High Levels Of Inhibitory Receptors Research Articles

TMIC-51. SEX-SPECIFIC T CELL BEHAVIOR DRIVES DIFFERENTIAL IMMUNE RESPONSES IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor and shows poor outcomes as a median survival is 12-18 months with current standard-of-care of therapy. GBM exhibits sex differences in incidence and overall survival, with males experiencing a higher incidence and a worse prognosis compared to females. However, the role of immune cells in GBM sex differences outside of myeloid cells remains poorly understood. Using a syngeneic mouse GBM model, we recapitulated the sex differences observed in patients, with shortened survival in male hosts compared to female hosts. These findings were not recapitulated in immuno-deficient mouse strains such as NSG and RAG1KO mice, suggesting a role for immune system, specifically T cells, in GBM sex differences. Flow cytometry analysis of tumor-infiltrating leukocytes revealed that more T cells were found in female tumors, whereas male tumors were enriched in macrophages. Additionally, more T cells in male tumors exhibited high levels of inhibitory receptors, whereas most female T cells were more functional as measured by expression of anti-tumor cytokines such as TNF, IFN-gamma, and granzyme B. A bone marrow chimera model (BMC) revealed that male T cells retained their phenotype in female hosts, whereas female T cell behavior was affected by the male environment. Yet survival analysis on BMC model and adoptive transfer model suggests strong immune cell-intrinsic effect on controlling tumor progression, further supported by in vitro T cell exhaustion model. Lastly, we found that males have more progenitor exhausted T cells, which led to better response to anti-PD1 treatment. Collectively, these results suggest that both cell-intrinsic and cell-extrinsic factors regulate T cell activity in a sex-specific manner, providing insights to develop sex-specific therapeutic approaches.

Sex-specific T cell behavior drives differential immune responses in mouse glioblastoma models

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor and has a median survival of 12–18 months with current standard-of-care of therapy. GBM sex differences in incidence and overall survival, with males experiencing a higher incidence and a worse prognosis compared to females. However, the role of immune cells in GBM sex differences outside of myeloid cells remains poorly understood. Using a syngeneic mouse GBM model, we recapitulated the sex differences observed in patients, with shortened survival in male hosts compared to female hosts. These findings were not recapitulated in immuno-deficient mouse strains such as NSG and RAG1KO mice, suggesting a role for immune cells, specifically T cells, in GBM sex differences. Flow cytometry analysis of tumor-infiltrating leukocytes revealed more T cells in female tumors, whereas male tumors were enriched in macrophages. Additionally, more T cells in male tumors exhibited high levels of inhibitory receptors, whereas most female T cells were more functional as measured by expression of anti-tumor cytokines such as TNF, IFN-gamma, and granzyme B. A bone marrow chimera model revealed that male T cells retained their phenotype in female hosts, whereas female T cell behavior was affected by the male environment. Furthermore, female T cells are more proliferative, with elevated expression of integrins compared to male T cells. Lastly, we found that males have more progenitor exhausted T cells, which led to better response to anti-PD1 treatment. Collectively, these results suggest that both cell-intrinsic and cell-extrinsic factors regulate T cell activity in a sex-specific manner, providing insights to develop sex-specific therapeutic approaches. Supported by grant from NIH (P01 CA245705)

Hallmark features of T cell dysfunction are established within hours after tumor antigen encounter

Abstract Tumor-specific CD8 T cells (TST) found in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is encoded by specific epigenetic and transcriptional programs and thought to be induced by chronic antigen stimulation. Using a genetic liver cancer mouse model, we followed the fate of naïve TST in tumor-bearing hosts. Strikingly, we found that within hours after encountering tumor antigen, TST lost effector function, expressed high levels of inhibitory receptors, and displayed chromatin peak accessibility changes previously characterized in late-stage dysfunction. This rapid onset challenges the paradigm that chronic antigen/T cell receptor stimulation is required to induce dysfunction/exhaustion. Moreover, dysfunction-associated epigenetic features were established even prior to cell division, opposing the prevailing notion that cell divisions are required for epigenetic remodeling and T cell differentiation. To determine whether these early dysfunction hallmarks were “imprinted”, we reisolated TST from malignant lesions and transferred them into antigen-free hosts. While loss of effector function was imprinted within hours of tumor exposure, other dysfunctional hallmarks, including PD1 expression, required several days of tumor exposure and additional chromatin remodeling to become stably encoded. Our study reveals the rapid induction and epigenetic remodeling which underlies TST commitment to the dysfunctional fate, highlighting the need to target early activation-induced pathways to improve anti-cancer T cell immunity. Supported by T32GM007347-43

Single-Cell Transcriptomic Analysis of Ecosystems in Papillary Thyroid Carcinoma Progression.

BackgroundDespite extensive research, the papillary thyroid carcinoma (PTC) ecosystem is poorly characterized and, in particular, locoregional progression. Available evidence supports that single-cell transcriptome sequencing (Sc-RNA seq) can dissect tumor ecosystems.MethodsTissue samples from one PTC patient, including matched primary tumor (Ca), lymph node (LN) metastasis, and paracancerous tissue (PCa), were subjected to Sc-RNA seq with 10×Genomics. Dual-label immunofluorescence and immunohistochemistry were used to confirm the existence of cell subtypes in a separate cohort.Results11,805 cell transcriptomes were profiled, cell landscapes of PTC were composed of malignant follicular epithelial cells (MFECs), CD8+ and CD4+ T cells, B cells, vascular endothelial cells, fibroblasts and cancer-associated fibroblasts (CAFs). Between Ca and LN ecosystems, the proportions of MFEC and interstitial cells were similar, less than 1/25(229/6,694, 361/3,895), while the proportion of normal follicular epithelial cells (NFECs) and interstitial cells was > 2 in PCa (455/171). NFECs in PCa formed a separate cluster, while MFECs in Ca and LN exhibited a profound transcriptional overlap, and the interstitial cells among these samples had an overall concordance in their identity and representation, albeit with some distinctions in terms of the cell percentage per subset. A fraction of the B cell subpopulation in Ca expressed inhibitory receptors, while their respective ligand genes were clearly transcribed in T cell and malignant epithelial cell clusters, while some CD8+ T cells in both Ca and LN produced high levels of inhibitory receptors whose respective ligands were overexpressed in some CD4+ T cells. Three CAF subtypes in Ca and LN were identified, which may be due to mutual transitions.ConclusionsOur data provide new insights into the PTC ecosystem and highlight the differences in ecosystems in PTC progression, which updates our understanding of PTC biology and will improve individualized patient treatment.

RNF5-deficiency inhibits CD8+ T cell exhaustion in chronic antigen stimulation

Abstract Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

Blockade of LAG-3 in PD-L1-Deficient Mice Enhances Clearance of Blood Stage Malaria Independent of Humoral Responses.

T cells expressing high levels of inhibitory receptors such as PD-1 and LAG-3 are a hallmark of chronic infections and cancer. Checkpoint blockade therapies targeting these receptors have been largely validated as promising strategies to restore exhausted T cell functions and clearance of chronic infections and tumors. The inability to develop long-term natural immunity in malaria-infected patients has been proposed to be at least partially accounted for by sustained expression of high levels of inhibitory receptors on T and B lymphocytes. While blockade or lack of PD-1/PD-L1 and/or LAG-3 was reported to promote better clearance of Plasmodium parasites in various mouse models, how exactly blockade of these pathways contributes to enhanced protection is not known. Herein, using the mouse model of non-lethal P. yoelii (Py) infection, we reveal that the kinetics of blood parasitemia as well as CD4+ T follicular helper (TFH) and germinal center (GC) B cell responses are indistinguishable between PD-1-/-, PD-L1-/- and WT mice. Yet, we also report that monoclonal antibody (mAb) blockade of LAG-3 in PD-L1-/- mice promotes accelerated control of blood parasite growth and clearance, consistent with prior therapeutic blockade experiments. However, neither CD4+ TFH and GC B cell responses, nor parasite-specific Ab serum titers and capacity to transfer protection differed. We also found that i) the majority of LAG-3+ cells are T cells, ii) selective depletion of CD4+ but not CD8+ T cells prevents anti-LAG-3-mediated protection, and iii) production of effector cytokines by CD4+ T cells is increased in anti-LAG-3-treated versus control mice. Thus, taken together, these results are consistent with a model in which blockade and/or deficiency of PD-L1 and LAG-3 on parasite-specific CD4+ T cells unleashes their ability to effectively clear blood parasites, independently from humoral responses.

Layilin augments integrin activation to promote antitumor immunity.

Tumor-infiltrating CD8+ T cells mediate antitumor immune responses. However, the mechanisms by which T cells remain poised to kill cancer cells despite expressing high levels of inhibitory receptors are unknown. Here, we report that layilin, a C-type lectin domain-containing membrane glycoprotein, is selectively expressed on highly activated, clonally expanded, but phenotypically exhausted CD8+T cells in human melanoma. Lineage-specific deletion of layilin on murine CD8+ T cells reduced their accumulation in tumors and increased tumor growth in vivo. Congruently, gene editing of LAYN in human CD8+ T cells reduced direct tumor cell killing ex vivo. On a molecular level, layilin colocalized with integrin αLβ2 (LFA-1) on T cells, and cross-linking layilin promoted the activated state of this integrin. Accordingly, LAYN deletion resulted in attenuated LFA-1-dependent cellular adhesion. Collectively, our results identify layilin as part of a molecular pathway in which exhausted or "dysfunctional" CD8+T cells enhance cellular adhesiveness to maintain their cytotoxic potential.

Decidual stromal cells promote the differentiation of CD56bright CD16- NK cells by secreting IL-24 in early pregnancy.

Decidual stromal cells (DSCs) are important origins of cytokines to modulate maternal-fetal immunotolerance and provide a feasible environment for embryo implantation and development. Interleukin (IL)-24 is a multifunctional cancer killing cytokine and a pleiotropic immunoregulator with complex potency according to tissue or cell types. Its role in establishment and maintenance of normal pregnancy is largely unknown. The aim of our study was to investigate the function and significance of IL-24 and its receptor in the coordination between DSCs and natural killer cells (NK) in early pregnancy. The levels of IL-24 in DSC, endometrial stromal cell (ESC), peripheral blood NK cells(pNK), or decidual NK cells(dNK) culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA), and the levels of IL-24 receptors were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry assays. The effect of IL-24 on the functions of decidual NK cells was analyzed by flow cytometry assays in vitro. The concentration of IL-24 in culture supernatant of DSCs was significantly higher than that of ESCs. Both eNK (endometrial NK cells) and dNK highly expressed IL-24 receptors (IL-20R1 and IL-22R1), especially on CD56dim eNK. However, there were extremely low levels of IL-20R1 and IL-22R1 on pNK. Recombinant human IL-24 or DSCs-secreted IL-24 downregulated the levels of CD16, Granzyme B, perforin, and interferon (IFN)-γ and upregulated the levels of inhibitory receptors killer-cell immunoglobulin-like receptor (KIR)2DL1 and KIR3DL1, or immunotolerant or angiogenic cytokines (eg, transforming growth factor (TGF)-β, IL-10, and IL-8), and elevated the percentage of CD56bright CD16- dNK in vitro. These data suggest that DSCs promote the differentiation of CD56bright CD16- NK with high levels of inhibitory receptors, immunotolerant, and angiogenic cytokines by secreting IL-24 during decidualization in early pregnancy.

IL-27 and TCR Stimulation Promote T Cell Expression of Multiple Inhibitory Receptors.

Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR.

Abstract 4669: Role of CD8+ tissue-resident memory T (TRM) cells in anti-tumor T-cell response and cancer immunotherapy

Abstract Tissue-resident memory T (TRM) cells, very often defined by the expression of CD103 integrin, correspond to a new subset of long-lived memory T lymphocytes that reside in tissues and are highly protective during localized reinfections. CD8+ TRM cells also frequently reside in human epithelial tumors and play an essential role in anti-tumor T-cell responses. In this context, our previous results indicated that the interaction of CD103 integrin with its ligand, the epithelial cell marker E-cadherin, favors adhesion of TRM cells to their specific epithelial cancer cells and participate to the potentiation of T-cell receptor (TCR)-mediated cytotoxic activity and cytokine production. We also demonstrated that an enhanced CD103+ tumor-infiltrating lymphocytes (TIL) subset correlates with improved early-stage non-small cell lung carcinoma (NSCLC) patient survival and increased intraepithelial T-lymphocyte infiltration. Notably, TRM cells are enriched with tumor-specific T cells and express high levels of inhibitory receptors, including PD-1 and Tim-3, making them an attractive target for cancer immunotherapy, such as checkpoint blockade. Moreover, we provided evidence that CD103 is involved in T-cell recruitment within epithelial tumor islets and in local early T-cell signaling. Our more recent results indicated that integrin-linked kinase (ILK) and paxillin adaptor protein are directly involved in CD103 bidirectional signaling events and defined the CD103 cytoplasmic domain that controls TRM adhesion, migratory behavior and TCR-mediated T-cell functions. Data emphasizing the contribution of CD8+/CD103+ TRM cells in promoting intra-tumoral cytotoxic T lymphocyte (CTL) responses and in success of cancer immunotherapy will be discussed. Citation Format: Fathia Mami-Chouaib. Role of CD8+ tissue-resident memory T (TRM) cells in anti-tumor T-cell response and cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4669.

Two Distinct Myeloid Subsets at the Term Human Fetal-Maternal Interface.

During pregnancy, immune cells infiltrate the placenta at different stages of fetal development. NK cells and macrophages are the most predominant cell types. These immune cells play pleiotropic roles, as they control spiral artery remodeling to ensure appropriate blood supply and maintain long-term tolerance to a true allograft; yet, they must be able to mount appropriate immune defenses to pathogens that may threaten the fetus. Whether the same cell type accomplishes all these tasks or if there are dedicated subsets remains controversial. Here, we identify and characterize two distinct subsets of myeloid cells that differ in their pro-inflammatory/regulatory capacity. While one subset predominantly produces the immune-modulating cytokine IL-10, the second subset has superior capacity to secrete pro-inflammatory mediators, such as IL-1β and IL-6. The putative regulatory myeloid cells also express high levels of inhibitory receptors and their ligands, including programmed cell death 1 (PD1) ligands. Importantly, a large fraction of CD8 and CD4 cells in normal term human placenta are PD1 positive, suggesting that the PD1/PD1 ligands axis might be critical to maintain tolerance during pregnancy.

Impact of antigen affinity on T cell dysfunction in solid tumors

Abstract Although tumor-specific T cells (TST) are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. TSTs express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying TST dysfunction remain poorly defined. T cell mediated immune response is triggered by T cell receptor (TCR) binding to peptide-major histocompatibility (pMHC) complex on the surface of cells. The affinity of TCR:pMHC interaction is a critical determinant of T cell expansion and effector function in acute infections, where T cells with high affinity interactions generally show superior function. However, little is known about how TCR:pMHC affinity impacts T cells activation, induction of dysfunction and susceptibility to immunotherapy in progressing tumors. To elucidate this, we generated tumor cell lines expressing altered peptide ligands derived from SV40 large T antigen epitope I (Tag) and recognized by Tag-specific transgenic CD8 T cells (TCRTag) with varying functional avidity. We found that tumor-infiltrating TCRTag encountering low and high affinity tumor antigens were equally activated and expressed similar levels of inhibitory receptors, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high affinity TCR:pMHC interactions led to complete loss of cytokine production, T cells with low affinity interactions remained functional, suggesting that TSTs with high affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming, with important implications for immunotherapy.

Abstract A093: Impact of antigen affinity on T cell dysfunction in solid tumors

Abstract Although tumor-specific CD8 T cells are found in human solid tumors, cancers progress, indicating that these T cells are dysfunctional. When naïve CD8 T cells encounter antigen in an inflammatory context (e.g. acute infection), a cell-intrinsic program is initiated that drives T cells to expand, produce effector cytokines, and differentiate into effector and memory T cells. In contrast, dysfunctional CD8 T cells in solid tumors express high levels of inhibitory receptors and fail to execute effector functions; however, the regulatory mechanisms underlying tumor-specific T cell dysfunction remain poorly defined. T cell-mediated immune responses are triggered by T cell receptor (TCR) binding to peptide-major histocompatibility complex (pMHC) on the surface of antigen presenting cells. The affinity of TCR:pMHC interaction is a critical determinant of CD8 T cell expansion and effector function in acute infections; while both low- and high-affinity antigens can activate naïve antigen-specific CD8 T cells, T cells with high-affinity TCR:pMHC interactions generally show superior effector function. However, little is known about how tumor antigen affinity impacts the activation of T cells, the induction of T cell dysfunction in progressing tumors, and T cell susceptibility to immunotherapeutic reprogramming. To investigate the role of TCR:pMHC affinity in T cell differentiation in progressing tumors, we generated tumor cell lines expressing altered peptide ligands (APL) derived from SV40 large T antigen epitope I (SV40-I). These APLs are recognized by SV40-I-specific transgenic CD8 T cells (TCRSV40-I) with varying functional avidity. We found that tumor-infiltrating TCRSV40-I encountering low- and high-affinity tumor antigens were equally activated and displayed similar expression levels of inhibitory receptors, such as PD1 and Lag3, suggesting that even very weak TCR ligations can induce a typical “exhaustion” phenotype. Strikingly, while high-affinity TCR:pMHC interactions led to complete loss of interferon gamma and tumor necrosis factor alpha production, T cells with low-affinity interactions remained functional and capable of producing high levels of these effector cytokines suggesting that contrary to our expectations, tumor-specific T cells with high-affinity TCR:pMHC interactions enter a profound state of dysfunction. Future experiments will test the impact of tumor antigen affinity on the efficacy of therapeutic reprogramming including checkpoint blockade, which will have important implications for immunotherapy. Citation Format: Mojdeh Shakiba, Mary Philip, Andrea Schietinger. Impact of antigen affinity on T cell dysfunction in solid tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A093.

Inhibitory receptors as targets for cancer immunotherapy

Inhibitory receptors expressed on T cells control immune responses while limiting autoimmunity. However, tumors can hijack these "checkpoints" for protection from immune attack. Tumor-specific T cells that exhibit an exhausted, unresponsive phenotype express high levels of inhibitory receptors including CTLA4, PD1, and LAG3, among others. Intratumoral regulatory T cells promote immunosuppression and also express multiple inhibitory receptors. Overcoming this inhibitory receptor-mediated immune tolerance has thus been a major focus of recent cancer immunotherapeutic developments. Here, we review how boosting the host's immune system by blocking inhibitory receptor signaling with antagonistic mAbs restores the capacity of T cells to drive durable antitumor immune responses. Clinical trials targeting the CTLA4 and PD1 pathways have shown durable effects in multiple tumor types. Many combinatorial therapies are currently being investigated with encouraging results that highlight enhanced antitumor immunogenicity and improved patient survival. Finally, we will discuss the ongoing identification and dissection of novel T-cell inhibitory receptor pathways, which could lead to the development of new combinatorial therapeutic approaches.

Primary human cytomegalovirus infection induces the expansion of virus-specific activated and atypical memory B cells.

Although neutralizing antibodies play a central role in the control of cytomegalovirus (CMV) dissemination, little is known about the response of B lymphocytes to primary human CMV infection. The proportion, phenotype, specificity, and functionality of B-cell subsets were studied in a cohort of pregnant women with primary CMV infection. CMV-seronegative pregnant women, as well as CMV-seronegative and CMV-seropositive healthy adults, were included as controls. Primary CMV infection was associated with a sustained expansion of activated (CD27(+)CD20(+)CD21(low)) and atypical (CD27(-)CD20(+)CD21(low)) memory B cells (MBCs). Both subsets expressed an effector phenotype, and their proportions were correlated with viremia. Activated MBCs expressed high levels of activation markers and included high frequencies of tumor necrosis α (TNF-α)-producing cells, whereas atypical MBCs expressed high levels of inhibitory receptors and had low TNF-α responses. Fluorescent-labeled antigen experiments indicated that activated and atypical MBCs were enriched in CMV-specific cells. Primary CMV infection mobilizes a large pool of memory B cells that includes activated and atypical MBCs. The functional regulation of CMV-specific MBCs may limit the production of antibodies and the control of viral dissemination.

ILT3+ ILT4+ Tolerogenic Endothelial Cells in Transplantation

T cells can recognize allogeneic major histocompatibility complex (MHC) antigens by two distinct routes: either directly as intact molecules or indirectly as processed peptides presented by syngeneic antigen-presenting cells (APC). The graft endothelium plays an important role in rejection eliciting and serving as a target of T cells activated via the direct and/or indirect allorecognition pathway. Recent evidence demonstrates, however, that endothelial cells are also endowed with the capacity to downregulate alloreactivity inducing tolerogenic responses. Similar to professional APC (such as dendritic cells), endothelial cells express high levels of inhibitory receptors (ILT3 and ILT4 in humans and PIR-B in rodents) and low levels of costimulatory and adhesion molecules upon interaction with allospecific CD8 T suppressor cells or exposure to inhibitory cytokines. Because of the importance of endothelial cells in the activation and control of T cell reactivity, understanding of their biology is crucial for the development of new strategies for induction of transplantation tolerance and treatment of cancer, chronic infection, and autoimmunity.