both diagnostically and therapeutically.

  View details for Web of Science ID A1984SF24800008

  View details for PubMedID 6371972

• PRODUCTION OF COLONY-STIMULATING FACTOR(S) FOR GRANULOCYTE-MACROPHAGE AND MULTIPOTENTIAL (GRANULOCYTE ERYTHROID MEGAKARYOCYTE MACROPHAGE) HEMATOPOIETIC PROGENITOR CELLS (CFU-GEMM) BY CLONAL LINES OF HUMAN IL-2-DEPENDENT LYMPHOCYTES-T EXPERIMENTAL HEMATOLOGY Greenberger, J. S., KRENSKY, A. M., Messner, H., Burakoff, S. J., WANDL, U., SAKAKEENY, M. A. 1984; 12 (9): 720-727

  Abstract

  Human T-lymphocyte lines that were selected for recognition of HLA-DR6 antigen and were dependent for growth in vitro on an added source of interleukin-2 (IL-2) were derived from the peripheral blood of normal individuals. Each was tested for production of a lymphokine(s) with properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) using as target cells nonadherent cells from human long-term bone marrow cultures (LTBMC) or fresh marrow. Each of eight T-lymphocyte lines that were OKT3, OKT4, and HLA-DR positive produced GM-CSF that stimulated colony formation by both LTBMC cells and fresh marrow. Individually examined single-cell-derived bone marrow colonies growing in T-cell GM-CSF contained peroxidase-positive neutrophils, and macrophage-monocytes (GM-CFUc). Supernatant from a single-cell-derived T-cell clonal line designated F1 stimulated formation of granulocyte-macrophage colonies, megakaryocyte colonies, macroscopic erythroid bursts, and multipotential colonies containing erythroid cells, megakaryocytes, neutrophilic and eosinophilic granulocytes, and monocyte-macrophages (CFU-GEMM) in the presence of added erythropoietin. These data indicate that human IL-2-responsive T-lymphocytes produce lymphokine(s) that stimulate proliferation of primitive as well as committed hematopoietic stem cells, and implicate human T-lymphocytes in regulation of human multipotential hematopoietic stem cells in vivo.

  View details for Web of Science ID A1984TQ98700008

  View details for PubMedID 6333354

• Experimental evidence for the potential usefulness of permanent IL-3-dependent multipotential hematopoietic progenitor cell lines in transfusion granulocyte support therapy: need for expansion of early passage cells. Kroc Foundation series Greenberger, J. S., SAKAKEENY, M. A., Krensky, A., Burakoff, S., Reid, D., Novak, T., Boggs, S. S. 1984; 18: 255-269

  View details for PubMedID 6152286

• INTERACTIONS OF LYMPHOCYTES-T WITH HUMAN VASCULAR ENDOTHELIAL-CELLS - ROLE OF ENDOTHELIAL-CELLS SURFACE-ANTIGENS IMMUNOBIOLOGY Pober, J. S., Gimbrone, M. A., Collins, T., Cotran, R. S., Ault, K. A., Fiers, W., KRENSKY, A. M., CLAYBERGER, C., Reiss, C. S., Burakoff, S. J. 1984; 168 (3-5): 483-494

  Abstract

  We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.

  View details for Web of Science ID A1984AAK9500030

  View details for PubMedID 6397428

• A MOLECULAR ANALYSIS OF THE CYTOLYTIC LYMPHOCYTE-T RESPONSE ADVANCES IN IMMUNOLOGY Burakoff, S. J., WEINBERGER, O., KRENSKY, A. M., Reiss, C. S. 1984; 36: 45-85

  View details for Web of Science ID A1984TN27000002

  View details for PubMedID 6239523

• LYMPHOCYTES RECOGNIZE HUMAN VASCULAR ENDOTHELIAL AND DERMAL FIBROBLAST IA ANTIGENS INDUCED BY RECOMBINANT IMMUNE INTERFERON NATURE Pober, J. S., Collins, T., Gimbrone, M. A., Cotran, R. S., Gitlin, J. D., Fiers, W., CLAYBERGER, C., KRENSKY, A. M., Burakoff, S. J., Reiss, C. S. 1983; 305 (5936): 726-729

  Abstract

  T-lymphocyte-mediated responses to the cellular components of blood vessels are important in rejection of allografts. The induction of cytolytic T lymphocytes (CTLs) depends on recognition of foreign class II major histocompatibility complex antigens (human HLA-DR, DC/DS, SB and others, collectively referred to as Ia) on the target cells whereas killing by CTLs usually depends on recognition of foreign class I antigens (HLA-A, B), although some alloreactive CTLs recognize foreign Ia instead of HLA-A, B (refs 5-8). The expression of Ia antigens has traditionally been regarded as restricted to immunological cell types, and the presence of class II antigen-bearing 'passenger' leukocytes in rodent organ grafts appears necessary for graft rejection. Recently, Ia antigens have been observed by immunofluorescence microscopy on human renal and dermal capillary endothelium. We have previously shown that human umbilical vein endothelial (HUVE) cells in standard culture conditions do not bear Ia antigens, but may be induced to do so by products of lectin- or alloantigen-activated T lymphocytes. Furthermore, we found that recombinant immune interferon (IFN-gamma), free of other lymphokines, is a potent inducer of Ia expression in HUVE cells. Here we report that IFN-gamma also induces Ia expression on human foreskin capillary endothelial (HFCE) cells, HUVE cells transformed by Simian virus 40 viral DNA (SV-HUVE cells) and human dermal fibroblast (HDF) cells in culture. Further, we present evidence that Ia present on HUVE cells and HDF cells can be functionally recognized by human T cells, resulting in a two-way interaction between T cells and mesenchymal cells that may be important in allograft rejection.

  View details for Web of Science ID A1983RM23900055

  View details for PubMedID 6415484

• CAUSES OF INCREASED RENAL ECHOGENICITY IN PEDIATRIC-PATIENTS PEDIATRICS KRENSKY, A. M., Reddish, J. M., Teele, R. L. 1983; 72 (6): 840-846

  Abstract

  Review of 2,700 abdominal ultrasonic examinations revealed 56 patients whose kidneys showed increased echogenicity. Echogenic kidneys were associated with medical renal disease in 94% of cases (30% glomerular, 48% tubulointerstitial, 16% end-stage) and with no detectable renal disease in 6% (three patients). Patterns of increased echogenicity and renal size were evaluated. Specific patterns occurred in end-stage renal disease and polycystic kidney disease. Other medical renal diseases had overlapping ultrasonographic features. Some generalizations could be made although increased echogenicity was often nonspecific.

  View details for Web of Science ID A1983RT62200015

  View details for PubMedID 6646928

• TUBULO-INTERSTITIAL NEPHRITIS ASSOCIATED WITH POLYOMAVIRUS (BK TYPE) INFECTION NEW ENGLAND JOURNAL OF MEDICINE Rosen, S., Harmon, W., KRENSKY, A. M., Edelson, P. J., PADGETT, B. L., Grinnell, B. W., Rubino, M. J., Walker, D. L. 1983; 308 (20): 1192-1196

  Abstract

  We studied viral injury to the kidney in a six-year-old boy with hyperimmunoglobulin M immunodeficiency who presented with irreversible acute renal failure and eventually died after five months of dialysis. Renal biopsy at the time of his presentation revealed a predominantly tubulo-interstitial process with numerous viral inclusions that were identified as polyomavirus. Urine cultures showed a massive viruria with BK-type, polyomavirus. The kidney disease was end stage, with persistence of BK virus identified by morphologic techniques and by culture. DNA hybridization analysis showed virus in low concentration in the lymph nodes, spleen, and lungs. The marked viruria, the high concentration of BK virus, and the extensive distribution of viral antigen throughout the kidney all suggest that infection with BK virus was the basis of the severe renal parenchymal injury.

  View details for Web of Science ID A1983QQ36300004

  View details for PubMedID 6302506

• HUMAN-LYMPHOCYTE FUNCTION ASSOCIATED ANTIGEN-1 (LFA-1) - IDENTIFICATION OF MULTIPLE ANTIGENIC EPITOPES AND THEIR RELATIONSHIP TO CTL-MEDIATED CYTO-TOXICITY JOURNAL OF IMMUNOLOGY Ware, C. F., SANCHEZMADRID, F., KRENSKY, A. M., Burakoff, S. J., STROMINGER, J. L., Springer, T. A. 1983; 131 (3): 1182-1188

  Abstract

  Human lymphocyte function-associated antigen (LFA)-1, a heterodimeric lymphocyte surface glycoprotein of 177,000 and 95,000 relative molecular weight has been implicated to function in the cytotoxic T lymphocyte (CTL) effector mechanism. Seven mouse hybridoma lines producing monoclonal antibodies (MAb) reactive with this structure were studied. Three unique and 3 partially over-lapping epitopes on human LFA-1 were defined by competitive cross inhibition binding assays using biosynthetically labeled anti-LFA-1 MAb. In contrast, of five rat antimouse LFA-1 MAb, all five recognized a common or shared epitope. An HLA-B7 specific human CTL line expressed 1.1 X 10(5) LFA-1 sites per cell with a direct saturation binding assay. Human CTL expressed two to four times more LFA-1 than peripheral blood lymphocytes or B and T lymphoblastoid cell lines. Titration of each of the anti-LFA-1 MAb in a 51chromium release cytolytic assay revealed quantitative differences in the ability of the different anti-LFA-1 MAb to block cytolysis indicating distinct functional and antigenic epitopes exist on the human LFA-1 molecule. Anti-LFA-1 MAb reversibly inhibited the CTL reaction by slowing the initial rate of cytolysis. These results suggest anti-LFA-1 MAb inhibit CTL function by specific blockade of a functionally relevant molecule.

  View details for Web of Science ID A1983RD69100023

  View details for PubMedID 6193178

• EPIDEMIC AND ENDEMIC HLA-B AND DR ASSOCIATIONS IN MUCOCUTANEOUS LYMPH-NODE SYNDROME HUMAN IMMUNOLOGY KRENSKY, A. M., Grady, S., SHANLEY, K. M., BERENBERG, W., YUNIS, E. J. 1983; 6 (2): 75-77

  Abstract

  HLA-A,B and DR antigens were evaluated in 51 patients with mucocutaneous lymph node syndrome (Kawasaki disease) during both endemic and epidemic periods in the Boston area. Although 80% of patients were HLA-B5 (70% HLA-Bw51) during endemic periods, there were no HLA-B5 individuals during the epidemic period. There was, however, an increased incidence of HLA-Bw44 during the Boston epidemic. Such different associations in endemic versus epidemic periods suggest possible different, as yet unknown, inciting agents.

  View details for Web of Science ID A1983QF45400002

  View details for PubMedID 6572621

• THE FUNCTIONAL-SIGNIFICANCE, DISTRIBUTION, AND STRUCTURE OF LFA-1, LFA-2, AND LFA-3 - CELL-SURFACE ANTIGENS ASSOCIATED WITH CTL-TARGET INTERACTIONS JOURNAL OF IMMUNOLOGY KRENSKY, A. M., SANCHEZMADRID, F., Robbins, E., Nagy, J. A., Springer, T. A., Burakoff, S. J. 1983; 131 (2): 611-616

  Abstract

  Three cell surface antigens associated with the cytolytic T lymphocyte(CTL)-target cell interaction were identified by generation of monoclonal antibodies (MAb) against OKT4+, HLA-DR-specific CTL and selection for inhibition of cytolysis in a 51Cr-release assay. These MAb block cytolysis by both OKT4+ and OKT8+ CTL and the proliferative responses to PHA and the mixed lymphocyte response (MLR). LFA-1 is an antigen widely distributed on lymphoid tissues and is composed of two polypeptides of 177,000 and 95,000 Mr on all cell types studied. Anti-LFA-1 MAb block NK cell-mediated cytolysis in addition to T lymphocyte-mediated cytotoxicity and proliferation. LFA-2 (Mr = 55,000 to 47,000), a determinant on the sheep red blood cell receptor, is expressed by T cells but not B cells and appears specific for T cell functions. LFA-3 (Mr = 60,000) is a widely distributed antigen present on both hematopoietic and nonhematopoietic tissues and appears to only be involved in T cell functions. MAb to LFA-1 and LFA-2 inhibit function by binding to effector cell surface molecules, whereas anti-LFA-3 MAb appear to block by binding to the target cells. Together with previously described molecules, LFA-1, LFA-2, and LFA-3 demonstrate the complexity of CTL-mediated cytotoxicity at the molecular level.

  View details for Web of Science ID A1983QZ47000017

  View details for PubMedID 6345670

• HEMOLYTIC UREMIC SYNDROME AND CRESCENTIC GLOMERULONEPHRITIS COMPLICATING CHILDHOOD NEPHROSIS CLINICAL NEPHROLOGY KRENSKY, A. M., Ingelfinger, J. R., Grupe, W. E., Rosen, S. 1983; 19 (2): 99-106

  Abstract

  Three children with nephrotic syndrome and focal segmental glomerulosclerosis and/or mesangial proliferation on renal biopsy developed the sudden onset of renal failure, microangiopathic hemolytic anemia, and thrombocytopenia. Two of the three children developed crescentic glomerulonephritis and never regained renal function while the third showed no change from his original histologic pattern and also developed chronic renal failure. These cases suggest an association between the lipoid nephrosis-focal segmental glomerulosclerosis group of glomerular diseases and crescentic glomerulonephritis, and may represent an unusual pathway in the evolution of childhood nephrosis.

  View details for Web of Science ID A1983QF07100008

  View details for PubMedID 6839557

• A DC-SPECIFIC CYTOLYTIC LYMPHOCYTE-T LINE IS OKT8+ JOURNAL OF IMMUNOLOGY KRENSKY, A. M., CLAYBERGER, C., Greenstein, J. L., CRIMMINS, M., Burakoff, S. J. 1983; 131 (6): 2777-2780

  Abstract

  A human cytotoxic T lymphocyte (CTL) line, A9, was generated by limiting dilution and was selected because of its apparent DC specificity. A9 is 100% OKT3+, 90% OKT4+, and 10% OKT8+, but by negative selection the CTL present are entirely OKT8+. These OKT8+ CTL are totally inhibitable by Genox 3.53, an anti-DC1 monoclonal antibody (mAb), and Leu-10, an anti-DC subgroup mAb, but are not inhibitable by a panel of anti-HLA-DR mAb. These CTL are also inhibitable by anti-OKT3 and anti-LFA-2 but not by OKT4 or OKT8 mAb. These findings extend previous studies that showed that OKT8+ CTL recognize HLA-A,B antigens, whereas OKT4+ CTL recognize HLA-DR and SB antigens. It is possible that an as yet undefined T cell surface molecule is involved in DC recognition.

  View details for Web of Science ID A1983RT38500031

  View details for PubMedID 6605989

• BASIC SCIENCE SEMINAR - CYTO-TOXIC LYMPHOCYTE-T RESPONSE TO HLA ANTIGENS JOURNAL OF PEDIATRICS KRENSKY, A. M., Burakoff, S. J. 1983; 102 (6): 814-819

  View details for Web of Science ID A1983QU32100002

  View details for PubMedID 6602213

• Cytotoxic T cells directed against HLA-DR antigens and their surface proteins. Diagnostic immunology Burakoff, S. J., CLAYBERGER, C., HEMLER, M., KRENSKY, A. M., Reiss, C. S., Robbins, E., Sanchez-Madrid, F., Springer, T. A., STROMINGER, J. L., Ware, C. F. 1983; 1 (3): 116-119

  Abstract

  The authors review their recent research involving the generation of cytotoxic T lymphocytes (CTL) directed against HLA-DR antigens. A mouse anti-human xenogeneic system first suggested that HLA-DR antigens could be recognized by CTL. Human allogeneic CTL specific for HLA-DR6 were generated and found to be OKT4+. The fact that these CTL were OKT4+ while anti HLA-A,B CTL were OKT8+ suggested that these T cell surface antigens may be involved in MHC antigen recognition; ie, they may be part of the T cell receptor. These OKT4+, HLA-DR specific CTL were further used to generate monoclonal antibodies (1) which block cytolysis and define novel antigens involved in the CTL-target interaction and (2) which define an antigenic complex on alloantigen activated T cells.

  View details for PubMedID 6238749

• SPECIFICITY OF OKT4+ CYTO-TOXIC LYMPHOCYTE-T CLONES JOURNAL OF IMMUNOLOGY KRENSKY, A. M., CLAYBERGER, C., Reiss, C. S., STROMINGER, J. L., Burakoff, S. J. 1982; 129 (5): 2001-2003

  Abstract

  OKT4+, HLA-DR-specific CTL were cloned by limiting dilution, and two clones were evaluated. One clone, B8, specifically recognized DR6 antigens, whereas another cloned, C6, recognized an Ia-like determinant on some DR 3, 5, and 6 target cells. Both clones were OKT3+, OKT4+, and OKT8-, and their cytolysis could be blocked by OKT3 and OKT4, but not OKT8, antibodies. A panel of monoclonal antibodies that recognize DR molecules blocked target cell recognition by these OKT+ CTL. Although cloned B8 recognized a DR6-specific determinant, clone C6 appeared to recognize a supratypic determinant that may be common to some DR molecules (e.g., MT2) or possibly another human Ia-like antigen (e.g., SB). The availability of OKT+ CTL clones should help to dissect the Ia-like antigens recognized by human T cells.

  View details for Web of Science ID A1982PM14700036

  View details for PubMedID 6181153

• TECHNIQUE FOR INFREQUENT ARTERIAL BLOOD-GAS MONITORING IN THE NEWBORN CLINICAL PEDIATRICS KRENSKY, A. M. 1982; 21 (1): 57-57

  View details for Web of Science ID A1982MZ10200011

  View details for PubMedID 7056004

• 3 DISTINCT ANTIGENS ASSOCIATED WITH HUMAN LYMPHOCYTE-T-MEDIATED CYTOLYSIS - LFA-1, LFA-2, AND LFA-3 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES SANCHEZMADRID, F., KRENSKY, A. M., Ware, C. F., Robbins, E., STROMINGER, J. L., Burakoff, S. J., Springer, T. A. 1982; 79 (23): 7489-7493

  Abstract

  Monoclonal antibodies were prepared to anti-HLA-DR cytolytic T lymphocytes (CTLs) and screened for inhibition of CTL-mediated killing. Binding of monoclonal antibodies to four types of molecules, LFA-1, LFA-2, LFA-3, and HLA-DR, inhibited killing, suggesting that these molecules participate in the CTL-target cell interaction. The antigens were characterized by immunoprecipitation, crosslinking, NaDodSO4/polyacrylamide gel electrophoresis, and immunofluorescence flow cytometry. The LFA-1 antigen contains alpha and beta polypeptide chains of Mr 177,000 and 95,000 that are noncovalently associated in an alpha 1 beta 1 structure. It is present on both B and T lymphocytes and marks subpopulations that differ in quantitative expression. Human LFA-1 appears to be the homologue of mouse LFA-1. Human LFA-2 is of Mr 49,000 with a minor component of Mr 36,000. It is expressed on CTL lines but not on a B-cell line and in peripheral blood preferentially on T lymphocytes. Human LFA-3 is of Mr 60,000 and is expressed on both B and T lymphocytes.

  View details for Web of Science ID A1982PT40700085

  View details for PubMedID 6984191

• IMMUNOREGULATORY ABNORMALITIES IN MUCOCUTANEOUS LYMPH-NODE SYNDROME CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Leung, D. Y., Siegel, R. L., Grady, S., Krensky, A., Meade, R., REINHERZ, E. L., Geha, R. S. 1982; 23 (1): 100-112

  View details for Web of Science ID A1982NG72100011

  View details for PubMedID 7047030

• LONG-TERM HUMAN CYTOLYTIC T-CELL LINES ALLOSPECIFIC FOR HLA-DR6 ANTIGEN ARE OKT4+ PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES KRENSKY, A. M., Reiss, C. S., Mier, J. W., STROMINGER, J. L., Burakoff, S. J. 1982; 79 (7): 2365-2369

  Abstract

  Human allospecific cytotoxic T lymphocyte lines were established by weekly stimulation of peripheral blood lymphocytes with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines in the presence of interleukin 2. The cytotoxic T lymphocyte lines were stimulated by either JY (which expresses HLA-A,B and -DR) or Daudi (which expresses HLA-DR but not -A,B antigens). Specificity of the effector cell lines was determined by antibody blocking and by patterns of cytolysis against a panel of target cell lines. The phenotype of the effector cells was identified by both negative (antibody and complement lysis) and positive (fluorescence-activated cell sorter) selection. The anti-JY lines only recognized targets bearing HLA-A2, B7 antigens and were composed exclusively of OKT4-8+ effector cells. The anti-Daudi lines, on the other hand, specifically recognized targets bearing DR6 and eventually contained only OKT4+8- cells. Thus, stimulation by Daudi cells can result in the generation of OKT4+ CTL lines that are allospecific for DR6 antigen.

  View details for Web of Science ID A1982NJ77700048

  View details for PubMedID 6980419

• GENERATION OF LONG-TERM HUMAN CYTOLYTIC CELL-LINES WITH PERSISTENT NATURAL-KILLER ACTIVITY JOURNAL OF IMMUNOLOGY KRENSKY, A. M., Ault, K. A., Reiss, C. S., STROMINGER, J. L., Burakoff, S. J. 1982; 129 (4): 1748-1751

  Abstract

  Human cell lines maintained by in vitro stimulation with the HLA-A, B-negative, DR-positive, Epstein Barr virus-transformed, lymphoblastoid cell line Daudi in the presence of conditioned medium demonstrated significant NK activity for over 6 wk in continuous culture. These cells lyse K562 and a broad panel of lymphoblastoid cell lines but do not lyse normal peripheral blood lymphocytes or pokeweed mitogen blasts. They possess the sheep red blood cell receptor but lack other T cell markers (Lyt-3+, OKT3-). Natural killer activity correlated with the presence of a Mac 1-positive subpopulation of cells present in these long-term lines.

  View details for Web of Science ID A1982PH18100074

  View details for PubMedID 6286775

• PERITONITIS IN CHILDHOOD NEPHROTIC SYNDROME - 1970-1980 AMERICAN JOURNAL OF DISEASES OF CHILDREN KRENSKY, A. M., Ingelfinger, J. R., Grupe, W. E. 1982; 136 (8): 732-736

  Abstract

  A retrospective review (1970 through 1980) of 351 children with idiopathic nephrotic syndrome disclosed 24 episodes of peritonitis in 19 patients. Twenty-six percent of the patients had more than one episode. Streptococcus pneumoniae was the most common agent (50%), but Escherichia coli remained important (25%). Four cases (16%) were culture-negative. Signs of peritoneal irritation were present in all patients, including the 16 children receiving corticosteroid therapy. No morphological subtype of nephrotic syndrome could be demonstrated to be at increased risk for the development of peritonitis. Significantly decreased IgG levels and an apparent susceptibility to pneumococcal infection among blacks may be important risk factors.

  View details for Web of Science ID A1982PE33800022

  View details for PubMedID 7048898

• ELEVATED NEPHROGENOUS CYCLIC ADENOSINE-MONOPHOSPHATE TO MONITOR EARLY RENAL OSTEODYSTROPHY CLINICAL NEPHROLOGY KRENSKY, A. M., Harmon, W. E., Ingelfinger, J. R., Kirkpatrick, J. A., Grupe, W. E. 1981; 16 (5): 245-250

  Abstract

  Radiographs, serum chemistries, parathyroid hormone (PTH), and nephrogenous cyclic adenosine monophosphate (cAMP) were evaluated in thirty-two children with normal serum creatinine, chronic renal insufficiency, chronic hemodialysis, and transplantation. Nephrogenous cAMP increases linearly with creatinine, and there is a good correlation (r = 0.89) between immunoreactive PTH (iPTH) and nephrogenous cAMP except for patients with severe renal insufficiency or requiring chronic hemodialysis. Elevated nephrogenous cAMP is evidence for metabolic bone disease earlier than usually recognized. Early measurements of iPTH and nephrogenous cAMP could ensure early therapeutic intervention which might alleviate renal osteodystrophy in chronic renal insufficiency and transplant patients.

  View details for Web of Science ID A1981MR87900005

  View details for PubMedID 6273034

• HLA ANTIGENS IN MUCOCUTANEOUS LYMPH-NODE SYNDROME IN NEW-ENGLAND PEDIATRICS KRENSKY, A. M., BERENBERG, W., Shanley, K., YUNIS, E. J. 1981; 67 (5): 741-743

  Abstract

  HLA antigens were evaluated in 27 patients with mucocutaneous lymph node syndrome (Kawasaki disease) in the Boston area. In contrast to previous Japanese studies, no increase of HLA-Bw22 was found. A significant increase (P less than .002), however, in HLA-Bw51 was found in the patients with mucocutaneous lymph node syndrome as compared to an appropriate control population. To our knowledge, this is only the second disease associated with HLA-B5 specificity. The observations illustrate the lack of unified genetic predisposition for a such disease in two ethnic groups, white and Japanese. Such findings may have genetic implications regarding interhuman variation in immune responsiveness.

  View details for Web of Science ID A1981LP33300037

  View details for PubMedID 7255009

• MUCOCUTANEOUS LYMPH-NODE SYNDROME AND HYDROPIC GALLBLADDERS PEDIATRICS KRENSKY, A. M. 1980; 66 (5): 814-814

  View details for Web of Science ID A1980KQ99600039

  View details for PubMedID 7432898

• STREPTOCOCCAL ANTIGENICITY IN MUCOCUTANEOUS LYMPH-NODE SYNDROME AND HYDROPIC GALLBLADDERS PEDIATRICS KRENSKY, A. M., Teele, R., Watkins, J., Bates, J. 1979; 64 (6): 979-980

  View details for Web of Science ID A1979HX49700046

  View details for PubMedID 390491

• IDENTIFICATION OF HETEROZYGOTE CARRIERS OF CONGENITAL ADRENAL-HYPERPLASIA BY RADIOIMMUNOASSAY OF SERUM 17-OH PROGESTERONE JOURNAL OF PEDIATRICS KRENSKY, A. M., Bongiovanni, A. M., Marino, J., Parks, J., Tenore, A. 1977; 90 (6): 930-933

  Abstract

  The response to administered adrenocorticotropin (ACTH, Cortrosyn) of 26 heterozygotes (parents of children with adrenal 21-hydroxylase deficiency) and of 14 controls are compared. The mean plasma levels of 4-pregnene-3, 20-dione-17, 21-diol (17-OH progesterone) were significantly greater in the heterozygotes 60 minutes (p less than 0.02) and 90 minutes (p less than 0.05) after stimulation with Cortrosyn than in controls. There is, however, considerable overlap. The results would indicate a partial enzyme deficiency in the parents of diseased subjects. There was no significant difference in the response of plasma cortisol.

  View details for Web of Science ID A1977DH24800009

  View details for PubMedID 859065