Insights into CD8 T Cell Activation and Exhaustion from a Mouse Gammaherpesvirus Model

The molecular basis of the failed immune response in chronic HBV: Therapeutic implications

Increased Frequency of TOX+ CD39+ TIGIT+ CD73- CD8+ T Cells in Patients with Newly Diagnosed AML

The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1(-/-) mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1(-/-) CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1(-/-) CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1(-/-) CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1(-/-) mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1(-/-) mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.

Limited efficiency of endogenous interleukin-7 levels in T cell reconstitution during HIV-1 infection: will exogenous interleukin-7 therapy work?

CD8+ T cells play a critical role in specific immunity. In recent years, cell therapy has been emerging rapidly. The specific cytotoxic capabilities of these cells enable them to precisely identify and kill cells presenting specific antigens. This has demonstrated promise in the treatment of autoimmune diseases and cancers, with wide-ranging applications and value. However, in some diseases, such as tumors and chronic infections, T cells may adopt an exhausted phenotype, resulting in a loss of cytotoxicity and limiting their further application. Epigenetics plays a significant role in the differentiation and regulation of gene expression in cells. There is extensive evidence indicating that epigenetic remodeling plays an important role in T cell exhaustion. Therefore, further understanding its role in CD8+ T cell function can provide insights into the programmatic regulation of CD8+ T cells from a genetic perspective and overcome these diseases. We attempted to describe the relationship between the activation, function, and exhaustion mechanisms of CD8+ T cells, as well as epigenetics. This understanding makes it possible for us to address the aforementioned issues.

In chronic infections and cancers, T lymphocytes (T cells) are exposed to persistent antigen or inflammatory signals. The condition is often associated with a decline in T-cell function: a state called “exhaustion”. T cell exhaustion is a state of T cell dysfunction characterized by increased expression of a series of inhibitory receptors (IRs), decreased effector function, and decreased cytokine secretion, accompanied by transcriptional and epigenetic changes and metabolic defects. The rise of immunotherapy, particularly the use of immune checkpoint inhibitors (ICIs), has dramatically changed the clinical treatment paradigm for patients. However, its low response rate, single target and high immunotoxicity limit its clinical application. The multiple immunomodulatory potential of traditional Chinese medicine (TCM) provides a new direction for improving the treatment of T cell exhaustion. Here, we review recent advances that have provided a clearer molecular understanding of T cell exhaustion, revealing the characteristics and causes of T cell exhaustion in persistent infections and cancers. In addition, this paper summarizes recent advances in improving T cell exhaustion in infectious diseases and cancer with the aim of providing a comprehensive and valuable source of information on TCM as an experimental study and their role in collaboration with ICIs therapy.

Aim: This study investigated the effects of successful hepatitis C virus (HCV) treatment with direct-acting antivirals (DAAs) on CD4+ T cell recovery in HIV/HCV coinfected immunological non-responders (INRs) to antiretroviral therapy (ART). The study assessed changes in CD4+ and CD8+ T cell counts, immune activation, inflammation, T cell exhaustion, and the size of the naïve CD4+ T cell pool following DAA therapy to determine whether HCV suppression enhances immune restoration in coinfected INRs. Methods: Three groups were analyzed: DAA-treated INRs (n = 9), untreated HIV/HCV coinfected INRs (n = 10), and healthy controls (n = 10). Plasma cytokine levels and viral loads were quantified using multiplex immunoassay and real-time PCR. Peripheral blood mononuclear cells were analyzed via flow cytometry to evaluate T cell subsets, activation (HLA-DR+CD38+), and exhaustion markers (PD-1, TIGIT). Results: Both INR groups showed significantly lower CD4+ T cell counts and elevated CD4+ T cell proliferation compared to controls, with no significant difference between DAA-treated and untreated patients. Deficits in naïve CD4+ T cells were observed in both INR groups but reached statistical significance only in untreated individuals. Activated CD4+ and CD8+ T cells and proinflammatory cytokines of IFN and IL-10 families remained elevated in INRs after DAA treatment. DAAs reduced PD-1 and TIGIT expression on CD4+ T cells, suggesting attenuated exhaustion, but did not alter exhausted T cell frequencies in CD4+ or CD8+ T cells. Conclusions: HCV coinfection in people living with HIV (PLWH) not only increases the risk of immunological non-response to ART but also has lasting impacts on the immune system. Even after successful HCV clearance with DAAs, INRs experience persistent immune dysregulation, including low CD4+ T cell counts, deficits in the naïve CD4+ T cell compartment, and ongoing inflammation. This indicates that HCV eradication alone is insufficient to reverse the long-term immune damage resulting from coinfection.

Multiple suppressive mechanisms within the tumor microenvironment (TME) are capable of blunting anti-tumor T cell responses. These include engagement of inhibitory receptors expressed in tumor-associated, exhausted CD8 T cells, such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT). While immune checkpoint blockade therapies aimed at reversing the dysfunctional state of tumor-associated T cells have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8 T cells to be reinvigorated by, for example, PD-1/PD-L1 blockade. As CD8 T cell exhaustion (or dysfunction) is a major therapeutic challenge, gaps in our understanding of cellular and molecular mechanisms underlying the T cell exhaustion (or dysfunction) program in cancer warrant further study of pathways that program T cells toward exhaustion (or dysfunction). Through comprehensive immune profiling of tumor-infiltrating T lymphocytes (TILs), we found that CD47 expression in CD8 TILs isolated from melanoma patients significantly correlates with expression of several checkpoint inhibitory molecules (e.g., TIM-3, PD-1 and LAG-3). Additionally, our re-analysis of single cell data from melanoma patients revealed that terminally exhausted T cells (Tex) and TCF7hi Tex precursor cells exhibit high levels of CD47 transcripts, suggesting phenotypic association of CD47 with T cell exhaustion. We confirmed our observations in murine B16-F10 melanoma where CD47 expression is significantly upregulated in exhausted CD8 TILs. We also show that CD47 functions as a negative regulator for T cell proliferation and function during T cell priming. To address the role of CD47 during the development of CD8 T cell exhaustion/dysfunction in cancer, we performed adoptive T cell transfer of the naïve-sorted Cd47+/+ (WT) and Cd47+/- (Het) antigen specific Pmel-1 CD8 T cells (but not Cd47-deficient Pmel-1 CD8 T cells as they would be subject to innate immune clearance) into B16 tumor-bearing mice and found that Cd47-Het Pmel-1 CD8 TILs, as compared to the Cd47-WT Pmel-1 CD8 TILs, exhibit less expression of exhaustion-related genes (e.g. Pdcd1, Lag3 and Tox), and increased expression of genes associated with T cell activation and proliferation (e.g. Mki67, Lck, Cd69, Gzma, Gzmk). We further confirmed that thrombospondin-1 (TSP-1), as an extracellular matrix protein and a ligand of CD47, contributes to driving the differentiation of CD8 T cells toward exhaustion. Our data highlight for the first time the potential of extracellular matrix protein TSP-1 in programming CD8 T cell exhaustion in cancer through its interaction with CD47 expressed on CD8 T cells. Citation Format: Chien-Huan Weng, Fadi Samaan, Sadna Budhu, Levi Mangarin, Sébastien Monette, Cailian Liu, Stephane Pourpe, Linda Hamadene, Hong Zhong, Xia Yang, David Schroder, Roberta Zappasodi, Pamela Holland, Jedd D. Wolchok, Taha Merghoub. Potential role of CD47 in T cell exhaustion program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6150.

Mutation of the CD28 Costimulatory Domain Confers Decreased CAR T Cell Exhaustion

Allogeneic stem cell transplantation (alloSCT) is a highly effective, curative therapy for leukemia yet does not provide a survival benefit above autologous SCT in patients with multiple myeloma (MM). To explore this, we developed preclinical models of SCT using C57Bl/6 recipient mice and either C57Bl/6 (ASCT) or C3H.SW (alloSCT) donor grafts. Importantly, these models recapitulated the clinical setting whereby alloSCT provided superior outcomes, compared to ASCT, in recipients bearing MLL-AF9-driven acute myeloid leukemia (AML) but not in those bearing Vk*MYC-MM. Interestingly, we found that MM-specific, T cell-mediated immunity was generated after ASCT, which failed due to MM-induced T cell exhaustion. MM relapse after ASCT could be prevented by TIGIT or PD-1 targeted immune checkpoint inhibition or via depletion of suppressive CSF1-R+ myeloid cells. Conversely, after alloSCT, T cell exhaustion was driven principally by alloantigen and CD8 T cells expressed high levels of PD-1, TIGIT and TIM-3. Furthermore, Vk*MYC myeloma exploited this alloantigen-driven T cell exhaustion via expression of high levels of PD-L1 and CD155 (the cognate ligands for PD-1 and TIGIT), which were expressed minimally on MLL-AF9 AML. To exploit alloSCT in MM, we used post-transplant cyclophosphamide (PT-Cy) to delete high affinity alloreactive T cells that generate the exhausted donor T cell pool. Subsequent administration of CD137 agonists enhanced T cell activation and cytolytic activity within bone marrow, without exacerbating GVHD. Thus, PT-Cy provides a platform for optimizing immunotherapy after alloSCT.

Background: Despite significant clinical benefit of checkpoint inhibitors (CPI) in some settings, unfortunately, the majority of patients still fail to respond and/or develop resistance. This is likely due to a complex set of factors including, but not limited to, the presence of immunosuppressive myeloid cells and/or T cell exhaustion due to chronic TCR activation in the absence of sufficient costimulation. ALPN-202, a variant CD80 vIgD™-Fc fusion protein that mediates PD-L1-dependent CD28 costimulation and blocks PD-L1 and CTLA-4, was designed to overcome several of these suppressive mechanisms. The objective of these studies was to measure effects of ALPN-202 on the suppression of T cell activation by M2c macrophages and to determine in vivo effects of ALPN-202 on T cell exhaustion in an adoptively transferred human TCR transgenic tumor model. Methods: M2c macrophages differentiated from primary monocytes with M-CSF and IL-10 were cocultured for 72hr with T cells, anti-CD3 and a titration of ALPN-202, anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies. Cytokine concentrations in the culture media were measured at 24 and 72 hrs, and T cells and macrophages characterized at 72 hrs by flow cytometry. To measure its anti-tumor activity and effect on T cell exhaustion in vivo, ALPN-202 was evaluated in a humanized model using anti-HPV E6 TCR-transduced human T cells transferred into immunodeficient NSG mice bearing HPV+ SCC152 squamous cell tumors stably expressing PD-L1. Tumor volume was measured twice weekly and on day 38 tumors were harvested, digested, and intratumoral T cells characterized for expression of exhaustion markers. Results: In the in vitro coculture assay, ALPN-202 increased T cell proliferation and production of IL-2, IFNγ, TNFα, GM-CSF, IL-6, and IL-21 significantly more potently than CPI alone. Additionally, the M2c macrophages in the presence of ALPN-202 displayed a dose-dependent elevation of MHC II, CD80, and CD86, indicative of a more pro-inflammatory, M1-like phenotype. In the SCC152 tumor model, ALPN-202 induced a more robust anti-tumor response than CPI while the intratumoral T cells expressed lower levels of exhaustion markers. Conclusions: As a dual checkpoint inhibitor and conditional CD28-costimulator, ALPN-202 induced robust and selective T cell costimulation in vitro which overcame M2c macrophage-mediated suppression more potently than CPI alone. Intriguingly, not only were T cells vigorously activated, but M2c macrophages transitioned to a more M1-like proinflammatory phenotype. In a humanized tumor model, ALPN-202 treatment resulted in potent anti-tumor activity with reduced signs of T cell exhaustion in the TME. The data suggest that by combining CD28 costimulation with CPI, ALPN-202 may provide a more robust and persistent anti-tumor T cell response compared to CPI alone. Citation Format: Mark F. Maurer, Siddarth Chandrasekaran, Katherine Lewis, Sherri Mudri, Kayla Kleist, Fariha Ahmed-Qadri, Chelsea Gudgeon, Steven D. Levin, Stacey R. Dillon, Kristine M. Swiderek, Stanford L. Peng. ALPN-202 combines checkpoint inhibition with conditional T cell costimulation to overcome T cell suppression by M2c macrophages and improve the durability of engineered T cell anti-tumor responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-085.

Antigen-specific CD8 T cells are central to the control of chronic infections and cancer, but persistent antigen stimulation results in T cell exhaustion. Exhausted CD8 T cells have decreased effector function and proliferative capacity, partly caused by overexpression of inhibitory receptors such as programmed cell death (PD)-1. Blockade of the PD-1 pathway has opened a new therapeutic avenue for reinvigorating T cell responses, with positive outcomes especially for patients with cancer. Other strategies to restore function in exhausted CD8 T cells are currently under evaluation-many in combination with PD-1-targeted therapy. Exhausted CD8 T cells comprise heterogeneous cell populations with unique differentiation and functional states. A subset of stem cell-like PD-1+ CD8 T cells responsible for the proliferative burst after PD-1 therapy has been recently described. A greater understanding of T cell exhaustion is imperative to establish rational immunotherapeutic interventions.

Purpose: Immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of cancer patients; however, robust and safe combination therapies are still needed. In the tumor microenvironment, prostaglandin E2 receptor type 4 (EP4) signaling has been implicated in both protumoral myeloid cell differentiation and cytotoxic T cell exhaustion. We evaluated the combination of the EP4 antagonist E7046 (clinical trial NCT02540291) with anti-PD1 or anti-CTLA4 in preclinical tumor models, and also interrogated the relationship between PGE2 pathway activation and cancer patient survival. Materials/Methods: Mouse syngeneic tumor models CT-26 and 4T1 were used for pharmacological investigation. GMP grade E7046 was administered to tumor-bearing animals by oral gavage. Co-culture of EG7-OVA and OT1 cells in an antigen-specific cytotoxic T cell (CTL) activation assay provided mechanistic insights. For translational validation, transcripts of five major genes involved in PGE2 synthesis, transport and degradation were compared between malignant and normal tissues across all TCGA tumor types, and correlation of their expression with overall survival was assessed. Results: In the CT26 tumor model, the combination of E7046 and anti-PD1 resulted in significantly more tumor-free animals compared with either agent alone. In the 4T1 tumor model, the combination of E7046 and anti-CTLA4 was also more effective in suppressing tumor growth and tumor rejection compared with anti-CTLA4 alone, and was accompanied by a markedly increased accumulation of GZMB+CD8T+ CTLs in the treated tumors. Consistent with those findings, addition of anti-PD1 antibody promoted OVA-specific CTL activation in vitro while addition of PGE2 strongly inhibited it, as measured by IFNγ secretion. Inclusion of E7046 dose-dependently reversed the PGE2-induced suppressive activity in the presence of anti-PD1 antibody. Among major human PGE2 pathway genes, TCGA analysis showed that PTGES1 was upregulated and HPGD downregulated across a broad range of tumor types. In contrast, COX1, COX2 and PGT showed less difference between malignant and normal tissues. Importantly, these differences of one or multiple PGE2 pathway genes were strongly associated with patient survival in certain cancer types. Conclusions: A subset of human cancer types displays upregulated PGE2 pathway that is associated with a poorer prognosis. PGE2-EP4 signaling potently suppresses antigen-specific CTL activation in the presence of PD1 signaling blockade. The combination of EP4 antagonist E7046 with either anti-PD1 or anti-CTLA4 demonstrated superior anti-tumor activity compared with anti-PD1 or anti-CTLA4 alone. This increased activity was accompanied by increased CTL activation. Citation Format: Diana I. Albu, David Verbel, Yuan Huang, Donna Kolber-Simonds, Zichun Wang, Xulong Wang, Zoltan Dezso, Christy Ingersoll, Kuan-Chun Huang, Janna Hutz, Mary Woodall-Jappe, Xingfeng Bao. Specific inhibition of PGE2-EP4 signaling by E7046 promotes anti-tumor activity of checkpoint blockade agents through boosting cytotoxic T cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4607. doi:10.1158/1538-7445.AM2017-4607

Rapid disappearance of antiviral CTL after transfusion into persistently infected individuals is a serious limitation of adoptive immunotherapy protocols. In the mouse model of persistent infection with lymphocytic choriomeningitis virus (LCMV) naive or immune virus-specific donor CD8+ T cells are exhausted after transfusion into carrier recipients with similar kinetics. Here we show that cotransfusion of immune CD4+ T cells prevents exhaustion of immune CD8+ T cells. Interestingly, cotransfer of primed B cells also prevented CD8+ T cell exhaustion in carriers even in the absence of T helper cells. This effect required the presence of immune B cells as repetitive treatment with hyperimmune serum led to the generation of antibody escape mutants. A combination of primed CD4+ T cells and primed B cells enhanced antiviral effects and prevented exhaustion also of naive CD8+ T cells. One key factor for prevention of CD8+ T cell exhaustion was the antiviral effect of the cotransfused cells thus reducing the time that CD8+ T cells are confronted with a high systemic viral load. These findings have implications for improving adoptive immunotherapy for persistent human viral infections.

T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Recently, a clearer picture of the functional and phenotypic profile of exhausted T cells has emerged and T cell exhaustion has been defined in many experimental and clinical settings. Although the pathways involved remain to be fully defined, advances in the molecular delineation of T cell exhaustion are clarifying the underlying causes of this state of differentiation and also suggest promising therapeutic opportunities.