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CD8+ T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6+ progenitor exhausted and Tim-3+ terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8+ T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3+ cells without altering Slamf6+ numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise, Ptpn2 deletion in CD8+ T cells enhanced Tim-3+ anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3+CD8+ T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8(+) tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8(+) TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8(+) TILs include a subpopulation of ‘progenitor exhausted’ cells that retain polyfunctionality, persist long term and differentiate into ‘terminally exhausted’ TILs. Consequently, progenitor exhausted CD8(+) TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8(+) T cells might be an important component of improving the response to checkpoint blockade.

Cancer-associated fibroblasts (CAFs) are generally associated with poor clinical outcome. CAFs support tumor growth in a variety of ways and can suppress antitumor immunity and response to immunotherapy. However, a precise understanding of CAF contributions to tumor growth and therapeutic response is lacking. Discrepancies in this field of study may stem from heterogeneity in the composition and function of fibroblasts in the tumor microenvironment. Furthermore, it remains unclear whether CAFs directly interact with and suppress T cells. Here, mouse and human breast tumors were used to examine stromal cells expressing fibroblast activation protein (FAP), a surface marker for CAFs. Two discrete populations of FAP+ mesenchymal cells were identified on the basis of podoplanin (PDPN) expression: a FAP+PDPN+ population of CAFs and a FAP+PDPN− population of cancer-associated pericytes (CAPs). Although both subsets expressed extracellular matrix molecules, the CAF transcriptome was enriched in genes associated with TGFβ signaling and fibrosis compared with CAPs. In addition, CAFs were enriched at the outer edge of the tumor, in close contact with T cells, whereas CAPs were localized around vessels. Finally, FAP+PDPN+ CAFs suppressed the proliferation of T cells in a nitric oxide–dependent manner, whereas FAP+PDPN− pericytes were not immunosuppressive. Collectively, these findings demonstrate that breast tumors contain multiple populations of FAP-expressing stromal cells of dichotomous function, phenotype, and location.

SUMMARYThe response of cytotoxic T cells to their cognate antigen involves rapid and broad changes in gene expression that are coupled with extensive chromatin remodeling. Here, we study the mechanisms by which the basic leucine zipper ATF-like transcription factor Batf helps regulate this process. Through genome-scale profiling, we observe critical roles for Batf in inducing transcriptional changes in stimulated naive cells, while affecting the chromatin at several levels, namely binding of key transcription factors, accessibility, and long range contacts. We identify a critical network of transcription factors that cooperate with Batf, including its binding partner Irf4, as well as Runx3 and T-bet, and demonstrate its synergistic activity in initiating aspects of the effector T cells’ transcriptional and epigenetic program in ectopically-induced fibroblasts. Our results provide a comprehensive resource for studying the epigenomic and transcriptomic landscape of effector differentiation of cytotoxic T cells.

The process of pyroptosis is mediated by inflammasomes and a downstream effector known as gasdermin D (GSDMD). Upon cleavage by inflammasome-associated caspases, the N-terminal domain of GSDMD forms membrane pores that promote cytolysis. Numerous proteins promote GSDMD cleavage, but none are known to be required for pore formation after GSDMD cleavage. Herein, we report a forward genetic screen that identified the Ragulator-Rag complex as being necessary for GSDMD pore formation and pyroptosis in macrophages. Mechanistic analysis revealed that Ragulator-Rag is not required for GSDMD cleavage upon inflammasome activation, but rather promotes GSDMD oligomerization in the plasma membrane. Defects in GSDMD oligomerization and pore formation can be rescued by mitochondrial poisons that stimulate reactive oxygen species (ROS) production, and ROS modulation impacts the ability of inflammasome pathways to promote pore formation downstream of GSDMD cleavage. These findings reveal an unexpected link between key regulators of immunity (inflammasome-GSDMD) and metabolism (Ragulator-Rag).

Both tumor- and host-derived PD-L1 can play critical roles in immunosuppression; differences in tumor immunogenicity appear to underlie their relative contributions. Juneja et al. show that in immunogenic MC38 tumors, PD-L1 on tumor cells dominates in suppressing tumor immunity by inhibiting CD8 T cell cytotoxicity., It is unclear whether PD-L1 on tumor cells is sufficient for tumor immune evasion or simply correlates with an inflamed tumor microenvironment. We used three mouse tumor models sensitive to PD-1 blockade to evaluate the significance of PD-L1 on tumor versus nontumor cells. PD-L1 on nontumor cells is critical for inhibiting antitumor immunity in B16 melanoma and a genetically engineered melanoma. In contrast, PD-L1 on MC38 colorectal adenocarcinoma cells is sufficient to suppress antitumor immunity, as deletion of PD-L1 on highly immunogenic MC38 tumor cells allows effective antitumor immunity. MC38-derived PD-L1 potently inhibited CD8+ T cell cytotoxicity. Wild-type MC38 cells outcompeted PD-L1–deleted MC38 cells in vivo, demonstrating tumor PD-L1 confers a selective advantage. Thus, both tumor- and host-derived PD-L1 can play critical roles in immunosuppression. Differences in tumor immunogenicity appear to underlie their relative importance. Our findings establish reduced cytotoxicity as a key mechanism by which tumor PD-L1 suppresses antitumor immunity and demonstrate that tumor PD-L1 is not just a marker of suppressed antitumor immunity.

Obesity is a major cancer risk factor, but how differences in systemic metabolism change the tumor microenvironment (TME) and impact anti-tumor immunity is not understood. Here, we demonstrate that high-fat diet (HFD)-induced obesity impairs CD8(+) T cell function in the murine TME, accelerating tumor growth. We generate a single-cell resolution atlas of cellular metabolism in the TME, detailing how it changes with diet-induced obesity. We find that tumor and CD8(+) T cells display distinct metabolic adaptations to obesity. Tumor cells increase fat uptake with HFD, whereas tumor-infiltrating CD8(+) T cells do not. These differential adaptations lead to altered fatty acid partitioning in HFD tumors, impairing CD8(+) T cell infiltration and function. Blocking metabolic reprogramming by tumor cells in obese mice improves anti-tumor immunity. Analysis of human cancers reveals similar transcriptional changes in CD8(+) T cell markers, suggesting interventions that exploit metabolism to improve cancer immunotherapy.

Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used noninvasive immuno-positron emission tomography (PET), using (89)Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8(+) T cells and CD11b(+) myeloid cells in response to anti–PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8(+) and CD11b(+) cells. Anti–PD-1 treatment mobilized CD8(+) T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8(+) T cells went on to complete resolution. All tumors contained CD11b(+) myeloid cells from the outset of treatment, with later recruitment of additional CD11b(+) cells. As tumors grew, the distribution of intratumoral CD11b(+) cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b(+) population in the center of the tumors. The changes in distribution of CD8(+) and CD11b(+) cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti–PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45(+) cells showed that CD11b(+) cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45(+) population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti–PD-1 treatment not only affects interactions of CD8(+) T cells with the tumor but also impacts the intratumoral myeloid compartment.

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8+ T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo., The use of functional genomics in primary immune cells has been limited by inefficient vector delivery and risk of perturbing cell states. Here the authors present CHimeric IMmune Editing (CHIME) for in vivo evaluation of gene function and pooled screening approaches.

Despite the significant clinical responses observed with checkpoint blockade, there is an urgent need to identify new therapeutic targets in immune cells for combination therapies. However, the use of functional genomic approaches in immune cells to discover immunotherapy targets is limited by inefficient vector delivery or perturbation of cell states. To circumvent these limitations, we developed CHIME: CHimeric IMmune Editing, a bone marrow chimeric CRISPR-Cas9 delivery system, to rapidly evaluate gene function in innate and adaptive immune cells in vivo without prior ex vivo manipulation. This approach enables efficient deletion of genes of interest in major immune lineages without altering immune development or function. To discover novel immunotherapy targets we performed an in vivo pooled genetic screen for negative regulators of CD8+ T-cell responses to LCMV Clone 13 viral infection. We found that deletion of the phosphatase Ptpn2 enhances CD8+ T-cell responses to chronic pathogens and cancer. In models of both chronic viral infection and transplantable tumors, Ptpn2 null CD8+ T cells expand more and show increased expression of effector genes compared to wild-type cells. Consistent with this, in the LCMV Clone 13 model, Ptpn2 deletion in CD8+ T cells affects the differentiation and expansion of exhausted subpopulations by increasing IFN-I signaling. Furthermore, deletion of Ptpn2 in the immune system induces a CD8+ T cell-dependent clearance of tumors and synergizes with PD-1 immune checkpoint blockade. Our results suggest that therapeutic inhibition of Ptpn2 in immune cells may enhance CD8+ T-cell effector function and mediate antitumor immunity to improve tumor control. More generally, these findings suggest that this genetic platform can enable rapid target discovery through pooled loss-of-function screening in immune cell lineages in vivo and presents a novel target for potential immune based therapies. Citation Format: Martin LaFleur, Thao Nguyen, Matthew Coxe, Brian Miller, Kathleen Yates, Jacob Gillis, Debattama Sen, John Doench, Nicholas Haining, Arlene Sharpe. CHIME screening identifies PTPN2 as a novel regulator of antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B47.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells.

T cell dysfunction in the tumor microenvironment (TME) is a hallmark of many cancers. Reinvigoration of T cell function by PD-1 checkpoint blockade can result in striking clinical responses, but is only effective in a minority of patients. The basis for T cell dysfunction in the TME, as well as the mechanisms by which anti-PD-1 therapy acts on dysfunctional T cells are not fully understood. Here we show that anti-PD-1 therapy acts on a specific subpopulation of CD8+ tumor-infiltrating lymphocytes (TILs) in melanoma mouse models, which can also be found in patients with melanoma. We find that dysfunctional CD8+ TILs possess canonical epigenetic and transcriptional features of T cell exhaustion, mirroring those seen in chronic viral infection. Similar to chronic viral infection, exhausted CD8+ TILs contain a subpopulation of “progenitor exhausted” T cells that have a distinct regulatory state. Progenitor exhausted TILs also have critical functional attributes that are not shared by the majority “terminally exhausted” TILs: they retain more polyfunctionality, persist following transfer into tumor-bearing mice, and differentiate to repopulate terminally exhausted TILs in the TME. As a result, progenitor exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Progenitor exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy but this occurs without reversion of their exhausted epigenetic state. Human melanomas contain CD8+ T cells with a progenitor exhausted phenotype and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Therefore, approaches to expand progenitor exhausted CD8+ T cells in the tumor microenvironment may be an important component of improving checkpoint blockade response. Citation Format: Brian C. Miller, Debattama R. Sen, Rose Al Abosy, Kevin Bi, Yamini V. Virkud, Martin W. LaFleur, Kathleen B. Yates, Ana Lako, Kristen Felt, Girish S. Naik, Michael Manos, Evisa Gjini, F. Stephen Hodi, Scott J. Rodig, Arlene H. Sharpe, W. Nicholas Haining. Functionally specialized subsets of exhausted CD8+ T cells mediate tumor control and differentially respond to checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2701.

Although PD-1 pathway inhibitors are revolutionizing cancer treatment, the mechanisms by which PD-1 regulates anti-tumor immunity are not fully understood. Following subcutaneous transplantation of MC38 adenocarcinoma tumor cells into mice, we show that complete loss of PD-1 selectively on CD8+ T cells improved metabolic activity and functions in the tumor microenvironment (TME). Since clinically PD-1 inhibitors likely act on T cells post-priming, we next deleted PD-1 after initial priming and restricted deletion to roughly 50% of cells. Loss of PD-1 led to T cell-intrinsic boosts in metabolism and CD8+ T cells that lost PD-1 after priming preferentially formed anti-tumor memory cells, suggesting PD-1 antagonizes memory formation. Unexpectedly, there was also a bystander effect that improved functions of PD-1 expressing CD8+ T cells in the TME. These data suggest that complete loss of PD-1 is not necessary for optimal tumor immunity, and that enhancing the functions of a subset of CD8+ T cells can promote an antitumor microenvironment and immunologic memory. Citation Format: Kristen E. Pauken, Vikram R. Juneja, Peter T. Sage, Martin W. LaFleur, Juhi R. Kuchroo, Alison Ringel, Noga Ron-Harel, Seth P. Maleri, Gordon J. Freeman, Nicolas Chevrier, Marcia C. Haigis, Arlene H. Sharpe. PD-1 modulation promotes antitumor immunity by improving metabolic fitness of both PD-1+ and PD-1- CD8+ T cells in the tumor [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A19.