Juliana Idoyaga

All Publications

• Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses. Nature immunology Sulczewski, F. B., Maqueda-Alfaro, R. A., Alcantara-Hernandez, M., Perez, O. A., Saravanan, S., Yun, T. J., Seong, D., Arroyo Hornero, R., Raquer-McKay, H. M., Esteva, E., Lanzar, Z. R., Leylek, R. A., Adams, N. M., Das, A., Rahman, A. H., Gottfried-Blackmore, A., Reizis, B., Idoyaga, J. 2023

  Abstract

  High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naive T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1beta secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.

  View details for DOI 10.1038/s41590-023-01545-7

  View details for PubMedID 37414907

• Rapid recruitment and IFN-I-mediated activation of monocytes dictate focal radiotherapy efficacy. Science immunology Tadepalli, S., Clements, D. R., Saravanan, S., Arroyo Hornero, R., Lüdtke, A., Blackmore, B., Paulo, J. A., Gottfried-Blackmore, A., Seong, D., Park, S., Chan, L., Kopecky, B. J., Liu, Z., Ginhoux, F., Lavine, K. J., Murphy, J. P., Mack, M., Graves, E. E., Idoyaga, J. 2023; 8 (84): eadd7446

  Abstract

  The recruitment of monocytes and their differentiation into immunosuppressive cells is associated with the low efficacy of preclinical nonconformal radiotherapy (RT) for tumors. However, nonconformal RT (non-CRT) does not mimic clinical practice, and little is known about the role of monocytes after RT modes used in patients, such as conformal RT (CRT). Here, we investigated the acute immune response induced by after CRT. Contrary to non-CRT approaches, we found that CRT induces a rapid and robust recruitment of monocytes to the tumor that minimally differentiate into tumor-associated macrophages or dendritic cells but instead up-regulate major histocompatibility complex II and costimulatory molecules. We found that these large numbers of infiltrating monocytes are responsible for activating effector polyfunctional CD8+ tumor-infiltrating lymphocytes that reduce tumor burden. Mechanistically, we show that monocyte-derived type I interferon is pivotal in promoting monocyte accumulation and immunostimulatory function in a positive feedback loop. We also demonstrate that monocyte accumulation in the tumor microenvironment is hindered when RT inadvertently affects healthy tissues, as occurs in non-CRT. Our results unravel the immunostimulatory function of monocytes during clinically relevant modes of RT and demonstrate that limiting the exposure of healthy tissues to radiation has a positive therapeutic effect on the overall antitumor immune response.

  View details for DOI 10.1126/sciimmunol.add7446

  View details for PubMedID 37294749

• Plasmacytoid dendritic cells: A dendritic cell in disguise. Molecular immunology Arroyo Hornero, R., Idoyaga, J. 2023; 159: 38-45

  Abstract

  Since their discovery, the identity of plasmacytoid dendritic cells (pDCs) has been at the center of a continuous dispute in the field, and their classification as dendritic cells (DCs) has been recently re-challenged. pDCs are different enough from the rest of the DC family members to be considered a lineage of cells on their own. Unlike the exclusive myeloid ontogeny of cDCs, pDCs may have dual origin developing from myeloid and lymphoid progenitors. Moreover, pDCs have the unique ability to quickly secrete abundant levels of type I interferon (IFN-I) in response to viral infections. In addition, pDCs undergo a differentiation process after pathogen recognition that allows them to activate T cells, a feature that has been shown to be independent of presumed contaminating cells. Here, we aim to provide an overview of the historic and current understanding of pDCs and argue that their classification as either lymphoid or myeloid may be an oversimplification. Instead, we propose that the capacity of pDCs to link the innate and adaptive immune response by directly sensing pathogens and activating adaptive immune responses justify their inclusion within the DC network.

  View details for DOI 10.1016/j.molimm.2023.05.007

  View details for PubMedID 37269733

• Reclassification of plasmacytoid dendritic cells as innate lymphocytes is premature. Nature reviews. Immunology Reizis, B., Idoyaga, J., Dalod, M., Barrat, F., Naik, S., Trinchieri, G., Tussiwand, R., Cella, M., Colonna, M. 2023

  View details for DOI 10.1038/s41577-023-00864-y

  View details for PubMedID 36959479

  View details for PubMedCentralID 2395256

• Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells. Immunity Feng, J., Pucella, J. N., Jang, G., Alcantara-Hernandez, M., Upadhaya, S., Adams, N. M., Khodadadi-Jamayran, A., Lau, C. M., Stoeckius, M., Hao, S., Smibert, P., Tsirigos, A., Idoyaga, J., Reizis, B. 2022

  Abstract

  Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.

  View details for DOI 10.1016/j.immuni.2022.01.016

  View details for PubMedID 35180378

• Alveolar macrophages and epithelial cells: The art of living together. The Journal of experimental medicine Clements, D., Idoyaga, J. 2021; 218 (10)

  Abstract

  In this issue of JEM, Gschwend et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20210745) reveal the indispensable role of alveolar epithelial cells type 2 in controlling the density of alveolar macrophages. This study highlights the intricate crosstalk that lung stroma and macrophages undergo to maintain homeostasis.

  View details for DOI 10.1084/jem.20211583

  View details for PubMedID 34491265

• Mass cytometry profiling of human dendritic cells in blood and tissues. Nature protocols Alcantara-Hernandez, M., Idoyaga, J. 2021

  Abstract

  The immune system comprises distinct functionally specialized cell populations, which can be characterized in depth by mass cytometry protein profiling. Unfortunately, the low-throughput nature of mass cytometry has made it challenging to analyze minor cell populations. This is the case for dendritic cells, which represent 0.2-2% of all immune cells in tissues and yet perform the critical task of initiating and modulating immune responses. Here, we provide an optimized step-by-step protocol for the characterization of well-known and emerging human dendritic cell populations in blood and tissues using mass cytometry. We provide detailed instructions for the generation of single-cell suspensions, sample enrichment, staining, acquisition and data analysis. We also include a barcoding option that reduces acquisition variability and allows the analysis of low numbers of dendritic cells, i.e., ~20,000. In contrast to other protocols, we emphasize the use of negative selection approaches to enrich for minor populations of immune cells while avoiding their activation. The entire procedure can be completed in 2-3 d and can be conveniently paused at several stages. The procedure described in this robust and reliable protocol allows the analysis of human dendritic cells in health and disease and during vaccination.

  View details for DOI 10.1038/s41596-021-00599-x

  View details for PubMedID 34480131

• Chromatin Landscape Underpinning Human Dendritic Cell Heterogeneity. Cell reports Leylek, R. n., Alcántara-Hernández, M. n., Granja, J. M., Chavez, M. n., Perez, K. n., Diaz, O. R., Li, R. n., Satpathy, A. T., Chang, H. Y., Idoyaga, J. n. 2020; 32 (12): 108180

  Abstract

  Human dendritic cells (DCs) comprise subsets with distinct phenotypic and functional characteristics, but the transcriptional programs that dictate their identity remain elusive. Here, we analyze global chromatin accessibility profiles across resting and stimulated human DC subsets by means of the assay for transposase-accessible chromatin using sequencing (ATAC-seq). We uncover specific regions of chromatin accessibility for each subset and transcriptional regulators of DC function. By comparing plasmacytoid DC responses to IFN-I-producing and non-IFN-I-producing conditions, we identify genetic programs related to their function. Finally, by intersecting chromatin accessibility with genome-wide association studies, we recognize DC subset-specific enrichment of heritability in autoimmune diseases. Our results unravel the basis of human DC subset heterogeneity and provide a framework for their analysis in disease pathogenesis.

  View details for DOI 10.1016/j.celrep.2020.108180

  View details for PubMedID 32966789

• The versatile plasmacytoid dendritic cell: Function, heterogeneity, and plasticity. International review of cell and molecular biology Leylek, R. n., Idoyaga, J. n. 2019; 349: 177–211

  Abstract

  Since their identification as the natural interferon-producing cell two decades ago, plasmacytoid dendritic cells (pDCs) have been attributed diverse functions in the immune response. Their most well characterized function is innate, i.e., their rapid and robust production of type-I interferon (IFN-I) in response to viruses. However, pDCs have also been implicated in antigen presentation, activation of adaptive immune responses and immunoregulation. The mechanisms by which pDCs enact these diverse functions are poorly understood. One central debate is whether these functions are carried out by different pDC subpopulations or by plasticity in the pDC compartment. This chapter summarizes the latest reports regarding pDC function, heterogeneity, cell conversion and environmentally influenced plasticity, as well as the role of pDCs in infection, autoimmunity and cancer.

  View details for DOI 10.1016/bs.ircmb.2019.10.002

  View details for PubMedID 31759431

• Integrated Cross-Species Analysis Identifies a Conserved Transitional Dendritic Cell Population. Cell reports Leylek, R. n., Alcántara-Hernández, M. n., Lanzar, Z. n., Lüdtke, A. n., Perez, O. A., Reizis, B. n., Idoyaga, J. n. 2019; 29 (11): 3736–50.e8

  Abstract

  Plasmacytoid dendritic cells (pDCs) are sensor cells with diverse immune functions, from type I interferon (IFN-I) production to antigen presentation, T cell activation, and tolerance. Regulation of these functions remains poorly understood but could be mediated by functionally specialized pDC subpopulations. We address pDC diversity using a high-dimensional single-cell approach: mass cytometry (CyTOF). Our analysis uncovers a murine pDC-like population that specializes in antigen presentation with limited capacity for IFN-I production. Using a multifaceted cross-species comparison, we show that this pDC-like population is the definitive murine equivalent of the recently described human AXL+ DCs, which we unify under the name transitional DCs (tDCs) given their continuum of pDC and cDC2 characteristics. tDCs share developmental traits with pDCs, as well as recruitment dynamics during viral infection. Altogether, we provide a framework for deciphering the function of pDCs and tDCs during diseases, which has the potential to open new avenues for therapeutic design.

  View details for DOI 10.1016/j.celrep.2019.11.042

  View details for PubMedID 31825848

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

  Abstract

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

  View details for PubMedID 29221729

• CDKN1A regulates Langerhans cell survival and promotes Treg cell generation upon exposure to ionizing irradiation. Nature neuroscience Price, J. G., Idoyaga, J., Salmon, H., Hogstad, B., Bigarella, C. L., Ghaffari, S., Leboeuf, M., Merad, M. 2015; 16 (10): 1060-1068

  Abstract

  Treatment with ionizing radiation (IR) can lead to the accumulation of tumor-infiltrating regulatory T cells (Treg cells) and subsequent resistance of tumors to radiotherapy. Here we focused on the contribution of the epidermal mononuclear phagocytes Langerhans cells (LCs) to this phenomenon because of their ability to resist depletion by high-dose IR. We found that LCs resisted apoptosis and rapidly repaired DNA damage after exposure to IR. In particular, we found that the cyclin-dependent kinase inhibitor CDKN1A (p21) was overexpressed in LCs and that Cdkn1a(-/-) LCs underwent apoptosis and accumulated DNA damage following IR treatment. Wild-type LCs upregulated major histocompatibility complex class II molecules, migrated to the draining lymph nodes and induced an increase in Treg cell numbers upon exposure to IR, but Cdkn1a(-/-) LCs did not. Our findings suggest a means for manipulating the resistance of LCs to IR to enhance the response of cutaneous tumors to radiotherapy.

  View details for DOI 10.1038/ni.3270

  View details for PubMedID 26343536

• Induction of innate and adaptive immunity by delivery of poly dA:dT to dendritic cells NATURE CHEMICAL BIOLOGY Barbuto, S., Idoyaga, J., Vila-Perello, M., Longhi, M. P., Breton, G., Steinman, R. M., Muir, T. W. 2013; 9 (4): 250-256

  Abstract

  Targeted delivery of antigens to dendritic cells (DCs) is a promising vaccination strategy. However, to ensure immunity, the approach depends on coadministration of an adjuvant. Here we ask whether targeting of both adjuvant and antigen to DCs is sufficient to induce immunity. Using a protein ligation method, we develop a general approach for linking the immune stimulant, poly dA:dT (pdA:dT), to a monoclonal antibody (mAb) specific for DEC205 (DEC). We show that DEC-specific mAbs deliver pdA:dT to DCs for the efficient production of type I interferon in human monocyte-derived DCs and in mice. Notably, adaptive T-cell immunity is elicited when mAbs specific for DEC-pdA:dT are used as the activation stimuli and are administered together with a DC-targeted antigen. Collectively, our studies indicate that DCs can integrate innate and adaptive immunity in vivo and suggest that dual delivery of antigen and adjuvant to DCs might be an efficient approach to vaccine development.

  View details for DOI 10.1038/NCHEMBIO.1186

  View details for Web of Science ID 000317025800015

  View details for PubMedID 23416331

• Specialized role of migratory dendritic cells in peripheral tolerance induction JOURNAL OF CLINICAL INVESTIGATION Idoyaga, J., Fiorese, C., Zbytnuik, L., Lubkin, A., Miller, J., Malissen, B., Mucida, D., Merad, M., Steinman, R. M. 2013; 123 (2): 844-854

  Abstract

  Harnessing DCs for immunotherapies in vivo requires the elucidation of the physiological role of distinct DC populations. Migratory DCs traffic from peripheral tissues to draining lymph nodes charged with tissue self antigens. We hypothesized that these DC populations have a specialized role in the maintenance of peripheral tolerance, specifically, to generate suppressive Foxp3+ Tregs. To examine the differential capacity of migratory DCs versus blood-derived lymphoid-resident DCs for Treg generation in vivo, we targeted a self antigen, myelin oligodendrocyte glycoprotein, using antibodies against cell surface receptors differentially expressed in these DC populations. Using this approach together with mouse models that lack specific DC populations, we found that migratory DCs have a superior ability to generate Tregs in vivo, which in turn drastically improve the outcome of experimental autoimmune encephalomyelitis. These results provide a rationale for the development of novel therapies targeting migratory DCs for the treatment of autoimmune diseases.

  View details for DOI 10.1172/JCI65260

  View details for Web of Science ID 000314553600037

  View details for PubMedID 23298832

• SnapShot: Dendritic Cells CELL Idoyaga, J., Steinman, R. M. 2011; 146 (4): 660-U186

  View details for DOI 10.1016/j.cell.2011.08.010

  View details for Web of Science ID 000294043600020

  View details for PubMedID 21854989

• Comparable T helper 1 (Th1) and CD8 T-cell immunity by targeting HIV gag p24 to CD8 dendritic cells within antibodies to Langerin, DEC205, and Clec9A PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Idoyaga, J., Lubkin, A., Fiorese, C., Lahoud, M. H., Caminschi, I., Huang, Y., Rodriguez, A., Clausen, B. E., Park, C. G., Trumpfheller, C., Steinman, R. M. 2011; 108 (6): 2384-2389

  Abstract

  Improved protein-based vaccines should facilitate the goal of effective vaccines against HIV and other pathogens. With respect to T cells, the efficiency of immunization, or "immunogenicity," is improved by targeting vaccine proteins to maturing dendritic cells (DCs) within mAbs to DC receptors. Here, we compared the capacity of Langerin/CD207, DEC205/CD205, and Clec9A receptors, each expressed on the CD8(+) DC subset in mice, to bring about immunization of microbial-specific T cells from the polyclonal repertoire, using HIV gag-p24 protein as an antigen. α-Langerin mAb targeted splenic CD8(+) DCs selectively in vivo, whereas α-DEC205 and α-Clec9A mAbs targeted additional cell types. When the mAb heavy chains were engineered to express gag-p24, the α-Langerin, α-DEC205, and α-Clec9A fusion mAbs given along with a maturation stimulus induced comparable levels of gag-specific T helper 1 (Th1) and CD8(+) T cells in BALB/c × C57BL/6 F1 mice. These immune T cells were more numerous than targeting the CD8(-) DC subset with α-DCIR2-gag-p24. In an in vivo assay in which gag-primed T cells were used to report the early stages of T-cell responses, α-Langerin, α-DEC205, and α-Clec9A also mediated cross-presentation to primed CD8(+) T cells if, in parallel to antigen uptake, the DCs were stimulated with α-CD40. α-Langerin, α-DEC205, and α-Clec9A targeting greatly enhanced T-cell immunization relative to nonbinding control mAb or nontargeted HIV gag-p24 protein. Therefore, when the appropriate subset of DCs is targeted with a vaccine protein, several different receptors expressed by that subset are able to initiate combined Th1 and CD8(+) immunity.

  View details for DOI 10.1073/pnas.1019547108

  View details for Web of Science ID 000287084500041

  View details for PubMedID 21262813

  View details for PubMedCentralID PMC3038758

• Features of the dendritic cell lineage IMMUNOLOGICAL REVIEWS Steinman, R. M., Idoyaga, J. 2010; 234: 5-17

  View details for Web of Science ID 000274813200001

  View details for PubMedID 20193008

• Antibody to Langerin/CD207 localizes large numbers of CD8 alpha(+) dendritic cells to the marginal zone of mouse spleen PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Idoyaga, J., Suda, N., Suda, K., Park, C. G., Steinman, R. M. 2009; 106 (5): 1524-1529

  Abstract

  Dendritic cells (DCs) are strategically positioned to take up antigens and initiate adaptive immunity. One DC subset expresses CD8alphaalpha in mice and is specialized to capture dying cells and process antigens for MHC class I "cross-presentation." Because CD8(+) DCs also express DEC205/CD205, which is localized to splenic T cell regions, it is thought that CD8(+) DCs also are restricted to T zones. Here, we used a new antibody to Langerin/CD207, which colabels isolated CD8(+) CD205(+) DCs, to immunolabel spleen sections. The mAb labeled discrete cells with high levels of CD11c and CD8. Surprisingly most CD207(+) profiles were in marginal zones surrounding splenic white pulp nodules, and only smaller numbers were in T cell areas, where CD205 colabeling was noted. Despite a marginal zone location, CD207(+) DCs lacked identifying molecules for 3 different types of macrophages, localized in proximity and, in contrast to macrophages, marginal zone DCs were poor scavengers of soluble and particulate substrates. After stimulation with microbial agonists, Langerin expression disappeared from the marginal zone at 6-12 h, but was greatly expanded in the T cell areas, and by 24-48 h, Langerin expression disappeared. Therefore, anti-Langerin antibodies localize a majority of CD8(+) DCs to non-T cell regions of mouse spleen, where they are distinct from adjacent macrophages.

  View details for DOI 10.1073/pnas.0812247106

  View details for Web of Science ID 000263074600044

  View details for PubMedID 19168629

• Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression. Nature methods Zengel, J., Wang, Y. X., Seo, J. W., Ning, K., Hamilton, J. N., Wu, B., Raie, M., Holbrook, C., Su, S., Clements, D. R., Pillay, S., Puschnik, A. S., Winslow, M. M., Idoyaga, J., Nagamine, C. M., Sun, Y., Mahajan, V. B., Ferrara, K. W., Blau, H. M., Carette, J. E. 2023

  Abstract

  The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

  View details for DOI 10.1038/s41592-023-01896-x

  View details for PubMedID 37291262

  View details for PubMedCentralID 3337962

• Invivo bioluminescence imaging of granzyme B activity in tumor response to cancer immunotherapy. Cell chemical biology Chen, M., Zhou, K., Dai, S., Tadepalli, S., Balakrishnan, P. B., Xie, J., Rami, F. E., Dai, T., Cui, L., Idoyaga, J., Rao, J. 2022

  Abstract

  Cancer immunotherapy has revolutionized the treatment of cancer, but only a small subset of patients benefits from this new treatment regime. Imaging tools are useful for early detection of tumor response to immunotherapy and probing the dynamic and complex immune system. Here, we report a bioluminescence probe (GBLI-2) for non-invasive, real-time, longitudinal imaging of granzyme B activity in tumors receiving immune checkpoint inhibitors. GBLI-2 is made of the mouse granzyme B tetrapeptide IEFD substrate conjugated to D-luciferin through a self-immolative group. GBLI-2 was evaluated for imaging the dynamics of the granzyme B activity and predicting therapeutic efficacy in a syngeneic mouse model of CT26 murine colorectal carcinoma. The GBLI-2 signal correlated with the change in the population of PD-1- and granzyme B-expressing CD8+ Tcells in tumors.

  View details for DOI 10.1016/j.chembiol.2022.08.006

  View details for PubMedID 36103874

• Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies. Advanced science (Weinheim, Baden-Wurttemberg, Germany) Correa, S., Meany, E. L., Gale, E. C., Klich, J. H., Saouaf, O. M., Mayer, A. T., Xiao, Z., Liong, C. S., Brown, R. A., Maikawa, C. L., Grosskopf, A. K., Mann, J. L., Idoyaga, J., Appel, E. A. 2022: e2103677

  Abstract

  When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, this study explored the ability of a slow-releasing injectable hydrogel depot to reduce dose-limiting toxicities of immunostimulatory CD40agonist (CD40a) while maintaining its potent anticancer efficacy. A previously described polymer-nanoparticle (PNP) hydrogel system is leveraged that exhibits shear-thinning and yield-stress properties that are hypothesized to improve locoregional delivery of CD40a immunotherapy. Using positron emission tomography, it is demonstrated that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

  View details for DOI 10.1002/advs.202103677

  View details for PubMedID 35975424

• The Tabula Sapiens: A multiple-organ, single-cell transcriptomic atlas of humans. Science (New York, N.Y.) Jones, R. C., Karkanias, J., Krasnow, M. A., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Pisco, A. O., Tan, S. Y., Travaglini, K. J., Xu, C., Alcántara-Hernández, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Crasta, S., Culver, R. N., Cvijović, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kolluru, S., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W. J., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Morri, M., Mrouj, K., Mukherjee, S., Muser, T., Neuhöfer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Pisco, A. O., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L. C., Subramaniam, V. R., Swarup, A., Swift, M., Travaglini, K. J., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgó, E., Martin, B. A., Ozawa, M. G., Silva, O., Tan, S. Y., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Detweiler, A. M., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., de Morree, A., Olivieri, J. E., Park, M., Pisco, A. O., Ravikumar, N., Salzman, J., Stanley, G., Swift, M., Tan, M., Tan, W., Tarashansky, A. J., Vanheusden, R., Vorperian, S. K., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Alcántara-Hernández, M., Antony, J., Chan, C. K., Chang, C. A., Colville, A., Crasta, S., Culver, R., Dethlefsen, L., Ezran, C., Gillich, A., Hang, Y., Ho, P. Y., Irwin, J. C., Jang, S., Kershner, A. M., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Liu, S., Loeb, G. B., Lu, W. J., Maltzman, J. S., Metzger, R. J., de Morree, A., Neuhöfer, P., Perez, K., Phansalkar, R., Qi, Z., Rao, P., Raquer-McKay, H., Sasagawa, K., Scott, B., Sinha, R., Song, H., Spencer, S. P., Swarup, A., Swift, M., Travaglini, K. J., Trimm, E., Veizades, S., Vijayakumar, S., Wang, B., Wang, W., Winkler, J., Xie, J., Yung, A. R., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Krasnow, M., Kuo, C. S., Nguyen, P., Quake, S. R., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wang, B., Wu, A., Wu, S. M., Wyss-Coray, T. 2022; 376 (6594): eabl4896

  Abstract

  Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

  View details for DOI 10.1126/science.abl4896

  View details for PubMedID 35549404

• Publisher Correction: Cell types of origin of the cell-free transcriptome. Nature biotechnology Vorperian, S. K., Moufarrej, M. N., Tabula Sapiens Consortium, Quake, S. R., Jones, R. C., Karkanias, J., Krasnow, M., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Tan, S. Y., Travaglini, K. J., Xu, C., Alcantara-Hernandez, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Culver, R. N., Cvijovic, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Mrouj, K., Mukherjee, S., Muser, T., Neuhofer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L., Subramaniam, V. R., Swarup, A., Swift, M., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgo, E., Martin, B. A., Ozawa, M. G., Silva, O., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Bierman, R., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., Olivieri, J. E., Park, M., Ravikumar, N., Stanley, G., Tan, W., Tarashansky, A. J., Vanheusden, R., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Culver, R., Dethlefsen, L., Ho, P., Liu, S., Maltzman, J. S., Metzger, R. J., Sasagawa, K., Sinha, R., Song, H., Wang, B., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Kuo, C. S., Nguyen, P., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wu, A., Wu, S. M., Wyss-Coray, T. 2022

  View details for DOI 10.1038/s41587-022-01293-3

  View details for PubMedID 35347330

• Cell types of origin of the cell-free transcriptome. Nature biotechnology Vorperian, S. K., Moufarrej, M. N., Tabula Sapiens Consortium, Quake, S. R., Jones, R. C., Karkanias, J., Krasnow, M., Pisco, A. O., Quake, S. R., Salzman, J., Yosef, N., Bulthaup, B., Brown, P., Harper, W., Hemenez, M., Ponnusamy, R., Salehi, A., Sanagavarapu, B. A., Spallino, E., Aaron, K. A., Concepcion, W., Gardner, J. M., Kelly, B., Neidlinger, N., Wang, Z., Crasta, S., Kolluru, S., Morri, M., Tan, S. Y., Travaglini, K. J., Xu, C., Alcantara-Hernandez, M., Almanzar, N., Antony, J., Beyersdorf, B., Burhan, D., Calcuttawala, K., Carter, M. M., Chan, C. K., Chang, C. A., Chang, S., Colville, A., Culver, R. N., Cvijovic, I., D'Amato, G., Ezran, C., Galdos, F. X., Gillich, A., Goodyer, W. R., Hang, Y., Hayashi, A., Houshdaran, S., Huang, X., Irwin, J. C., Jang, S., Juanico, J. V., Kershner, A. M., Kim, S., Kiss, B., Kong, W., Kumar, M. E., Kuo, A. H., Leylek, R., Li, B., Loeb, G. B., Lu, W., Mantri, S., Markovic, M., McAlpine, P. L., de Morree, A., Mrouj, K., Mukherjee, S., Muser, T., Neuhofer, P., Nguyen, T. D., Perez, K., Phansalkar, R., Puluca, N., Qi, Z., Rao, P., Raquer-McKay, H., Schaum, N., Scott, B., Seddighzadeh, B., Segal, J., Sen, S., Sikandar, S., Spencer, S. P., Steffes, L., Subramaniam, V. R., Swarup, A., Swift, M., Van Treuren, W., Trimm, E., Veizades, S., Vijayakumar, S., Vo, K. C., Vorperian, S. K., Wang, W., Weinstein, H. N., Winkler, J., Wu, T. T., Xie, J., Yung, A. R., Zhang, Y., Detweiler, A. M., Mekonen, H., Neff, N. F., Sit, R. V., Tan, M., Yan, J., Bean, G. R., Charu, V., Forgo, E., Martin, B. A., Ozawa, M. G., Silva, O., Toland, A., Vemuri, V. N., Afik, S., Awayan, K., Bierman, R., Botvinnik, O. B., Byrne, A., Chen, M., Dehghannasiri, R., Gayoso, A., Granados, A. A., Li, Q., Mahmoudabadi, G., McGeever, A., Olivieri, J. E., Park, M., Ravikumar, N., Stanley, G., Tan, W., Tarashansky, A. J., Vanheusden, R., Wang, P., Wang, S., Xing, G., Xu, C., Yosef, N., Culver, R., Dethlefsen, L., Ho, P., Liu, S., Maltzman, J. S., Metzger, R. J., Sasagawa, K., Sinha, R., Song, H., Wang, B., Artandi, S. E., Beachy, P. A., Clarke, M. F., Giudice, L. C., Huang, F. W., Huang, K. C., Idoyaga, J., Kim, S. K., Kuo, C. S., Nguyen, P., Rando, T. A., Red-Horse, K., Reiter, J., Relman, D. A., Sonnenburg, J. L., Wu, A., Wu, S. M., Wyss-Coray, T. 2022

  Abstract

  Cell-free RNA from liquid biopsies can be analyzed to determine disease tissue of origin. We extend this concept to identify cell types of origin using the Tabula Sapiens transcriptomic cell atlas as well as individual tissue transcriptomic cell atlases in combination with the Human Protein Atlas RNA consensus dataset. We define cell type signature scores, which allow the inference of cell types that contribute to cell-free RNA for a variety of diseases.

  View details for DOI 10.1038/s41587-021-01188-9

  View details for PubMedID 35132263

• RNA splicing programs define tissue compartments and cell types at single-cell resolution ELIFE Olivieri, J., Dehghannasiri, R., Wang, P. L., Jang, S., de Morree, A., Tan, S. Y., Ming, J., Wu, A., Consortium, T., Quake, S. R., Krasnow, M. A., Salzman, J. 2021; 10

  View details for DOI 10.7554/eLife.70692.sa2

  View details for Web of Science ID 000715795700001

• Prolonged Codelivery of Hemagglutinin and a TLR7/8 Agonist in a Supramolecular Polymer-Nanoparticle Hydrogel Enhances Potency and Breadth of Influenza Vaccination. ACS biomaterials science & engineering Roth, G. A., Saouaf, O. M., Smith, A. A., Gale, E. C., Hernandez, M. A., Idoyaga, J., Appel, E. A. 2021

  Abstract

  The sustained release of vaccine cargo has been shown to improve humoral immune responses to challenging pathogens such as influenza. Extended codelivery of antigen and adjuvant prolongs germinal center reactions, thus improving antibody affinity maturation and the ability to neutralize the target pathogen. Here, we develop an injectable, physically cross-linked polymer-nanoparticle (PNP) hydrogel system to prolong the local codelivery of hemagglutinin and a toll-like receptor 7/8 agonist (TLR7/8a) adjuvant. By tethering the TLR7/8a to a NP motif within the hydrogels (TLR7/8a-NP), the dynamic mesh of the PNP hydrogels enables codiffusion of the adjuvant and protein antigen (hemagglutinin), therefore enabling sustained codelivery of these two physicochemically distinct molecules. We show that subcutaneous delivery of PNP hydrogels carrying hemagglutinin and TLR7/8a-NP in mice improves the magnitude and duration of antibody titers in response to a single injection vaccination compared to clinically used adjuvants. Furthermore, the PNP gel-based slow delivery of influenza vaccines led to increased breadth of antibody responses against future influenza variants, including a future pandemic variant, compared to clinical adjuvants. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

  View details for DOI 10.1021/acsbiomaterials.0c01496

  View details for PubMedID 33404236

• Skin dendritic cells in melanoma are key for successful checkpoint blockade therapy. Journal for immunotherapy of cancer Prokopi, A., Tripp, C. H., Tummers, B., Hornsteiner, F., Spoeck, S., Crawford, J. C., Clements, D. R., Efremova, M., Hutter, K., Bellmann, L., Cappellano, G., Cadilha, B. L., Kobold, S., Boon, L., Ortner, D., Trajanoski, Z., Chen, S., de Gruijl, T. D., Idoyaga, J., Green, D. R., Stoitzner, P. 2021; 9 (1)

  Abstract

  BACKGROUND: Immunotherapy with checkpoint inhibitors has shown impressive results in patients with melanoma, but still many do not benefit from this line of treatment. A lack of tumor-infiltrating T cells is a common reason for therapy failure but also a loss of intratumoral dendritic cells (DCs) has been described.METHODS: We used the transgenic tg(Grm1)EPv melanoma mouse strain that develops spontaneous, slow-growing tumors to perform immunological analysis during tumor progression. With flow cytometry, the frequencies of DCs and T cells at different tumor stages and the expression of the inhibitory molecules programmed cell death protein-1 (PD-1) and T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells were analyzed. This was complemented with RNA-sequencing (RNA-seq) and real-time quantitative PCR (RT-qPCR) analysis to investigate the immune status of the tumors. To boost DC numbers and function, we administered Fms-related tyrosine 3 ligand (Flt3L) plus an adjuvant mix of polyI:C and anti-CD40. To enhance T cell function, we tested several checkpoint blockade antibodies. Immunological alterations were characterized in tumor and tumor-draining lymph nodes (LNs) by flow cytometry, CyTOF, microarray and RT-qPCR to understand how immune cells can control tumor growth. The specific role of migratory skin DCs was investigated by coculture of sorted DC subsets with melanoma-specific CD8+ T cells.RESULTS: Our study revealed that tumor progression is characterized by upregulation of checkpoint molecules and a gradual loss of the dermal conventional DC (cDC) 2 subset. Monotherapy with checkpoint blockade could not restore antitumor immunity, whereas boosting DC numbers and activation increased tumor immunogenicity. This was reflected by higher numbers of activated cDC1 and cDC2 as well as CD4+ andCD8+ T cells in treated tumors. At the same time, the DC boost approach reinforced migratory dermal DC subsets to prime gp100-specific CD8+ T cells in tumor-draining LNs that expressed PD-1/TIM-3 and produced interferon gamma (IFNgamma)/tumor necrosis factor alpha (TNFalpha). As a consequence, the combination of the DC boost with antibodies against PD-1 and TIM-3 released the brake from T cells, leading to improved function within the tumors and delayed tumor growth.CONCLUSIONS: Our results set forth the importance of skin DC in cancer immunotherapy, and demonstrates that restoring DC function is key to enhancing tumor immunogenicity and subsequently responsiveness to checkpoint blockade therapy.

  View details for DOI 10.1136/jitc-2020-000832

  View details for PubMedID 33408092

• Gastric Mucosal Immune Profiling and Dysregulation in Idiopathic Gastroparesis. Clinical and translational gastroenterology Gottfried-Blackmore, A. n., Namkoong, H. n., Adler, E. n., Martin, B. n., Gubatan, J. n., Fernandez-Becker, N. n., Clarke, J. O., Idoyaga, J. n., Nguyen, L. n., Habtezion, A. n. 2021; 12 (5): e00349

  Abstract

  It is unclear how immune perturbations may influence the pathogenesis of idiopathic gastroparesis, a prevalent functional disorder of the stomach which lacks animal models. Several studies have noted altered immune characteristics in the deep gastric muscle layer associated with gastroparesis, but data are lacking for the mucosal layer, which is endoscopically accessible. We hypothesized that immune dysregulation is present in the gastroduodenal mucosa in idiopathic gastroparesis and that specific immune profiles are associated with gastroparesis clinical parameters.In this cross-sectional prospective case-control study, routine endoscopic biopsies were used for comprehensive immune profiling by flow cytometry, multicytokine array, and gene expression in 3 segments of the stomach and the duodenal bulb. Associations of immune endpoints with clinical parameters of gastroparesis were also explored.The gastric mucosa displayed large regional variation of distinct immune profiles. Furthermore, several-fold increases in innate and adaptive immune cells were found in gastroparesis. Various immune cell types showed positive correlations with duration of disease, proton pump inhibitor dosing, and delayed gastric emptying.This initial observational study showed immune compartmentalization of the human stomach mucosa and significant immune dysregulation at the level of leukocyte infiltration in idiopathic gastroparesis patients that extends to the duodenum. Select immune cells, such as macrophages, may correlate with clinicopathological traits of gastroparesis. This work supports further mucosal studies to advance our understanding of gastroparesis pathophysiology.

  View details for DOI 10.14309/ctg.0000000000000349

  View details for PubMedID 33979305

• ImmGen at 15 NATURE IMMUNOLOGY Aguilar, S., Aguilar, O., Allan, R., Amir, E., Angeli, V., Artyomov, M. N., Asinovski, N., Astarita, J., Austen, K., Bajpai, G., Barrett, N., Baysoy, A., Benoist, C., Bellemare-Pelletier, A., Berg, B., Best, A., Bezman, N., Blair, D., Blander, J. M., Bogunovic, M., Brennan, P., Brenner, M., Brown, B., Buechler, M., Buenrostro, J., Casanova, M., Choi, K., Chow, A., Chudnovskiy, A., Cipoletta, D., Cohen, N., Collins, J. J., Colonna, M., Cook, A., Costello, J., Cremasco, V., Crowl, T., Crozat, K., Cruse, R., D'Angelo, J., Dalod, M., Davis, S., Demiralp, C., Deng, T., Desai, J., Desland, F., Dhainaut, M., Ding, J., Doedens, A., Dominguez, C., Doran, G., Dress, R., Dustin, M., Dwyer, D., Dzhagalov, I., Elpek, K., Ergun, A., Ericson, J., Esomonu, E., Fairfax, K., Fletcher, A., Frascoli, M., Fuchs, A., Gainullina, A., Gal-Oz, S., Gallagher, M., Gautier, E., Gazit, R., Gibbings, S., Giraud, M., Ginhoux, F., Goldrath, A., Gotthardt, D., Gray, D., Greter, M., Grieshaber-Bouyer, R., Guilliams, M., Haidermota, S., Hardy, R., Hashimoto, D., Helft, J., Hendricks, D., Heng, T., Hill, J., Hyatt, G., Idoyaga, J., Jakubzick, C., Jarjoura, J., Jepson, D., Jia, B., Jianu, R., Johanson, T., Jordan, S., Jojic, V., Jordan, S., Kamimura, Y., Kana, V., Kang, J., Kapoor, V., Kenigsberg, E., Kent, A., Kim, C., Kim, E., Kim, F., Kim, J., Kim, K., Kiner, E., Knell, J., Koller, D., Kozinn, L., Krchma, K., Kreslavsky, T., Kronenberg, M., Kwan, W., Laidlaw, D., Lam, V., Lanier, L., Laplace, C., Lareau, C., Lavin, Y., Lavine, K. J., Leader, A., Leboeuf, M., Lee, J., Lee, J., Li, B., Li, H., Li, Y., Lionakis, M. S., Luche, H., Lynch, L., Magen, A., Maier, B., Malhotra, D., Malhotra, N., Malissen, M., Maslova, A., Mathis, D., McFarland, A., Merad, M., Meunier, E., Miller, J., Milner, J., Mingueneau, M., Min-Oo, G., Monach, P., Moodley, D., Mortha, A., Morvan, M., Mostafavi, S., Muller, S., Muus, C., Nabekura, T., Rao, T., Narang, V., Narayan, K., Ner-Gaon, H., Nguyen, Q., Nigrovic, P. A., Novakovsky, G., Nutt, S., Omilusik, K., Ortiz-Lopez, A., Paidassi, H., Paik, H., Painter, M., Paynich, M., Peng, V., Potempa, M., Pradhan, R., Price, J., Qi, Y., Qi, Y., Quon, S., Ramirez, R., Ramanan, D., Randolph, G., Regev, A., Rhoads, A., Robinette, M., Rose, S., Rossi, D., Rothamel, K., Sachidanandam, R., Sathe, P., Scott, C., Seddu, K., See, P., Sergushichev, A., Shaw, L., Shay, T., Shemesh, A., Shinton, S., Shyer, J., Sieweke, M., Smillie, C., Spel, L., Spidale, N., Stifano, G., Subramanian, A., Sun, J., Sylvia, K., Tellier, J., This, S., Tomasello, E., Todorov, H., Turley, S., Vijaykumar, B., Wagers, A., Wakamatsu, E., Wang, C., Wang, P. L., Wroblewska, A., Wu, J., Yang, E., Yang, L., Yim, A., Yng, L., Yoshida, H., Yu, B., Zhou, Y., Zhu, Y., Ziemkiewicz, C., Immunological Genome Project 2020; 21 (7): 700–703

  View details for DOI 10.1038/s41590-020-0687-4

  View details for Web of Science ID 000542863800003

  View details for PubMedID 32577013

• Landscape of coordinated immune responses to H1N1 challenge in humans. The Journal of clinical investigation Rahil, Z. n., Leylek, R. n., Schürch, C. M., Chen, H. n., Bjornson-Hooper, Z. n., Christensen, S. R., Gherardini, P. F., Bhate, S. S., Spitzer, M. H., Fragiadakis, G. K., Mukherjee, N. n., Kim, N. n., Jiang, S. n., Yo, J. n., Gaudilliere, B. n., Affrime, M. n., Bock, B. n., Hensley, S. E., Idoyaga, J. n., Aghaeepour, N. n., Kim, K. n., Nolan, G. P., McIlwain, D. R. 2020

  Abstract

  Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.

  View details for DOI 10.1172/JCI137265

  View details for PubMedID 33044226

• Injectable Hydrogels for Sustained Codelivery of Subunit Vaccines Enhance Humoral Immunity. ACS central science Roth, G. A., Gale, E. C., Alcántara-Hernández, M. n., Luo, W. n., Axpe, E. n., Verma, R. n., Yin, Q. n., Yu, A. C., Lopez Hernandez, H. n., Maikawa, C. L., Smith, A. A., Davis, M. M., Pulendran, B. n., Idoyaga, J. n., Appel, E. A. 2020; 6 (10): 1800–1812

  Abstract

  Vaccines aim to elicit a robust, yet targeted, immune response. Failure of a vaccine to elicit such a response arises in part from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the codelivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique delivery characteristics, whereby physicochemically distinct compounds (such as antigen and adjuvant) could be codelivered over the course of weeks. When administered in mice, hydrogel-based sustained vaccine exposure enhanced the magnitude, duration, and quality of the humoral immune response compared to standard PBS bolus administration of the same model vaccine. We report that the creation of a local inflammatory niche within the hydrogel, coupled with sustained exposure of vaccine cargo, enhanced the magnitude and duration of germinal center responses in the lymph nodes. This strengthened germinal center response promoted greater antibody affinity maturation, resulting in a more than 1000-fold increase in antigen-specific antibody affinity in comparison to bolus immunization. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of subunit vaccines.

  View details for DOI 10.1021/acscentsci.0c00732

  View details for PubMedID 33145416

  View details for PubMedCentralID PMC7596866

• A Nanoparticle Platform for Improved Potency, Stability, and Adjuvanticity of Poly(I:C) ADVANCED THERAPEUTICS Gale, E. C., Roth, G. A., Smith, A. A., Alcantara-Hernandez, M., Idoyaga, J., Appel, E. A. 2019

  View details for DOI 10.1002/adtp.201900174

  View details for Web of Science ID 000502045100001

• Nursing Markedly Protects Postpartum Mice From Stroke: Associated Central and Peripheral Neuroimmune Changes and a Role for Oxytocin FRONTIERS IN NEUROSCIENCE Stary, C. M., Xu, L., Voloboueva, L. A., Alcantara-Hernandez, M., Arvola, O. J., Idoyaga, J., Giffard, R. G. 2019; 13

  View details for DOI 10.3389/fnins.2019.00609

  View details for Web of Science ID 000474791200001

• Nursing Markedly Protects Postpartum Mice From Stroke: Associated Central and Peripheral Neuroimmune Changes and a Role for Oxytocin. Frontiers in neuroscience Stary, C. M., Xu, L., Voloboueva, L. A., Alcántara-Hernández, M., Arvola, O. J., Idoyaga, J., Giffard, R. G. 2019; 13: 609

  Abstract

  Recent studies demonstrate significant neuroimmune changes in postpartum females, a period that also carries an increased risk of stroke. Oxytocin, a major hormone upregulated in the brains of nursing mothers, has been shown to both modulate neuroinflammation and protect against stroke. In the present study we assessed whether and how nursing modulates the neuroimmune response and injury after stroke. We observed that postpartum nursing mice were markedly protected from 1 h of transient middle cerebral artery occlusion (MCAO) relative to either non-pregnant/non-postpartum or non-nursing (pups removed) postpartum females. Nursing mice also expressed reduced levels of pro-inflammatory cytokines, had decreased migration of blood leukocytes into the brain following MCAO, and displayed peripheral neuroimmune changes characterized by increased spleen weight and increased fraction of spleen monocytes. Intranasal oxytocin treatment in non-pregnant females in part recapitulated the protective and anti-inflammatory effects associated with nursing. In summary, the results of the present study demonstrate that nursing in the postpartum period provides relative protection against transient ischemic stroke associated with decreased brain leukocytes and increased splenic monocytes. These effects appear to be regulated, at least in part, by oxytocin.

  View details for DOI 10.3389/fnins.2019.00609

  View details for PubMedID 31354401

  View details for PubMedCentralID PMC6637858

• Opposing T cell responses in experimental autoimmune encephalomyelitis. Nature Saligrama, N. n., Zhao, F. n., Sikora, M. J., Serratelli, W. S., Fernandes, R. A., Louis, D. M., Yao, W. n., Ji, X. n., Idoyaga, J. n., Mahajan, V. B., Steinmetz, L. M., Chien, Y. H., Hauser, S. L., Oksenberg, J. R., Garcia, K. C., Davis, M. M. 2019

  Abstract

  Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

  View details for DOI 10.1038/s41586-019-1467-x

  View details for PubMedID 31391585

• The Nontoxic Cholera B Subunit Is a Potent Adjuvant for Intradermal DC-Targeted Vaccination. Frontiers in immunology Antonio-Herrera, L., Badillo-Godinez, O., Medina-Contreras, O., Tepale-Segura, A., García-Lozano, A., Gutierrez-Xicotencatl, L., Soldevila, G., Esquivel-Guadarrama, F. R., Idoyaga, J., Bonifaz, L. C. 2018; 9: 2212

  Abstract

  CD4+ T cells are major players in the immune response against several diseases; including AIDS, leishmaniasis, tuberculosis, influenza and cancer. Their activation has been successfully achieved by administering antigen coupled with antibodies, against DC-specific receptors in combination with adjuvants. Unfortunately, most of the adjuvants used so far in experimental models are unsuitable for human use. Therefore, human DC-targeted vaccination awaits the description of potent, yet nontoxic adjuvants. The nontoxic cholera B subunit (CTB) can be safely used in humans and it has the potential to activate CD4+ T cell responses. However, it remains unclear whether CTB can promote DC activation and can act as an adjuvant for DC-targeted antigens. Here, we evaluated the CTB's capacity to activate DCs and CD4+ T cell responses, and to generate long-lasting protective immunity. Intradermal (i.d.) administration of CTB promoted late and prolonged activation and accumulation of skin and lymphoid-resident DCs. When CTB was co-administered with anti-DEC205-OVA, it promoted CD4+ T cell expansion, differentiation, and infiltration to peripheral nonlymphoid tissues, i.e., the skin, lungs and intestine. Indeed, CTB promoted a polyfunctional CD4+ T cell response, including the priming of Th1 and Th17 cells, as well as resident memory T (RM) cell differentiation in peripheral nonlymphoid tissues. It is worth noting that CTB together with a DC-targeted antigen promoted local and systemic protection against experimental melanoma and murine rotavirus. We conclude that CTB administered i.d. can be used as an adjuvant to DC-targeted antigens for the induction of broad CD4+ T cell responses as well as for promoting long-lasting protective immunity.

  View details for DOI 10.3389/fimmu.2018.02212

  View details for PubMedID 30319653

  View details for PubMedCentralID PMC6171476

• The Nontoxic Cholera B Subunit Is a Potent Adjuvant for Intradermal DC-Targeted Vaccination FRONTIERS IN IMMUNOLOGY Antonio-Herrera, L., Badillo-Godinez, O., Medina-Contreras, O., Tepale-Segura, A., Garcia-Lozano, A., Gutierrez-Xicotencatl, L., Soldevila, G., Esquivel-Guadarrama, F. R., Idoyaga, J., Bonifaz, L. C. 2018; 9

  View details for DOI 10.3389/fimmu.2018.02212

  View details for Web of Science ID 000445778500002

• Vaccination-induced skin-resident memory CD8(+) T cells mediate strong protection against cutaneous melanoma ONCOIMMUNOLOGY Galvez-Cancino, F., Lopez, E., Menares, E., Diaz, X., Flores, C., Caceres, P., Hidalgo, S., Chovar, O., Alcantara-Hernandez, M., Borgna, V., Varas-Godoy, M., Salazar-Onfray, F., Idoyaga, J., Lladser, A. 2018; 7 (7): e1442163

  Abstract

  Memory CD8+ T cell responses have the potential to mediate long-lasting protection against cancers. Resident memory CD8+ T (Trm) cells stably reside in non-lymphoid tissues and mediate superior innate and adaptive immunity against pathogens. Emerging evidence indicates that Trm cells develop in human solid cancers and play a key role in controlling tumor growth. However, the specific contribution of Trm cells to anti-tumor immunity is incompletely understood. Moreover, clinically applicable vaccination strategies that efficiently establish Trm cell responses remain largely unexplored and are expected to strongly protect against tumors. Here we demonstrated that a single intradermal administration of gene- or protein-based vaccines efficiently induces specific Trm cell responses against models of tumor-specific and self-antigens, which accumulated in vaccinated and distant non-vaccinated skin. Vaccination-induced Trm cells were largely resistant to in vivo intravascular staining and antibody-dependent depletion. Intradermal, but not intraperitoneal vaccination, generated memory precursors expressing skin-homing molecules in circulation and Trm cells in skin. Interestingly, vaccination-induced Trm cell responses strongly suppressed the growth of B16F10 melanoma, independently of circulating memory CD8+ T cells, and were able to infiltrate tumors. This work highlights the therapeutic potential of vaccination-induced Trm cell responses to achieve potent protection against skin malignancies.

  View details for PubMedID 29900048

• Ebola virus infection kinetics in chimeric mice reveal a key role of T cells as barriers for virus dissemination. Scientific reports Lüdtke, A., Ruibal, P., Wozniak, D. M., Pallasch, E., Wurr, S., Bockholt, S., Gómez-Medina, S., Qiu, X., Kobinger, G. P., Rodríguez, E., Günther, S., Krasemann, S., Idoyaga, J., Oestereich, L., Muñoz-Fontela, C. 2017; 7: 43776-?

  Abstract

  Ebola virus (EBOV) causes severe systemic disease in humans and non-human primates characterized by high levels of viremia and virus titers in peripheral organs. The natural portals of virus entry are the mucosal surfaces and the skin where macrophages and dendritic cells (DCs) are primary EBOV targets. Due to the migratory properties of DCs, EBOV infection of these cells has been proposed as a necessary step for virus dissemination via draining lymph nodes and blood. Here we utilize chimeric mice with competent hematopoietic-driven immunity, to show that EBOV primarily infects CD11b(+) DCs in non-lymphoid and lymphoid tissues, but spares the main cross-presenting CD103(+) DC subset. Furthermore, depletion of CD8 and CD4 T cells resulted in loss of early control of virus replication, viremia and fatal Ebola virus disease (EVD). Thus, our findings point out at T cell function as a key determinant of EVD progress and outcome.

  View details for DOI 10.1038/srep43776

  View details for PubMedID 28256637

  View details for PubMedCentralID PMC5335601

• Pseudogenization of the Secreted Effector Gene sseI Confers Rapid Systemic Dissemination of S. Typhimurium ST313 within Migratory Dendritic Cells. Cell host & microbe Carden, S. E., Walker, G. T., Honeycutt, J., Lugo, K., Pham, T., Jacobson, A., Bouley, D., Idoyaga, J., Tsolis, R. M., Monack, D. 2017; 21 (2): 182-194

  Abstract

  Genome degradation correlates with host adaptation and systemic disease in Salmonella. Most lineages of the S. enterica subspecies Typhimurium cause gastroenteritis in humans; however, the recently emerged ST313 lineage II pathovar commonly causes systemic bacteremia in sub-Saharan Africa. ST313 lineage II displays genome degradation compared to gastroenteritis-associated lineages; yet, the mechanisms and causal genetic differences mediating these infection phenotypes are largely unknown. We find that the ST313 isolate D23580 hyperdisseminates from the gut to systemic sites, such as the mesenteric lymph nodes (MLNs), via CD11b(+) migratory dendritic cells (DCs). This hyperdissemination was facilitated by the loss of sseI, which encodes an effector that inhibits DC migration in gastroenteritis-associated isolates. Expressing functional SseI in D23580 reduced the number of infected migratory DCs and bacteria in the MLN. Our study reveals a mechanism linking pseudogenization of effectors with the evolution of niche adaptation in a bacterial pathogen.

  View details for DOI 10.1016/j.chom.2017.01.009

  View details for PubMedID 28182950

  View details for PubMedCentralID PMC5325708

• T-cell immunodominance. European journal of immunology Cruz, J. L., Pérez-Girón, J. V., Lüdtke, A., Gómez-Medina, S., Ruibal, P., Idoyaga, J., Muñoz-Fontela, C. 2017; 47 (2): 345-352

  Abstract

  Influenza virus infection triggers an increase in the number of monocyte-derived dendritic cells (moDCs) in the respiratory tract, but the role of these cells during antiviral immunity is still unclear. Here we show that during influenza infection, moDCs dominate the late activation of CD8(+) T cells and trigger the switch in immunodominance of the CD8(+) T-cell response from acidic polymerase specificity to nucleoprotein specificity. Abrogation of monocyte recruitment or depletion of moDCs strongly compromised host resistance to secondary influenza challenge. These findings underscore a novel function of moDCs in the antiviral response to influenza virus, and have important implications for vaccine design.

  View details for DOI 10.1002/eji.201646523

  View details for PubMedID 27859043

  View details for PubMedCentralID PMC5324604

• Monocyte-derived dendritic cells enhance protection against secondary influenza challenge by controlling the switch in CD8(+) T-cell immunodominance EUROPEAN JOURNAL OF IMMUNOLOGY Cruz, J. L., Perez-Giron, J., Luedtke, A., Gomez-Medina, S., Ruibal, P., Idoyaga, J., Munoz-Fontela, C. 2017; 47 (2): 345-352

  Abstract

  Influenza virus infection triggers an increase in the number of monocyte-derived dendritic cells (moDCs) in the respiratory tract, but the role of these cells during antiviral immunity is still unclear. Here we show that during influenza infection, moDCs dominate the late activation of CD8(+) T cells and trigger the switch in immunodominance of the CD8(+) T-cell response from acidic polymerase specificity to nucleoprotein specificity. Abrogation of monocyte recruitment or depletion of moDCs strongly compromised host resistance to secondary influenza challenge. These findings underscore a novel function of moDCs in the antiviral response to influenza virus, and have important implications for vaccine design.

  View details for DOI 10.1002/eji.201646523

  View details for Web of Science ID 000394839800014

  View details for PubMedCentralID PMC5324604

• Ebola Virus Disease Is Characterized by Poor Activation and Reduced Levels of Circulating CD16+ Monocytes. journal of infectious diseases Lüdtke, A., Ruibal, P., Becker-Ziaja, B., Rottstegge, M., Wozniak, D. M., Cabeza-Cabrerizo, M., Thorenz, A., Weller, R., Kerber, R., Idoyaga, J., Magassouba, N., Gabriel, M., Günther, S., Oestereich, L., Muñoz-Fontela, C. 2016; 214: S275-S280

  Abstract

  A number of previous studies have identified antigen-presenting cells (APCs) as key targets of Ebola virus (EBOV), but the role of APCs in human Ebola virus disease (EVD) is not known. We have evaluated the phenotype and kinetics of monocytes, neutrophils, and dendritic cells (DCs) in peripheral blood of patients for whom EVD was diagnosed by the European Mobile Laboratory in Guinea. Acute EVD was characterized by reduced levels of circulating nonclassical CD16(+) monocytes with a poor activation profile. In survivors, CD16(+) monocytes were activated during recovery, coincident with viral clearance, suggesting an important role of this cell subset in EVD pathophysiology.

  View details for PubMedID 27521367

• Expansion and Activation of CD103(+) Dendritic Cell Progenitors at the Tumor Site Enhances Tumor Responses to Therapeutic PD-L1 and BRAF Inhibition IMMUNITY Salmon, H., Idoyaga, J., Rahman, A., Leboeuf, M., Remark, R., Jordan, S., Casanova-Acebes, M., Khudoynazarova, M., Agudo, J., Tung, N., Chakarov, S., Rivera, C., Hogstad, B., Bosenberg, M., Hashimoto, D., Gnjatic, S., Bhardwaj, N., Palucka, A. K., Brown, B. D., Brody, J., Ginhoux, F., Merad, M. 2016; 44 (4): 924-938

  Abstract

  Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.

  View details for DOI 10.1016/j.immuni.2016.03.012

  View details for Web of Science ID 000374444300022

  View details for PubMedID 27096321

• Reply to: "Subverting misconceptions about radiation therapy". Nature immunology Price, J. G., Idoyaga, J., Merad, M. 2016; 17 (4): 345-346

  View details for DOI 10.1038/ni.3376

  View details for PubMedID 27002832

• ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis. PloS one Silva-Sánchez, A., Meza-Pérez, S., Flores-Langarica, A., Donis-Maturano, L., Estrada-García, I., Calderón-Amador, J., Hernández-Pando, R., Idoyaga, J., Steinman, R. M., Flores-Romo, L. 2015; 10 (4)

  Abstract

  Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

  View details for DOI 10.1371/journal.pone.0124828

  View details for PubMedID 25915045

• Activation of Toll-like Receptor-2 by Endogenous Matrix Metalloproteinase-2 Modulates Dendritic-Cell-Mediated Inflammatory Responses CELL REPORTS Godefroy, E., Gallois, A., Idoyaga, J., Merad, M., Tung, N., Monu, N., Saenger, Y., Fu, Y., Ravindran, R., Pulendran, B., Jotereau, F., Trombetta, S., Bhardwaj, N. 2014; 9 (5): 1856-1870

  Abstract

  Matrix metalloproteinase-2 (MMP-2) is involved in several physiological mechanisms, including wound healing and tumor progression. We show that MMP-2 directly stimulates dendritic cells (DCs) to both upregulate OX40L on the cell surface and secrete inflammatory cytokines. The mechanism underlying DC activation includes physical association with Toll-like receptor-2 (TLR2), leading to NF-κB activation, OX40L upregulation on DCs, and ensuing TH2 differentiation. Significantly, MMP-2 polarizes T cells toward type 2 responses in vivo, in a TLR2-dependent manner. MMP-2-dependent type 2 polarization may represent a key immune regulatory mechanism for protection against a broad array of disorders, such as inflammatory, infectious, and autoimmune diseases, which can be hijacked by tumors to evade immunity.

  View details for DOI 10.1016/j.celrep.2014.10.067

  View details for Web of Science ID 000346851900027

  View details for PubMedID 25466255

  View details for PubMedCentralID PMC4336179

• Murine Langerin(+) dermal dendritic cells prime CD8(+) T cells while Langerhans cells induce cross-tolerance EMBO MOLECULAR MEDICINE Flacher, V., Tripp, C. H., Mairhofer, D. G., Steinman, R. M., Stoitzner, P., Idoyaga, J., Romani, N. 2014; 6 (9): 1191-1204

  Abstract

  Skin dendritic cells (DCs) control the immunogenicity of cutaneously administered vaccines. Antigens targeted to DCs via the C-type lectin Langerin/CD207 are cross-presented to CD8(+) T cells in vivo. We investigated the relative roles of Langerhans cells (LCs) and Langerin(+) dermal DCs (dDCs) in different vaccination settings. Poly(I:C) and anti-CD40 agonist antibody promoted cytotoxic responses upon intradermal immunization with ovalbumin (OVA)-coupled anti-Langerin antibodies (Langerin/OVA). This correlated with CD70 upregulation in Langerin(+) dDCs, but not LCs. In chimeric mice where Langerin targeting was restricted to dDCs, CD8(+) T-cell memory was enhanced. Conversely, providing Langerin/OVA exclusively to LCs failed to prime cytotoxicity, despite initial antigen cross-presentation to CD8(+) T cells. Langerin/OVA combined with imiquimod could not prime CD8(+) T cells and resulted in poor cytotoxicity in subsequent responses. This tolerance induction required targeting and maturation of LCs. Altogether, Langerin(+) dDCs prime long-lasting cytotoxic responses, while cross-presentation by LCs negatively influences CD8(+) T-cell priming. Moreover, this highlights that DCs exposed to TLR agonists can still induce tolerance and supports the existence of qualitatively different DC maturation programs.

  View details for Web of Science ID 000341700700008

  View details for PubMedID 25085878

• BRAF-V600E expression in precursor versus differentiated dendritic cells defines clinically distinct LCH risk groups JOURNAL OF EXPERIMENTAL MEDICINE Berres, M., Lim, K. P., Peters, T., Price, J., Takizawa, H., Salmon, H., Idoyaga, J., Ruzo, A., Lupo, P. J., Hicks, M. J., Shih, A., Simko, S. J., Abhyankar, H., Chakraborty, R., Leboeuf, M., Beltrao, M., Lira, S. A., Heym, K. M., Bigley, V., Collin, M., Manz, M. G., McClain, K., Merad, M., Allen, C. E. 2014; 211 (4): 669-683

  Abstract

  Langerhans cell histiocytosis (LCH) is a clonal disorder with elusive etiology, characterized by the accumulation of CD207(+) dendritic cells (DCs) in inflammatory lesions. Recurrent BRAF-V600E mutations have been reported in LCH. In this study, lesions from 100 patients were genotyped, and 64% carried the BRAF-V600E mutation within infiltrating CD207(+) DCs. BRAF-V600E expression in tissue DCs did not define specific clinical risk groups but was associated with increased risk of recurrence. Strikingly, we found that patients with active, high-risk LCH also carried BRAF-V600E in circulating CD11c(+) and CD14(+) fractions and in bone marrow (BM) CD34(+) hematopoietic cell progenitors, whereas the mutation was restricted to lesional CD207(+) DC in low-risk LCH patients. Importantly, BRAF-V600E expression in DCs was sufficient to drive LCH-like disease in mice. Consistent with our findings in humans, expression of BRAF-V600E in BM DC progenitors recapitulated many features of the human high-risk LCH, whereas BRAF-V600E expression in differentiated DCs more closely resembled low-risk LCH. We therefore propose classification of LCH as a myeloid neoplasia and hypothesize that high-risk LCH arises from somatic mutation of a hematopoietic progenitor, whereas low-risk disease arises from somatic mutation of tissue-restricted precursor DCs.

  View details for DOI 10.1084/jem.20130977

  View details for Web of Science ID 000334917400009

  View details for PubMedID 24638167

• Targeting Leishmania major Antigens to Dendritic Cells In Vivo Induces Protective Immunity. PloS one Matos, I., Mizenina, O., Lubkin, A., Steinman, R. M., Idoyaga, J. 2013; 8 (6): e67453

  Abstract

  Efficient vaccination against the parasite Leishmania major, the causative agent of human cutaneous leishmaniasis, requires development of type 1 T-helper (Th1) CD4(+) T cell immunity. Because of their unique capacity to initiate and modulate immune responses, dendritic cells (DCs) are attractive targets for development of novel vaccines. In this study, for the first time, we investigated the capacity of a DC-targeted vaccine to induce protective responses against L. major. To this end, we genetically engineered the N-terminal portion of the stress-inducible 1 protein of L. major (LmSTI1a) into anti-DEC205/CD205 (DEC) monoclonal antibody (mAb) and thereby delivered the conjugated protein to DEC(+) DCs in situ in the intact animal. Delivery of LmSTI1a to adjuvant-matured DCs increased the frequency of antigen-specific CD4(+) T cells producing IFN-γ(+), IL-2(+), and TNF-α(+) in two different strains of mice (C57BL/6 and Balb/c), while such responses were not observed with the same doses of a control Ig-LmSTI1a mAb without receptor affinity or with non-targeted LmSTI1a protein. Using a peptide library for LmSTI1a, we identified at least two distinct CD4(+) T cell mimetopes in each MHC class II haplotype, consistent with the induction of broad immunity. When we compared T cell immune responses generated after targeting DCs with LmSTI1a or other L. major antigens, including LACK (Leishmania receptor for activated C kinase) and LeIF (Leishmania eukaryotic ribosomal elongation and initiation factor 4a), we found that LmSTI1a was superior for generation of IFN-γ-producing CD4(+) T cells, which correlated with higher protection of susceptible Balb/c mice to a challenge with L. major. For the first time, this study demonstrates the potential of a DC-targeted vaccine as a novel approach for cutaneous leishmaniasis, an increasing public health concern that has no currently available effective treatment.

  View details for DOI 10.1371/journal.pone.0067453

  View details for PubMedID 23840706

  View details for PubMedCentralID PMC3694010

• Streamlined Expressed Protein Ligation Using Split Inteins JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Vila-Perello, M., Liu, Z., Shah, N. H., Willis, J. A., Idoyaga, J., Muir, T. W. 2013; 135 (1): 286-292

  Abstract

  Chemically modified proteins are invaluable tools for studying the molecular details of biological processes, and they also hold great potential as new therapeutic agents. Several methods have been developed for the site-specific modification of proteins, one of the most widely used being expressed protein ligation (EPL) in which a recombinant α-thioester is ligated to an N-terminal Cys-containing peptide. Despite the widespread use of EPL, the generation and isolation of the required recombinant protein α-thioesters remain challenging. We describe here a new method for the preparation and purification of recombinant protein α-thioesters using engineered versions of naturally split DnaE inteins. This family of autoprocessing enzymes is closely related to the inteins currently used for protein α-thioester generation, but they feature faster kinetics and are split into two inactive polypeptides that need to associate to become active. Taking advantage of the strong affinity between the two split intein fragments, we devised a streamlined procedure for the purification and generation of protein α-thioesters from cell lysates and applied this strategy for the semisynthesis of a variety of proteins including an acetylated histone and a site-specifically modified monoclonal antibody.

  View details for DOI 10.1021/ja309126m

  View details for Web of Science ID 000313143000048

  View details for PubMedID 23265282

• Consortium biology in immunology: the perspective from the Immunological Genome Project NATURE REVIEWS IMMUNOLOGY Benoist, C., Lanier, L., Merad, M., Mathis, D. 2012; 12 (10): 734-740

  Abstract

  Although the field has a long collaborative tradition, immunology has made less use than genetics of 'consortium biology', wherein groups of investigators together tackle large integrated questions or problems. However, immunology is naturally suited to large-scale integrative and systems-level approaches, owing to the multicellular and adaptive nature of the cells it encompasses. Here, we discuss the value and drawbacks of this organization of research, in the context of the long-running 'big science' debate, and consider the opportunities that may exist for the immunology community. We position this analysis in light of our own experience, both positive and negative, as participants of the Immunological Genome Project.

  View details for DOI 10.1038/nri3300

  View details for Web of Science ID 000309263800014

  View details for PubMedID 22955842

• Zinc finger transcription factor zDC is a negative regulator required to prevent activation of classical dendritic cells in the steady state JOURNAL OF EXPERIMENTAL MEDICINE Meredith, M. M., Liu, K., Kamphorst, A. O., Idoyaga, J., Yamane, A., Guermonprez, P., Rihn, S., Yao, K., Silva, I. T., Oliveira, T. Y., Skokos, D., Casellas, R., Nussenzweig, M. C. 2012; 209 (9): 1583-1593

  Abstract

  Classical dendritic cells (cDCs) process and present antigens to T cells. Under steady-state conditions, antigen presentation by cDCs induces tolerance. In contrast, during infection or inflammation, cDCs become activated, express higher levels of cell surface MHC molecules, and induce strong adaptive immune responses. We recently identified a cDC-restricted zinc finger transcription factor, zDC (also known as Zbtb46 or Btbd4), that is not expressed by other immune cell populations, including plasmacytoid DCs, monocytes, or macrophages. We define the zDC consensus DNA binding motif and the genes regulated by zDC using chromatin immunoprecipitation and deep sequencing. By deleting zDC from the mouse genome, we show that zDC is primarily a negative regulator of cDC gene expression. zDC deficiency alters the cDC subset composition in the spleen in favor of CD8(+) DCs, up-regulates activation pathways in steady-state cDCs, including elevated MHC II expression, and enhances cDC production of vascular endothelial growth factor leading to increased vascularization of skin-draining lymph nodes. Consistent with these observations, zDC protein expression is rapidly down-regulated after TLR stimulation. Thus, zDC is a TLR-responsive, cDC-specific transcriptional repressor that is in part responsible for preventing cDC maturation in the steady state.

  View details for DOI 10.1084/jem.20121003

  View details for Web of Science ID 000308423900006

  View details for PubMedID 22851594

• Expression of the zinc finger transcription factor zDC (Zbtb46, Btbd4) defines the classical dendritic cell lineage JOURNAL OF EXPERIMENTAL MEDICINE Meredith, M. M., Liu, K., Darrasse-Jeze, G., Kamphorst, A. O., Schreiber, H. A., Guermonprez, P., Idoyaga, J., Cheong, C., Yao, K., Niec, R. E., Nussenzweig, M. C. 2012; 209 (6): 1153-1165

  Abstract

  Classical dendritic cells (cDCs), monocytes, and plasmacytoid DCs (pDCs) arise from a common bone marrow precursor (macrophage and DC progenitors [MDPs]) and express many of the same surface markers, including CD11c. We describe a previously uncharacterized zinc finger transcription factor, zDC (Zbtb46, Btbd4), which is specifically expressed by cDCs and committed cDC precursors but not by monocytes, pDCs, or other immune cell populations. We inserted diphtheria toxin (DT) receptor (DTR) cDNA into the 3' UTR of the zDC locus to serve as an indicator of zDC expression and as a means to specifically deplete cDCs. Mice bearing this knockin express DTR in cDCs but not other immune cell populations, and DT injection into zDC-DTR bone marrow chimeras results in cDC depletion. In contrast to previously characterized CD11c-DTR mice, non-cDCs, including pDCs, monocytes, macrophages, and NK cells, were spared after DT injection in zDC-DTR mice. We compared immune responses to Toxoplasma gondii and MO4 melanoma in DT-treated zDC- and CD11c-DTR mice and found that immunity was only partially impaired in zDC-DTR mice. Our results indicate that CD11c-expressing non-cDCs make significant contributions to initiating immunity to parasites and tumors.

  View details for DOI 10.1084/jem.20112675

  View details for Web of Science ID 000304907800010

  View details for PubMedID 22615130

• Dll4-Notch signaling in Flt3-independent dendritic cell development and autoimmunity in mice JOURNAL OF EXPERIMENTAL MEDICINE Billiard, F., Lobry, C., Darrasse-Jeze, G., Waite, J., Liu, X., Mouquet, H., Danave, A., Tait, M., Idoyaga, J., Leboeuf, M., Kyratsous, C. A., Burton, J., Kalter, J., Klinakis, A., Zhang, W., Thurston, G., Merad, M., Steinman, R. M., Murphy, A. J., Yancopoulos, G. D., Aifantis, I., Skokos, D. 2012; 209 (5): 1011-1028

  Abstract

  Delta-like ligand 4 (Dll4)-Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4-Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg) cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural T(reg) cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4(-)CD8(-)c-kit(+)CD44(+)CD25(-) (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4(+) T cells into T(reg) cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of T(reg) cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a T(reg) cell-mediated mechanism and inhibits CD8(+) T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased T(reg) cell numbers in the pancreas-draining lymph nodes. These results identify Dll4-Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated T(reg) cell homeostasis and autoimmunity.

  View details for DOI 10.1084/jem.20111615

  View details for Web of Science ID 000303684300014

  View details for PubMedID 22547652

• Skin Langerin(+) Dendritic Cells Transport Intradermally Injected Anti-DEC-205 Antibodies but Are Not Essential for Subsequent Cytotoxic CD8(+) T Cell Responses JOURNAL OF IMMUNOLOGY Flacher, V., Tripp, C. H., Haid, B., Kissenpfennig, A., Malissen, B., Stoitzner, P., Idoyaga, J., Romani, N. 2012; 188 (5): 2146-2155

  Abstract

  Incorporation of Ags by dendritic cells (DCs) increases when Ags are targeted to endocytic receptors by mAbs. We have previously demonstrated in the mouse that mAbs against C-type lectins administered intradermally are taken up by epidermal Langerhans cells (LCs), dermal Langerin(neg) DCs, and dermal Langerin(+) DCs in situ. However, the relative contribution of these skin DC subsets to the induction of immune responses after Ag targeting has not been addressed in vivo. We show in this study that murine epidermal LCs and dermal DCs transport intradermally injected mAbs against the lectin receptor DEC-205/CD205 in vivo. Skin DCs targeted in situ with mAbs migrated through lymphatic vessels in steady state and inflammation. In the skin-draining lymph nodes, targeting mAbs were found in resident CD8α(+) DCs and in migrating skin DCs. More than 70% of targeted DCs expressed Langerin, including dermal Langerin(+) DCs and LCs. Numbers of targeted skin DCs in the nodes increased 2-3-fold when skin was topically inflamed by the TLR7 agonist imiquimod. Complete removal of the site where OVA-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against OVA peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin(+) skin DCs in Langerin-DTR knock-in mice did not affect such responses independently of the adjuvant chosen. Thus, in cutaneous immunization strategies where Ag is targeted to DCs, Langerin(+) skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerin(neg) dermal DCs and CD8α(+) DCs are sufficient to subsequent CD8(+) T cell responses.

  View details for DOI 10.4049/jimmunol.1004120

  View details for Web of Science ID 000300610800014

  View details for PubMedID 22291181

• Treml4, an Ig Superfamily Member, Mediates Presentation of Several Antigens to T Cells In Vivo, Including Protective Immunity to HER2 Protein JOURNAL OF IMMUNOLOGY Hemmi, H., Zaidi, N., Wang, B., Matos, I., Fiorese, C., Lubkin, A., Zbytnuik, L., Suda, K., Zhang, K., Noda, M., Kaisho, T., Steinman, R. M., Idoyaga, J. 2012; 188 (3): 1147-1155

  Abstract

  Members of the triggering expressed on myeloid cells (Trem) receptor family fine-tune inflammatory responses. We previously identified one of these receptors, called Treml4, expressed mainly in the spleen, as well as at high levels by CD8α(+) dendritic cells and macrophages. Like other Trem family members, Treml4 has an Ig-like extracellular domain and a short cytoplasmic tail that associates with the adaptor DAP12. To follow up on our initial results that Treml4-Fc fusion proteins bind necrotic cells, we generated a knockout mouse to assess the role of Treml4 in the uptake and presentation of dying cells in vivo. Loss of Treml4 expression did not impair uptake of dying cells by CD8α(+) dendritic cells or cross-presentation of cell-associated Ag to CD8(+) T cells, suggesting overlapping function between Treml4 and other receptors in vivo. To further investigate Treml4 function, we took advantage of a newly generated mAb against Treml4 and engineered its H chain to express three different Ags (i.e., OVA, HIV GAGp24, and the extracellular domain of the breast cancer protein HER2). OVA directed to Treml4 was efficiently presented to CD8(+) and CD4(+) T cells in vivo. Anti-Treml4-GAGp24 mAbs, given along with a maturation stimulus, induced Th1 Ag-specific responses that were not observed in Treml4 knockout mice. Also, HER2 targeting using anti-Treml4 mAbs elicited combined CD4(+) and CD8(+) T cell immunity, and both T cells participated in resistance to a transplantable tumor. Therefore, Treml4 participates in Ag presentation in vivo, and targeting Ags with anti-Treml4 Abs enhances immunization of otherwise naive mice.

  View details for DOI 10.4049/jimmunol.1102541

  View details for Web of Science ID 000299690200029

  View details for PubMedID 22210914

• Dendritic cell-targeted protein vaccines: a novel approach to induce T-cell immunity JOURNAL OF INTERNAL MEDICINE Trumpfheller, C., Longhi, M. P., Caskey, M., Idoyaga, J., Bozzacco, L., Keler, T., Schlesinger, S. J., Steinman, R. M. 2012; 271 (2): 183-192

  Abstract

  Current vaccines primarily work by inducing protective antibodies. However, in many infections like HIV, malaria and tuberculosis as well as cancers, there remains a need for durable and protective T-cell immunity. Here, we summarize our efforts to develop a safe T-cell-based protein vaccine that exploits the pivotal role of dendritic cells (DC) in initiating adaptive immunity. Focusing on HIV, gag-p24 protein antigen is introduced into a monoclonal antibody (mAb) that efficiently and specifically targets the DEC-205 antigen uptake receptor on DC. When administered together with synthetic double-stranded RNA, polyriboinosinic:polyribocytidylic acid (poly IC) or its analogue poly IC stabilized with carboxymethylcellulose and poly-L-lysine (poly ICLC), as adjuvant, HIV gag-p24 within anti-DEC-205 mAb is highly immunogenic in mice, rhesus macaques, and in ongoing research, healthy human volunteers. Human subjects form both T- and B-cell responses to DC-targeted protein. Thus, DC-targeted protein vaccines are a potential new vaccine platform, either alone or in combination with highly attenuated viral vectors, to induce integrated immune responses against microbial or cancer antigens, with improved ease of manufacturing and clinical use.

  View details for DOI 10.1111/j.1365-2796.2011.02496.x

  View details for Web of Science ID 000299157900011

  View details for PubMedID 22126373

• Microbial Stimulation Fully Differentiates Monocytes to DC-SIGN/CD209(+) Dendritic Cells for Immune T Cell Areas CELL Cheong, C., Matos, I., Choi, J., Dandamudi, D. B., Shrestha, E., Longhi, M. P., Jeffrey, K. L., Anthony, R. M., Kluger, C., Nchinda, G., Koh, H., Rodriguez, A., Idoyaga, J., Pack, M., Velinzon, K., Park, C. G., Steinman, R. M. 2010; 143 (3): 416-429

  Abstract

  Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCs.

  View details for DOI 10.1016/j.cell.2010.09.039

  View details for Web of Science ID 000283603900015

  View details for PubMedID 21029863

• Targeting of antigens to skin dendritic cells: possibilities to enhance vaccine efficacy IMMUNOLOGY AND CELL BIOLOGY Romani, N., Thurnher, M., Idoyaga, J., Steinman, R. M., Flacher, V. 2010; 88 (4): 424-430

  Abstract

  Vaccinations in medicine are commonly administered through the skin. Therefore, the vaccine is immunologically processed by antigen-presenting cells of the skin. There is recent evidence that the clinically less often used intradermal route is effective; in cases even superior to the conventional subcutaneous or intramuscular route. Professional antigen-presenting cells of the skin comprise epidermal Langerhans cells (CD207/langerin(+)), dermal langerin(-) and dermal langerin(+) dendritic cells (DCs). In human skin, langerin(-) dermal DCs can be further subdivided on the basis of their reciprocal CD1a and CD14 expression. The relative contributions of these subsets to the generation of immunity or tolerance are still unclear. Langerhans cells in human skin seem to be specialized for induction of cytotoxic T lymphocytes. Likewise, mouse Langerhans cells are capable of cross-presentation and of protecting against experimental tumours. It is desirable to harness these properties for immunotherapy. A promising strategy to dramatically improve the outcome of vaccinations is 'antigen targeting'. Thereby, the vaccine is delivered directly and selectively to defined types of skin DCs. Targeting is achieved by means of coupling antigen to antibodies that recognize cell surface receptors on DCs. This approach is being widely explored. Little is known, however, about the events that take place in the skin and the DCs subsets involved therein. This topic will be discussed in this article.

  View details for DOI 10.1038/icb.2010.39

  View details for Web of Science ID 000277442500014

  View details for PubMedID 20368713

  View details for PubMedCentralID PMC2907485

• Epidermal Langerhans Cells Rapidly Capture and Present Antigens from C-Type Lectin-Targeting Antibodies Deposited in the Dermis JOURNAL OF INVESTIGATIVE DERMATOLOGY Flacher, V., Tripp, C. H., Stoitzner, P., Haid, B., Ebner, S., Del Frari, B., Koch, F., Park, C. G., Steinman, R. M., Idoyaga, J., Romani, N. 2010; 130 (3): 755-762

  Abstract

  Antigen-presenting cells can capture antigens that are deposited in the skin, including vaccines given subcutaneously. These include different dendritic cells (DCs) such as epidermal Langerhans cells (LCs), dermal DCs, and dermal langerin+ DCs. To evaluate access of dermal antigens to skin DCs, we used mAb to two C-type lectin endocytic receptors, DEC-205/CD205 and langerin/CD207. When applied to murine and human skin explant cultures, these mAbs were efficiently taken up by epidermal LCs. In addition, anti-DEC-205 targeted langerin+ CD103+ and langerin- CD103- mouse dermal DCs. Unexpectedly, intradermal injection of either mAb, but not isotype control, resulted in strong and rapid labeling of LCs in situ, implying that large molecules can diffuse through the basement membrane into the epidermis. Epidermal LCs targeted in vivo by ovalbumin-coupled anti-DEC-205 potently presented antigen to CD4+ and CD8+ T cells in vitro. However, to our surprise, LCs targeted through langerin were unable to trigger T-cell proliferation. Thus, epidermal LCs have a major function in uptake of lectin-binding antibodies under standard vaccination conditions.

  View details for DOI 10.1038/jid.2009.343

  View details for Web of Science ID 000275017600019

  View details for PubMedID 19890348

• Acute in vivo exposure to interferon-gamma enables resident brain dendritic cells to become effective antigen presenting cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gottfried-Blackmore, A., Kaunzner, U. W., Idoyaga, J., Felger, J. C., McEwen, B. S., Bulloch, K. 2009; 106 (49): 20918-20923

  Abstract

  Dendritic cells (DC) are the professional antigen presenting cells (APC) that bridge the innate and adaptive immune system. Previously, in a CD11c/EYFP transgenic mouse developed to study DC functions, we anatomically mapped and phenotypically characterized a discrete population of EYFP(+) cells within the microglia that we termed brain dendritic cells (bDC). In this study, we advanced our knowledge of the function of these cells in the CD11c/EYFP transgenic mouse and its chimeras, using acute stimuli of stereotaxically inoculated IFNgamma or IL-4 into the CNS. The administration of IFNgamma increased the number of EYFP(+)bDC but did not recruit peripheral DC into the CNS. IFNgamma, but not IL-4, upregulated the expression levels of major histocompatibility class II (MHC-II). In addition, IFNgamma-activated EYFP(+)bDC induced antigen-specific naïve CD4 T cells to proliferate and secrete Th1/Th17 cytokines. Activated bDC were also able to stimulate naïve CD8 T cells. Collectively, these data reveal the Th1 cytokine IFNgamma, but not the Th2 cytokine IL4, induces bDC to up-regulate MHC-II and become competent APC.

  View details for DOI 10.1073/pnas.0911509106

  View details for Web of Science ID 000272553000066

  View details for PubMedID 19906988

  View details for PubMedCentralID PMC2791588

• Dendritic cells require a systemic type I interferon response to mature and induce CD4(+) Th1 immunity with poly IC as adjuvant JOURNAL OF EXPERIMENTAL MEDICINE Longhi, M. P., Trumpfheller, C., Idoyaga, J., Caskey, M., Matos, I., Kluger, C., Salazar, A. M., Colonna, M., Steinman, R. M. 2009; 206 (7): 1589-1602

  Abstract

  Relative to several other toll-like receptor (TLR) agonists, we found polyinosinic:polycytidylic acid (poly IC) to be the most effective adjuvant for Th1 CD4(+) T cell responses to a dendritic cell (DC)-targeted HIV gag protein vaccine in mice. To identify mechanisms for adjuvant action in the intact animal and the polyclonal T cell repertoire, we found poly IC to be the most effective inducer of type I interferon (IFN), which was produced by DEC-205(+) DCs, monocytes, and stromal cells. Antibody blocking or deletion of type I IFN receptor showed that IFN was essential for DC maturation and development of CD4(+) immunity. The IFN-AR receptor was directly required for DCs to respond to poly IC. STAT 1 was also essential, in keeping with the type I IFN requirement, but not type II IFN or IL-12 p40. Induction of type I IFN was mda5 dependent, but DCs additionally used TLR3. In bone marrow chimeras, radioresistant and, likely, nonhematopoietic cells were the main source of IFN, but mda5 was required in both marrow-derived and radioresistant host cells for adaptive responses. Therefore, the adjuvant action of poly IC requires a widespread innate type I IFN response that directly links antigen presentation by DCs to adaptive immunity.

  View details for DOI 10.1084/jem.20090247

  View details for Web of Science ID 000267738700014

  View details for PubMedID 19564349

• A New Triggering Receptor Expressed on Myeloid Cells (Trem) Family Member, Trem-Like 4, Binds to Dead Cells and Is a DNAX Activation Protein 12-Linked Marker for Subsets of Mouse Macrophages and Dendritic Cells JOURNAL OF IMMUNOLOGY Hemmi, H., Idoyaga, J., Suda, K., Suda, N., Kennedy, K., Noda, M., Aderem, A., Steinman, R. M. 2009; 182 (3): 1278-1286

  Abstract

  Dendritic cells (DCs) are professional APCs that can control immune responses against self and altered self, typically foreign, determinants. DCs can be divided into several subsets, including CD8alpha(+) and CD8alpha(-) DCs. These subsets possess specific functions. For example, mouse splenic CD8alpha(+), but not CD8alpha(-) DCs selectively take up dying cells and cross-present cell-associated Ags to naive T cells. In this study, we identified genes that were more expressed in CD8alpha(+) than CD8alpha(-) DCs by microarray analysis. Only one of these genes, when the extracellular domains were linked to human IgG Fc domain, could bind to late apoptotic or necrotic cells. This gene was a new member of the triggering receptor expressed on myeloid cells (Trem) family, Trem-like 4 (Treml4). Treml4 mRNA and protein, the latter detected with a new mAb, were predominantly expressed in spleen. Treml4, like other Trem family members, could associate with the adaptor molecule DNAX activation protein 12 kDa, but neither DNAX activation protein 10 kDa nor FcRgamma. Consistent with the microarray data, we confirmed that Treml4 protein was more expressed on CD8alpha(+) than CD8alpha(-) DCs, and we also found that Treml4 was expressed at high levels on splenic macrophages in spleen, particularly red pulp and marginal metallophilic macrophages. In addition, Treml4 expression on DCs was not changed after maturation induced by TLR ligands. Thus, Treml4 is a new Trem family molecule that is abundantly expressed on CD8alpha(+) DCs and subsets of splenic resident macrophages, and can recognize dead cells by different types of phagocytes in spleen.

  View details for Web of Science ID 000262842100010

  View details for PubMedID 19155473

• Cutting edge: Langerin/CD207 receptor on dendritic cells mediates efficient antigen presentation on MHC I and II products in vivo JOURNAL OF IMMUNOLOGY Idoyaga, J., Cheong, C., Suda, K., Suda, N., Kim, J. Y., Lee, H., Park, C. G. 2008; 180 (6): 3647-3650

  Abstract

  The targeted delivery of Ags to dendritic cell (DCs) in vivo greatly improves the efficiency of Ag presentation to T cells and allows an analysis of receptor function. To evaluate the function of Langerin/CD207, a receptor expressed by subsets of DCs that frequently coexpress the DEC205/CD205 receptor, we genetically introduced OVA into the C terminus of anti-receptor Ab H chains. Taking advantage of the new L31 mAb to the extracellular domain of mouse Langerin, we find that the hybrid Ab targets appropriate DC subsets in draining lymph nodes and spleen. OVA is then presented efficiently to CD8(+) and CD4(+) T cells in vivo, which undergo 4-8 cycles of division in 3 days. Peptide MHC I and II complexes persist for days. Dose response studies indicate only modest differences between Langerin and DEC receptors in these functions. Thus, Langerin effectively mediates Ag presentation.

  View details for Web of Science ID 000257506600006

  View details for PubMedID 18322168

• Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection JOURNAL OF IMMUNOLOGICAL METHODS Park, S. H., Cheong, C., Idoyaga, J., Kim, J. Y., Choi, J., Do, Y., Lee, H., Jo, J. H., Oh, Y., Im, W., Steinman, R. M., Park, C. G. 2008; 331 (1-2): 27-38

  Abstract

  Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS (E. coliOmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.

  View details for DOI 10.1016/j.jim.2007.10.012

  View details for Web of Science ID 000254059400003

  View details for PubMedID 18054954

• Tumor cells prevent mouse dendritic cell maturation induced by TLR ligands CANCER IMMUNOLOGY IMMUNOTHERAPY Idoyaga, J., Moreno, J., Bonifaz, L. 2007; 56 (8): 1237-1250

  Abstract

  Tumor cells can evade the immune system through several mechanisms, one of which is to block DC maturation. It has been suggested that signaling via Toll-like receptors (TLR) may be involved in the induction of prophylactic anti-cancer immunity and in the treatment of established tumors. In the present study we found that high numbers of tumor cells interfere with BMDC activation induced by the TLR ligands LPS and poly IC. Tumor cells blocked TLR3- and TLR4-mediated induction of MHCII and the co-stimulatory molecules CD40 and CD86, as well as the cytokines IL-12, TNF-alpha and IL-6. Importantly, tumor cells induced inhibitory molecules (B7-DC, B7-H1 and CD80) on spleen DC in vivo and on BMDC, even in the presence of TLR ligands. Moreover, after a long exposure with tumor cells, purified BMDC were unable to respond to a second challenge with TLR ligands. The failure of tumor exposed-BMDC to express co-stimulatory molecules and cytokines in the presence of TLR ligands has implications for the future development of DC-based cancer immune therapies using TLR ligands as adjuvants for the activation of DC.

  View details for DOI 10.1007/s00262-006-0275-y

  View details for Web of Science ID 000246561900011

  View details for PubMedID 17237931

• Production of monoclonal antibodies that recognize the extracellular domain of mouse Langerin/CD207 JOURNAL OF IMMUNOLOGICAL METHODS Cheong, C., Idoyaga, J., Do, Y., Pack, M., Park, S. H., Lee, H., Kang, Y., Choi, J., Kim, J. Y., Bonito, A., Inaba, K., Yamazaki, S., Steinman, R. M., Park, C. G. 2007; 324 (1-2): 48-62

  Abstract

  Langerin CD207 is a type II transmembrane protein. It is responsible for the formation of Birbeck granules, which are intracellular organelles within Langerhans cells, the dendritic cells of stratified squamous epithelia like the epidermis. Because current anti-CD207 antibodies have limitations, we prepared new monoclonals by immunizing rats with the extracellular region of mouse Langerin followed by a boost with enriched Langerhans cells (LCs). We secured a large panel of mAbs, most of which reacted with the carboxy terminal carbohydrate recognition domain. These mAbs could be used to immunoblot and immunoprecipitate mouse Langerin and to stain the cell surface and intracellular pools of CD207 by FACS analysis. Labeling of Birbeck granules was also achieved by immunoelectron microscopy. Anti-CD207 identified LCs in the epidermis and skin draining lymph nodes of BALB/c and C57BL/6 mice, but BALB/c mice had an additional Langerin(+) population in spleen, thymus and mesenteric lymph node. This additional subset had higher levels of CD8 and CD205 than epidermal LCs, and also had a less mature phenotype, i.e., lower MHC II, CD40 and CD86. Subcutaneous injection of IgG but not IgM forms of these new anti-CD207 mAbs led to rapid and selective labeling of the Langerin(+) cells in skin draining lymph nodes as well as spleen. The new IgG anti-CD207 mAbs should be useful for further research on LCs and dendritic cells including an evaluation of the consequences of antigen delivery within anti-CD207 mAbs in vivo.

  View details for DOI 10.1016/j.jim.2007.05.001

  View details for Web of Science ID 000248516400005

  View details for PubMedID 17553520

  View details for PubMedCentralID PMC2700064

• Innate NKT lymphocytes confer superior adaptive immunity via tumor-capturing dendritic cells JOURNAL OF EXPERIMENTAL MEDICINE Liu, K., Idoyaga, J., Charalambous, A., Fujii, S., Bonito, A., Mordoh, J., Wainstok, R., Bai, X. F., Liu, Y., Steinman, R. M. 2005; 202 (11): 1507-1516

  Abstract

  If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex-negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid alpha-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic alphaCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance.

  View details for DOI 10.1084/jem.20050956

  View details for Web of Science ID 000233753900007

  View details for PubMedID 16330814

• Dendritic cells charged with apoptotic tumor cells induce long-lived protective CD4(+) and CD8(+) T cell immunity against B16 melanoma JOURNAL OF IMMUNOLOGY Goldszmid, R. S., Idoyaga, J., Bravo, A. I., Steinman, R., Mordoh, J., Wainstok, R. 2003; 171 (11): 5940-5947

  Abstract

  Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4(+) and CD8(+) T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to approximately 0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4(+) and CD8(+) T cells were efficiently primed in vaccinated animals, as evidenced by IFN-gamma secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8(+) T cells in vitro, and cytolytic activity against tyrosinase-related protein 2(180-188)-pulsed target cells was observed in vivo. When either CD4(+) or CD8(+) T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.

  View details for Web of Science ID 000186767200038

  View details for PubMedID 14634105