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Supplementary Figure and Table Legends

Supplementary Figures

Supplementary Table S1

Supplementary Table S2

All dose-response curves. Dose-response curves for all library compounds

Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type–specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity–promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell–specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.

Supplementary Table S3

Metabolic constraints in the tumor microenvironment constitute a barrier to effective antitumor immunity and similarities in the metabolic properties of T cells and cancer cells impede the specific therapeutic targeting of metabolism in either population. To identify distinct metabolic vulnerabilities of CD8+ T cells and cancer cells, we developed a high-throughput in vitro pharmacologic screening platform and used it to measure the cell type–specific sensitivities of activated CD8+ T cells and B16 melanoma cells to a wide array of metabolic perturbations during antigen-specific killing of cancer cells by CD8+ T cells. We illustrated the applicability of this screening platform by showing that CD8+ T cells were more sensitive to ferroptosis induction by inhibitors of glutathione peroxidase 4 (GPX4) than B16 and MC38 cancer cells. Overexpression of ferroptosis suppressor protein 1 (FSP1) or cytosolic GPX4 yielded ferroptosis-resistant CD8+ T cells without compromising their function, while genetic deletion of the ferroptosis sensitivity–promoting enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4) protected CD8+ T cells from ferroptosis but impaired antitumor CD8+ T-cell responses. Our screen also revealed high T cell–specific vulnerabilities for compounds targeting NAD+ metabolism or autophagy and endoplasmic reticulum (ER) stress pathways. We focused the current screening effort on metabolic agents. However, this in vitro screening platform may also be valuable for rapid testing of other types of compounds to identify regulators of antitumor CD8+ T-cell function and potential therapeutic targets.

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.

T cell exhaustion describes an acquired dysfunction common in settings of cancer and chronic viral infection. Despite clinical efforts to rescue exhaustion, the fundamental mechanisms specifying this state, and the potential for reprogramming exhausted T cells, remain poorly understood. We profiled accessible chromatin in chronic viral infection to show that exhausted CD8+ cells acquire a state-specific landscape of enhancers that profoundly differs from functional memory. By comparing antigen-specific T cells in several contexts of T cell dysfunction, we found that CD8+ tumor infiltrating lymphocytes share significant epigenetic and transcriptional features with chronic viral infection, highlighting that T cell exhaustion is a fundamental adaptation to settings of chronic stimulation. Critically, we identify a core epigenetic signature, independent of disease-specific milieu, that can act as a precise biomarker of the exhausted state. Comparison of mouse cells to those isolated from patients infected with HCV or HIV showed that the core epigenetic program of exhaustion is conserved across species. Importantly, curative therapy, which reduces viral antigen load, as well as checkpoint blockade immunotherapy, which reduces inhibitory T cell signaling, failed to reverse the exhausted epigenetic profile. T cell exhaustion is therefore a stable epigenetic state that is not rescued by common treatment modalities. We then sought new strategies to modulate T cell exhaustion. We identified a novel candidate enhancer near the PD-1 gene that is unique to exhausted CD8+ T cells and a component of the core epigenetic program. Using Cas9-mediated genome editing, we generated a novel mouse strain with germ-line deletion of this region to characterize the role of this enhancer in vivo. We observed 2-3-fold enrichment of PD-1 enhancer-null cells over control cells in chronic infection, suggesting that CD8+ T cells in these mice might be less prone to exhaustion. Importantly, PD-1 enhancer-null cells had increased persistence without any deficits in functionality as has been described with the full PD-1 gene knock-out. As a result, deletion of the PD-1 enhancer gave rise to significantly higher numbers of IFNg+ immunotherapy-responsive T cells compared to both WT and PD-1 gene ablation. These data suggest that deletion of a state-specific enhancer in immune cells can promote unique functional capacities from those observed with the full gene knock-out. Next, we wanted to understand the role of this enhancer in regulating CD8+ T cell responses in the tumor microenvironment. We found that PD-1 enhancer-null mice exhibit slower tumor growth and increased survival when challenged with either B16-ova melanoma or LLC-ova lung carcinoma. Moreover, PD-1 enhancer-null CD8+ T cells outcompete WT cells in the tumor and preferentially differentiate into functional effectors. The establishment of a core program of T cell exhaustion and increased insight into its epigenetic modulation has crucial implications for the future of immunotherapy. Furthermore, our work suggests that perturbing exhaustion-specific enhancers in T cells could be used to prevent sustained expression of inhibitory genes without damaging the gene locus itself in CAR-T based clinical trials, where there is intense interest in engineering against T cell exhaustion. Citation Format: Debattama Sen, Sarah A. Weiss, Brian C. Miller, Kathleen B. Yates, Martin W. Lafleur, Arlene H. Sharpe, W. Nicholas Haining. Disrupting enhancers within the core epigenetic program of exhaustion improves CD8+ T cell responses and enhances tumor control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG04.

T-cell exhaustion describes an acquired dysfunction common in settings of cancer and chronic viral infection. Despite clinical efforts to rescue exhaustion, the fundamental mechanisms specifying this state, and the potential for reprogramming exhausted T cells, remain poorly understood. We profiled accessible chromatin in chronic viral infection to show that exhausted CD8+ cells acquire a state-specific landscape of enhancers that profoundly differs from functional memory. Critically, CD8+ tumor-infiltrating lymphocytes shared significant epigenetic and transcriptional features with chronic viral infection, suggesting that T-cell exhaustion is a fundamental adaptation to settings of chronic stimulation. Comparison of mouse cells to those isolated from patients infected with HCV or HIV showed that the core epigenetic program of exhaustion is conserved across species. Importantly, curative therapy, which reduces viral antigen load, as well as anti-PD-1 immunotherapy, which reduces inhibitory T-cell signaling, failed to reverse the exhausted epigenetic profile. T-cell exhaustion is therefore a stable epigenetic state that is not rescued by common treatment modalities. We then sought new strategies to modulate T-cell exhaustion. We used Cas9-mediated genome editing to generate a novel mouse strain with germline deletion of a core exhaustion-associated enhancer near PD-1. We observed 2- to 3-fold enrichment in vivo of PD-1 enhancer-null cells over control cells in chronic infection, suggesting that CD8+ T cells in these mice might be less prone to exhaustion. PD-1 enhancer-null mice also exhibited slower tumor growth and increased survival when challenged with B16-ova melanoma. The establishment of a core program of T-cell exhaustion and increased insight into its epigenetic modulation has crucial implications for the future of immunotherapy and rational engineering of T cells for clinical use. This abstract is also being presented as Poster A3. Citation Format: Debattama R. Sen, Sarah A. Weiss, Brian C. Miller, Pierre Tonnerre, Rose Al Abosy, Kathleen B. Yates, Kevin Bi, Martin W. Lafleur, David Wolski, Lauer Georg, Arlene H. Sharpe, W. Nicholas Haining. Disrupting enhancers within the core epigenetic program of exhaustion improves T-cell responses and enhances tumor control [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 PR6.

Although PD-1 pathway inhibitors are revolutionizing cancer treatment, the mechanisms by which PD-1 regulates antitumor immunity are not fully understood. To determine PD-1 functions on different cell types, we used PD-1 conditional knockout mice. Subcutaneous transplantation of MC38 adenocarcinoma tumor cells in mice with complete deletion of PD-1 selectively on CD8+ T cells improved CD8+ T functions in the tumor microenvironment (TME). CD8+ T cells were required for the protective effects of PD-1 deletion in this model. To assess whether loss of PD-1 on all cells was necessary for improved antitumor immunity, we restricted PD-1 deletion to only half of the T-cell population. We hypothesized that a cell-intrinsic loss of PD-1 was necessary for improved T-cell fitness and effector functions. Here, deletion of PD-1 indeed led to T cell-intrinsic boosts in function. Unexpectedly, however, 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, a finding that has important implications for applying PD-1-based immunotherapies to cancer patients. This abstract is also being presented as Poster B82. Citation Format: Kristen E. Pauken, Vikram R. Juneja, Alison Ringel, Jared H. Rowe, Kelly P. Burke, Peter T. Sage, Martin W. LaFleur, Juhi R. Kuchroo, Noga Ron-Harel, Seth Maleri, Jaclyn M. Long, Gordon J. Freeman, Nicolas Chevrier, Marcia C. Haigis, Arlene H. Sharpe. Loss of PD-1 promotes antitumor immunity by improving functions of both PD-1+ and PD-1- CD8+ T cells in the tumor microenvironment [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 PR9.

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.

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 mechanisms by which anti-PD-1 therapy acts on exhausted 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. Exhausted CD8+ TILs contain a subpopulation of “progenitor exhausted” T cells with 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. Melanoma patients with a higher percentage of progenitor exhausted cells have a longer duration of response to 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 Virkud, Martin W. LaFleur, Kathleen B. Yates, Ana Lako, Kristen Felt, Girish S. Naik, Michael Manos, Evisa Gjini, Jeffrey J. Ishizuka, F. Stephen Hodi, Scott J. Rodig, Arlene H. Sharpe, W. Nicholas Haining. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade [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 A83.

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.

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 effective only 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 as well as 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 “stem-like exhausted” T-cells that have a distinct regulatory state. Stem-like 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, stem-like exhausted CD8+ TILs are better able to control tumor growth than terminally exhausted cells. Stem-like exhausted, but not terminally exhausted, CD8+ TILs can respond to anti-PD-1 therapy without reversion of their exhausted epigenetic state. CD8+ T-cells with a stem-like exhausted phenotype can be found in human melanoma samples and patients with a higher fraction of this subpopulation in their tumors have a significantly longer duration of response to combination checkpoint blockade therapy. Responsiveness to checkpoint blockade is therefore restricted to a subpopulation of exhausted TILs that retain specific functional properties which enable them to control tumors. Approaches to expand stem-like exhausted CD8+ T-cells in the tumor microenvironment may be an important component of improving checkpoint blockade response. Citation Format: Debattama R. Sen, Brian C. Miller, Rose Al Abosy, Kevin Bi, Martin W. LaFleur, Kathleen B. Yates, Ana Lako, Kristen D. Felt, Girish S. Naik, Michael Manos, Evisa Gjini, Yamini V. Virkud, Stephen Hodi, Scott J. Rodig, Arlene H. Sharpe, W. Nicholas Haining. Functionally specialized subsets of exhausted CD8+ T-cells mediate tumor control and response to checkpoint blockade [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 A216.

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.