Co-expression Of Multiple Inhibitory Receptors Research Articles

Pre-Transplant Exhausted Marrow Infiltrating T Lymphocytes in MRD Negative Patients with Acute Leukemia Predict Early Relapse Following Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplant (alloSCT) is curative for patients with high-risk acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Despite this, relapse remains the leading cause of treatment failure. The use of flow cytometry to identify aberrant leukemia-associated immunophenotypes is an established method of detecting minimal residual disease (MRD). MRD status immediately prior to transplant remains one of the single best predictors of post-transplant outcome. Unfortunately, not all MRD(-) patients maintain remission post-transplant. Therefore, we sought to find additional predictors of post-transplant relapse. T cell exhaustion occurs following continuous exposure to antigen and leads to sustained up-regulation of multiple inhibitory receptors, progressive loss of function, and expression of a distinct transcriptional state. Bachireddy et al recently reported that exhausted CD8+ marrow infiltrating lymphocytes (MILs) were a novel predictor of response to donor lymphocyte infusion (DLI) for patients with relapsed chronic myelogenous leukemia (CML). Based on this principle, we hypothesized that exhausted CD8+MILs may also be predictive of outcome for MRD(-) patients prior to alloSCT.We matched pre-transplant samples from 17 MRD(-) patients who maintained remission for greater than 2 years post-transplant to 11 MRD(-) patients who suffered relapse within 2 years of transplant. Patients had either ALL or AML and did not differ by demographic details. MILs were evaluated for exhaustion using 2 multicolor flow cytometry panels which collectively included a viability dye, T cell markers (CD3 and CD8) surface inhibitory receptors (PD-1, Tim-3, and TIGIT), transcription factors (T-bet and Eomes), and intracellular cytokine staining (IL-2, TNF-α, and IFN-γ) in response to CD3/CD28 bead stimulation.There was no difference in the percentage of total T cells (CD3), the CD8+ T cells (CD3+CD8+), or CD8+ T cell subsets between the 2 groups. While there was also no difference in the mean fluorescent intensity (MFI) of Tim-3, both PD-1 and TIGIT were significantly elevated on the CD8+ MILs of MRD(-) patients that eventually relapsed (p=0.01 and p=0.03, respectively). Among CD8+ MILs, cells with the highest PD-1 expression (PD-1High) were significantly elevated in the relapse group (p=0.006), as was co-expression of multiple inhibitory receptors on single cells (p=0.01) (Figure A,B). In contrast, the proportion of CD8+ MILs lacking all 3 of these inhibitory receptors was significantly higher among the MRD(-) group that maintained remission (p=0.01) (Figure C). Interestingly, CD8+ MILs in the remission group with dual PD-1+TIGIT+ expression or expressing PD-1High were more likely to be central memory cells (both p=0.05), while these same immunophenotypes were significantly more often effector memory cells in patients who went on to relapse (p=0.03 and p=0.02, respectively).As noted above, inhibitory receptor expression alone does not define exhaustion. We further assessed the CD8+ inhibitory receptor expressing MILs in the relapse group for other phenotypic evidence of exhaustion by evaluating their function and transcription factor expression. Cells with either PD-1+TIGIT+ dual expression or PD-1High were directly compared to cells that lacked inhibitory receptors. The dual PD-1+TIGIT+ cells had impaired ability to produce TNF-α and IFN-γ (both with p<0.0001), while PD-1High cells produced significantly less TNF-α (p<0.0001), IFN-γ (P<0.0001) and IL-2 (p=0.0001). Moreover, both sets of MILs had significantly decreased polyfunctional cytokine production compared to cells without these surface receptors (p < 0.0001) (Figure D). Furthermore, transcription factor expression was also altered among inhibitory receptor expressing cells. They exhibited both increased early exhaustion as evidenced by high T-bet expression in PD-1 expressing cells as well as terminal exhaustion indicated by significantly elevated Eomes expression on PD-1High expressing cells (Figure E,F).Collectively, this study suggests that the presence of exhaustion in CD8+ MILs in acute leukemia patients who are MRD(-) prior to transplant may be at higher risk for post-transplant relapse. If validated, this data may be used to further risk-stratify patients and/or direct pre- or post-transplant therapy, including the potential use of checkpoint blockade. [Display omitted] DisclosuresBachanova:Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Wagner:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding; Celegene: Consultancy.

Landscape of Exhausted Virus-Specific CD8 T Cells in Chronic LCMV Infection

A hallmark of chronic infections is the presence of exhausted CD8 Tcells, characterized by a distinct transcriptional program compared with functional effector or memory cells, co-expression of multiple inhibitory receptors, and impaired effector function, mainly driven by recurrent Tcell receptor engagement. In the context of chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, most studies focused on studying splenic virus-specific CD8 Tcells. Here, we provide a detailed characterization of exhausted CD8 Tcells isolated from six different tissues during established LCMV infection, using single-cell RNA sequencing. Our data reveal that exhausted cells are heterogeneous, adopt organ-specific transcriptomic profiles, and can be divided into five main functional subpopulations: advanced exhaustion, effector-like, intermediate, proliferating, or memory-like. Adoptive transfer experiments showed that these phenotypes are plastic, suggesting that the tissue microenvironment has a major impact in shaping the phenotype and function of virus-specific CD8 Tcells during chronic infection.

Abstract A197: The HMG transcription factor TOX induces a transcriptional and epigenetic program of CD8+ T-cell exhaustion in chronic infection and cancer

Abstract The protective capacity of the adaptive immune system relies on efficient and coordinated transitions between cellular fates. Following initial activation by specific antigen, naïve CD8+ T-cells proliferate extensively and undergo a highly orchestrated program of molecular rewiring and differentiation into effector CD8+ T-cells (TEFF) that can mediate protection through cytotoxicity and production of inflammatory cytokines. If the infection or antigen is cleared, most of this TEFF pool dies, but a subset persists, undergoing additional differentiation to form a pool of long-lived, self-renewing memory T-cells (TMEM) capable of mounting rapid recall responses. In contrast, during chronic infections or cancer, when T-cell stimulation persists, this program of functional T-cell differentiation is diverted and T-cells fail to sustain robust effector functions, instead becoming exhausted. Exhausted CD8+ T-cells (TEX) may balance limited pathogen or tumor control while restraining damaging immunopathology, but the consequence of restrained functionality is disease persistence and possible progression. Though first described in mice infected with lymphocytic choriomeningitis virus (LCMV), it is now clear that T-cell exhaustion is a common feature of many chronic infections as well as a variety of cancers in both mice and humans. Indeed, TEX are highly therapeutically relevant since these cells are a major target of checkpoint blockade mediated immune reinvigoration in human cancer patients. T-cell exhaustion is characterized by the progressive decline in effector function including the hierarchical loss of inflammatory cytokine production (IL-2, TNFa, IFNg). TEX also sustain high co-expression of multiple inhibitory receptors (PD-1, LAG3, TIGIT, CD160, TIM-3, 2B4), have reduced glycolytic and oxidative phosphorylation capacity, and impaired proliferation and survival. Underlying these major differences in TEX compared to TEFF and TMEM, is a distinct transcriptional program highlighted by altered use of key transcription factors and altered transcriptional circuits. Moreover, recent epigenetic analyses revealed that TEX differ from TEFF and TMEM by ~6,000 open chromatin regions, similar to differences between other major hematopoietic lineages, suggesting that TEX are not simply a state of activation of TEFF or TMEM, but rather are a distinct immune lineage. Yet the mechanisms that initiate this TEX fate commitment and epigenetic and transcriptional programming have thus far remained poorly understood, and exhaustion-specific TFs or transcriptional programming activities have remained elusive. Here we identify the HMG-box protein TOX as a master regulator of the TEX lineage. We find that robust TOX expression is limited to chronic infections and cancers in both mice and humans. TOX is largely dispensable for TEFF and TMEM formation, but in the absence of TOX, TEX do not form. Instead, TOX-deficiency results in the accumulation and subsequent rapid deletion of TEFF cells. TOX is induced by calcineurin and, specifically, NFAT2 and then operates in a feed-forward loop to become calcineurin independent and is durably expressed at a high level in TEX, but not TEFF or TMEM. TOX interacts with histone modifying enzyme proteins including the HBO1 complex and the acetyl-transferase Kat7, providing a mechanism for TEX lineage-specific epigenetic changes. Thus, high and sustained induction of TOX causes lineage commitment to TEX by translating persisting TCR stimulation cues into a distinct TEX transcriptional and epigenetic developmental program. Citation Format: Omar Khan, Josephine R. Giles, Sierra McDonald, E. John Wherry. The HMG transcription factor TOX induces a transcriptional and epigenetic program of CD8+ T-cell exhaustion in chronic infection and cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A197.

Abstract LB-111: Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion

Abstract In patients with chronic viral infection or cancer, continuous antigen exposure results in T cell exhaustion, which is characterized by sustained co-expression of multiple inhibitory receptors (ex. PD-1, TIM-3, LAG-3) and a hierarchical loss of effector function. Human T cells expressing a high-affinity anti-GD2 chimeric antigen receptor (CAR, HA.28z) develop phenotypic and functional hallmarks of exhaustion due to clustering of surface CAR and tonic signaling in the absence of antigen. In the present study, we exploited the HA.28z CAR as a model to characterize human T cell exhaustion and assess its potential for reversibility. CyTOF analyses of dysfunctional HA.28z CAR T cells demonstrated a distinct phenotype in which canonical exhaustion markers, T cell differentiation markers, and effector transcription factors were differentially expressed compared to CD19.28z CAR T cells that do not tonically signal. To interrogate the reversibility of T cell exhaustion, we engineered a regulatable HA.28z CAR by fusing a destabilization domain to the C-terminus of the CAR that rapidly induces CAR protein degradation. Addition of a small molecule stabilizes the protein and results in CAR surface expression. Using this model, we modulated the duration and intensity of tonic signaling by precisely modulating CAR surface expression. We hypothesized that transient cessation of tonic signaling would allow exhausted CAR T cells to recover and regain effector function. Following onset of exhaustion, elimination of CAR surface expression for 4 days resulted in a profound reversal of the exhausted phenotype. CyTOF analyses of reinvigorated cells indicated enhanced T cell memory formation, diminished surface marker expression of multiple exhaustion markers (ex. PD-1, TIM-3, LAG-3, CTLA-4, CD39) and reduced expression of transcription factors T-bet and Blimp-1. Furthermore, RNA sequencing of exhausted and reinvigorated HA.28z CAR T cells revealed a global molecular reprogramming upon removal of tonic CAR signaling, suggesting that CAR T cell exhaustion is functionally reversible. Upon CAR re-expression and co-culture with tumor antigen, reinvigorated HA.28z CAR T cells exhibited augmented killing and cytokine secretion compared to exhausted cells that continuously expressed surface CAR during expansion. In vivo studies assessing CAR T cell exhaustion reversibility using this model are currently being evaluated. In summary, tuning CAR surface expression offers a novel strategy to augment both the safety and efficacy of CAR T cell therapy. Moreover, these studies suggest that transient “rest” in T cells experiencing chronic antigen stimulation may be one mechanism by which exhaustion is prevented or reversed. Citation Format: Evan W. Weber, Rachel C. Lynn, Meena Malipatlolla, Elena Sotillo, Peng Xu, Crystal L. Mackall. Precise regulation of CAR signaling prevents and reverses CAR T cell exhaustion [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 LB-111.

The binding of an anti-PD-1 antibody to Fc\u03b3R\u0399 has a profound impact on its biological functions

Antibodies targeting PD-1 have been demonstrated durable anti-cancer activity in certain cancer types. However, the anti-PD-1 antibodies are less or not efficacious in many situations, which might be attributed to co-expression of multiple inhibitory receptors or presence of immunosuppressive cells in the tumor microenvironment. Most of the anti-PD-1 antibodies used in clinical studies are of IgG4 isotype with the S228P mutation (IgG4S228P). The functional impact by the interaction of anti-PD-1 IgG4S228P antibody with Fc gamma receptors (FcγRs) is poorly understood. To assess the effects, we generated a pair of anti-PD-1 antibodies: BGB-A317/IgG4S228P and BGB-A317/IgG4-variant (abbreviated as BGB-A317), with the same variable regions but two different IgG4 Fc-hinge sequences. There was no significant difference between these two antibodies in binding to PD-1. However, BGB-A317/IgG4S228P binds to human FcγRI with high affinity and mediates crosslinking between PD-1 and FcγRI. In contrast, BGB-A317 does neither. Further cell-based assays showed that such crosslinking could reverse the function of an anti-PD-1 antibody from blocking to activating. More importantly, the crosslinking induces FcγRI+ macrophages to phagocytose PD-1+ T cells. In a mouse model transplanted with allogeneic human cancer cells and PBMCs, BGB-A317 showed significant tumor growth inhibition, whereas BGB-A317/IgG4S228P had no such inhibition. Immunohistochemistry study revealed an inverse correlation between FcγRI+ murine macrophage infiltration and the density of CD8+PD-1+ human T cells within tumors in the BGB-A317/IgG4S228P-treated group. These evidences suggested that FcγRI+ binding and crosslinking had negative impact on the anti-PD-1 antibody-mediated anti-cancer activity.

Downregulated IL-21 Response and T Follicular Helper Cell Exhaustion Correlate with Compromised CD8 T Cell Immunity during Chronic Toxoplasmosis.

CD8 T cells are important for maintaining the chronicity of Toxoplasma gondii infection. In a T. gondii encephalitis susceptible model, we recently demonstrated that CD4 T cells play an essential helper role in the maintenance of the effector response and CD8 T cell dysfunctionality was linked to CD4 T cell exhaustion. However, CD4 T cells are constituted of different subsets with various functions and the population(s) providing help to the CD8 T cells has not yet been determined. In the present study, T follicular helper cells (Tfh), which are known to be essential for B cell maturation and are one of the main sources of IL-21, were significantly increased during chronic toxoplasmosis. However, at week 7 p.i., when CD8 T cells are exhausted, the Tfh population exhibited increased expression of several inhibitory receptors and levels of IL-21 in the serum were decreased. The importance of IL-21 in the maintenance of CD8 T cells function after T. gondii infection was further demonstrated in IL-21R KO mouse model. Interestingly, while CD8 T cells from both knockout (KO) and wild-type mice expressed similar levels of PD-1, animals with defective IL-21 signaling exhibited lower polyfunctionality than wild-type controls. This reduced polyfunctional ability observed in CD8 T cells from KO mice was associated with a significant increase in other inhibitory receptors like Tim-3, LAG-3, and 2B4. Furthermore, the animals exhibited greater signs of Toxoplasma reactivation manifested by the reduced number of cysts and increased expression of tachyzoite (replicative form of the parasite) specific genes (SAG1 and ENO2) in the brain. Also, IL-21R KO mice displayed a higher frequency of tachyzoite-infected monocytes in the blood and spleen. Our findings suggest the importance of Tfh and IL-21 during chronic toxoplasmosis and establish a critical role for this cytokine in regulating CD8 T cell dysfunction by preventing the co-expression of multiple inhibitory receptors during chronic parasitic infection.

Early Effector T Lymphocytes Coexpress Multiple Inhibitory Receptors in Primary Non-Small Cell Lung Cancer.

Clinical efficacy of PD-1/PD-L1 targeting relies upon the reactivation of tumor-specific but functionally impaired PD-1+ T cells present before therapy. Thus, analyzing early-stage primary tumors may reveal the presence of T cells that are not yet functionally impaired. In this study, we report that activated (HLA-DR+) T cells with an effector memory (TEM) profile are enriched in such lesions. Tumor-infiltrating lymphocytes coexpressed PD-1 with the inhibitory receptors TIM-3, CTLA-4, LAG-3, and TIGIT, but also displayed a recently activated, nonexhausted phenotype. We also identified a subset of CD8+PD-1+FOXP3+ T lymphocytes at the earliest phase of functional differentiation after priming, termed "early effector cells" (EEC), which also exhibited an activated nonexhausted phenotype, but was less differentiated and associated with coexpression of multiple inhibitory receptors. In response to autologous tumor, EECs upregulated CD107a, produced IL2 and IFNγ, and were competent for differentiation. The identification of EECs marked by inhibitory receptor expression at tumor sites will enable investigations of early stages of adaptive antitumor immunity, as well as support the rationale for administering immunotherapy in early-stage non-small cell lung cancer. Cancer Res; 77(4); 851-61. ©2016 AACR.

Blockade of Programmed Death 1 Augments the Ability of Human T Cells Engineered to Target NY-ESO-1 to Control Tumor Growth after Adoptive Transfer.

Tumor-infiltrating lymphocytes (TILs) become hypofunctional, although the mechanisms are not clear. Our goal was to generate a model of human tumor-induced TIL hypofunction to study mechanisms and to test anti-human therapeutics. We transduced human T cells with a published, optimized T-cell receptor (TCR) that is directed to a peptide within the cancer testis antigen, NY-ESO-1. After demonstrating antigen-specific in vitro activity, these cells were used to target a human lung cancer line that expressed NY-ESO-1 in the appropriate HLA context growing in immunodeficient mice. The ability of anti-PD1 antibody to augment efficacy was tested. Injection of transgenic T cells had some antitumor activity, but did not eliminate the tumors. The injected T cells became profoundly hypofunctional accompanied by upregulation of PD1, Tim3, and Lag3 with coexpression of multiple inhibitory receptors in a high percentage of cells. This model allowed us to test reagents targeted specifically to human T cells. We found that injections of an anti-PD1 antibody in combination with T cells led to decreased TIL hypofunction and augmented the efficacy of the adoptively transferred T cells. This model offers a platform for preclinical testing of adjuvant immunotherapeutics targeted to human T cells prior to transition to the bedside. Because the model employs engineering of human T cells with a TCR clone instead of a CAR, it allows for study of the biology of tumor-reactive TILs that signal through an endogenous TCR. The lessons learned from TCR-engineered TILs can thus be applied to tumor-reactive TILs.

Progression of Lung Cancer Is Associated with Increased Dysfunction of T Cells Defined by Coexpression of Multiple Inhibitory Receptors.

Dysfunctional T cells present in malignant lesions are characterized by a sustained and highly diverse expression of inhibitory receptors, also referred to as immune checkpoints. Yet, their relative functional significance in different cancer types remains incompletely understood. In this study, we provide a comprehensive characterization of the diversity and expression patterns of inhibitory receptors on tumor-infiltrating T cells from patients with non-small cell lung cancer. In spite of the large heterogeneity observed in the amount of PD-1, Tim-3, CTLA-4, LAG-3, and BTLA expressed on intratumoral CD8(+) T cells from 32 patients, a clear correlation was established between increased expression of these inhibitory coreceptors and progression of the disease. Notably, the latter was accompanied by a progressively impaired capacity of T cells to respond to polyclonal activation. Coexpression of several inhibitory receptors was gradually acquired, with early PD-1 and late LAG-3/BTLA expression. PD-1 blockade was able to restore T-cell function only in a subset of patients. A high percentage of PD-1(hi) T cells was correlated with poor restoration of T-cell function upon PD-1 blockade. Of note, PD-1(hi) expression marked a particularly dysfunctional T-cell subset characterized by coexpression of multiple inhibitory receptors and thus may assist in identifying patients likely to respond to inhibitory receptor-specific antibodies. Overall, these data may provide a framework for future personalized T-cell-based therapies aiming at restoration of tumor-infiltrating lymphocyte effector functions.

PP 5.6 - Co-expression of multiple inhibitory receptors on CD8 + T cells in viremic and ART-suppressed HIV-1(+) individuals

PP 5.6 - Co-expression of multiple inhibitory receptors on CD8 + T cells in viremic and ART-suppressed HIV-1(+) individuals

Hierarchy of inhibitory receptor expression by Hepatitis B Virus (HBV)-specific CD8+ T cells in chronic HBV infection

Virus-specific CD8+ T cells are thought to play a crucial role in the immunobiology of HBV infection. In chronic HBV infection, up-regulation of several inhibitory signaling pathways by HBV-specific CD8+ T cells is thought to contribute to viral persistence. However, the relative contribution of the individual inhibitory pathways has not been addressed so far. In this study, we determined the coexpression of multiple inhibitory receptors (PD–1, 2B4, CD160, KLRG1 and Tim–3) ex vivo by HBV-specific CD8+ T cells from 17 patients (16 HBeAg neg., 1 HBeAg pos.) with significant HBV-specific T cell frequencies and compared it to CD8+ T cells specific for other viruses (FLU, CMV, HCV). The differentiation stage of virus-specific CD8+ T cells was analyzed by CCR7, CD27 and CD45RA expression. We found that the coexpression of inhibitory receptors was associated with intermediate T cell differentiation stages. Further, HBV-specific CD8+ T cells expressed higher amounts of PD–1 and 2B4 compared to CD160, KLRG1 and Tim–3. PD–1+2B4+ HBV-specific CD8+ T cells were enriched intrahepatically compared to the peripheral blood. The expression pattern of inhibitory receptors clearly differed between HBV-specific (high PD–1 and 2B4 expression) and Influenza- (high Tim–3 expression) or CMV-specific CD8+ T cells (high 2B4 and KLRG1 expression) but showed a similarly high expression of PD–1 and 2B4 with HCV-specific CD8+ T cells, suggesting a common hierarchy of inhibitory receptor expression between CD8+ T cells specific for hepatitis B and C viruses. These results suggest a hierarchy of inhibitory receptor expression by HBV-specific CD8+ T cells dominated by PD–1 and 2B4. They also suggest a rationale for the therapeutic targeting of these inhibitory receptor pathways in chronic HBV infection.

Mechanisms of costimulation

The immune system has the remarkable ability to defend against a diversity of microbial pathogens, yet not respond to self. The discovery of the T-cell antigen receptor (TCR) provided an explanation for the specificity and diversity of T-cell responses. Yet, functional studies of Lafferty and colleagues (1) suggested that antigen alone was not sufficient to drive the activation of naive T cells and led to the two signal concept of T-cell activation. According to this model, productive T-cell activation requires a first signal provided by the interaction of antigenic peptide/major histocompatibility complex (MHC) with the TCR and a second, antigen-independent co-signal, the ‘costimulatory signal’. In addition, seminal studies of Jenkins and Schwartz (2, 3) demonstrated that TCR-mediated activation of T cells in the absence of costimulation resulted in antigen-specific unresponsiveness (termed T-cell anergy), rendering the T cells unable to respond to subsequent exposure to antigen. Thus, costimulation was postulated to have a pivotal role in determining whether the outcome of T-cell encounter with antigen would be activation or anergy. The critical role of costimulation in regulating the immune response has given impetus to the study of costimulation and resulted in incredible growth of this field, since an understanding of costimulation is of fundamental and therapeutic interest. Early studies defined the function of the CD28 receptor as a stimulatory receptor for the activation of naive T cells (2), and identified the B7 family members, B7-1 (CD80) and B7-2 (CD86) as its ligands (5–7). The interaction between CD28 and B7-1/B7-2 fulfilled many of the requirements for the costimulatory signal postulated by Lafferty, Schwarz, and colleagues (1). The CD28 homolog cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (3) was found to be a higher affinity binding partner for B7-1 and B7-2, as compared to CD28 (4). It was presumed that CTLA-4 would also be a stimulatory receptor; however, the dramatic fatal inflammatory phenotype of CTLA-4 deficient (−/−) mice revealed the critical inhibitory function of CTLA-4 (10, 11). Furthermore, the phenotype of CTLA-4−/− mice demonstrated that T-cell costimulatory receptors could deliver inhibitory, as well as stimulatory, second signals for T-cell responses, and provided the first indication that inhibitory second signals (co-inhibitory) could regulate T-cell tolerance. While the B7-1/B7-2:CD28/CTLA-4 pathway is the best characterized T-cell costimulatory pathway, there are now many additional costimulatory pathways. These pathways fall into two major families: the immunoglobulin (Ig) superfamily, which includes the B7/CD28, CD2, and T-cell Ig and mucin domain (TIM) families, and the tumor necrosis family (TNF)/TNF receptor (TNFR) family. Reviews in this issue of Immunol Rev discuss the functions of pathways within all of these families. These articles not only review the current understanding of costimulation on the fundamental level, but also the therapeutic opportunities and challenges of targeting costimulatory pathways for treating autoimmunity, graft rejection, cancer and infectious diseases. In this introduction, I provide perspectives on key functions of costimulatory pathways, highlighting recent advances, and discuss how this progress is changing our view of the functions of T-cell costimulation.

Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection

T cell exhaustion often occurs during chronic infections and prevents optimal viral control. The molecular pathways involved in T cell exhaustion, however, remain poorly understood. We demonstrate that exhausted CD8+ T cells are subject to complex layers of negative regulation due to co-expression of multiple inhibitory receptors. Exhausted CD8+ T cells expressed up to 7 inhibitory receptors. Co-expression of multiple distinct inhibitory receptors correlated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by diverse inhibitory pathways was non-redundant since blockade of PD-1 and LAG-3 simultaneously in vivo synergistically improved T cell responses and reduced viral load. Thus, CD8+ T cell responses during chronic viral infections are regulated by complex patterns of co-expressed inhibitory receptors.