Publications

2020
Pauken KE, Godec J, Odorizzi PM, Brown KE, Yates KB, Ngiow SF, Burke KP, Freeman GJ, Haining WN, Wherry EJ, et al. The PD-1 Pathway Regulates Development and Function of Memory CD8+ T Cells following Respiratory Viral Infection. Cell Reports. 2020;31 (13) :107827.Abstract

The PD-1 pathway regulates dysfunctional T cells in chronic infection and cancer, but the role of this pathway during acute infection remains less clear. Here, we demonstrate that PD-1 signals are needed for optimal memory. Mice deficient in the PD-1 pathway exhibit impaired CD8+ T cell memory following acute influenza infection, including reduced virus-specific CD8+ T cell numbers and compromised recall responses. PD-1 blockade during priming leads to similar differences early post-infection but without the defect in memory formation, suggesting that timing and/or duration of PD-1 blockade could be tailored to modulate host responses. Our studies reveal a role for PD-1 as an integrator of CD8+ T cell signals that promotes CD8+ T cell memory formation and suggest PD-1 continues to fine-tune CD8+ T cells after they migrate into nonlymphoid tissues. These findings have important implications for PD-1-based immunotherapy, in which PD-1 inhibition may influence memory responses in patients.

PubMed DOI

LaFleur MW, Miller BC, Sharpe AH. Prevention of CAR-T-cell dysfunction. Nat Biomed Eng. 2020;4 (1) :16-17.Abstract

The overexpression of the transcription factor c-Jun improves chimeric-antigen-receptor T-cell functionality and enhances the killing of low-antigen-expressing liquid and solid cancers in mice.

PubMed DOI

2019
Brown FD, Sen DR, LaFleur MW, Godec J, Lukacs-Kornek V, Danial NN, Manning BD, Sharpe AH, Haining WN, Turley SJ. Fibroblastic reticular cells enhance T cell metabolism and survival via epigenetic remodeling. Nature Immunology. 2019;20 (12) :1668-1680.Abstract

Lymph node fibroblastic reticular cells (FRCs) respond to signals from activated T cells by releasing nitric oxide, which inhibits T cell proliferation and restricts the size of the expanding T cell pool. Whether interactions with FRCs also support the function or differentiation of activated CD8+ T cells is not known. Here we report that encounters with FRCs enhanced cytokine production and remodeled chromatin accessibility in newly activated CD8+ T cells via interleukin-6. These epigenetic changes facilitated metabolic reprogramming and amplified the activity of pro-survival pathways through differential transcription factor activity. Accordingly, FRC conditioning significantly enhanced the persistence of virus-specific CD8+ T cells in vivo and augmented their differentiation into tissue-resident memory T cells. Our study demonstrates that FRCs play a role beyond restricting T cell expansion—they can also shape the fate and function of CD8+ T cells.

PubMed DOI

Ron-Harel N, Ghergurovich JM, Notarangelo G, LaFleur MW, Tsubosaka Y, Sharpe AH, Rabinowitz JD, Haigis MC. T Cell Activation Depends on Extracellular Alanine. Cell Reports. 2019;28 (12) :3011-3021.Abstract

T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation.

PubMed DOI

Rashidian M, LaFleur MW, Verschoor VL, Dongre A, Zhang Y, Nguyen TH, Kolifrath S, Aref AR, Lau CJ, Paweletz CP, et al. Immuno-PET identifies the myeloid compartment as a key contributor to the outcome of the antitumor response under PD-1 blockade. PNAS. 2019;116 (34) :16971-16980.Abstract

Immunotherapy, especially blockade of the PD-1/PD-L1 and CTLA-4 axes, has resulted in durable responses in a range of cancers. However, responses remain heterogeneous among patients. Treatment outcome results from changes in the tumor microenvironment imposed by such blockade. Here, we use immuno-PET and single-cell RNA sequencing to increase our understanding of the dynamics of immune cells and their functional status in the tumor microenvironment in response to PD-1 blockade. Our data provide insights into the dynamics of CD8+ T cells and the functional status of the myeloid compartment in response to PD-1 blockade.

PubMed DOI

LaFleur MW, Nguyen TH, Coxe MA, Miller BC, Yates KB, Gaudiano EF, Sen DR, Gillis JE, Al Abosy R, Freeman GJ, et al. PTPN2 regulates the generation of exhausted CD8+ T cell subpopulations and restrains tumor immunity. Nature Immunology. 2019;20 (10) :1335-1347.Abstract

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.

PubMed DOI

Pauken KE, Dougan M, Rose NR, Lichtman AH, Sharpe AH. Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities. Trends Immunol. 2019;40 (6) :511-523.Abstract

Oncology has recently undergone a revolutionary change with widespread adoption of immunotherapy for many cancers. Immunotherapy using monoclonal antibodies against checkpoint molecules, including programmed death (PD)-1, PD ligand (PD-L)1, and cytotoxic T lymphocyte-associated antigen (CTLA)-4, is effective in a significant subset of patients. However, immune-related adverse events (irAEs) have emerged as frequent complications of checkpoint blockade, likely due to the physiological role of checkpoint pathways in regulating adaptive immunity and preventing autoimmunity. As immunotherapy becomes more common, a better understanding of the etiology of irAEs and ways to limit these events is needed. At the same time, studying these new therapy-related disorders provides an opportunity to better understand naturally occurring human autoimmune and inflammatory disorders, with the potential to improve therapies for cancer and autoimmune diseases.

PubMed DOI

Hou S, Clement RL, Diallo A, Blazar BR, Rudensky AY, Sharpe AH, Sage PT. FoxP3 and Ezh2 regulate Tfr cell suppressive function and transcriptional program. J Exp Med. 2019;216 (3) :605-620.Abstract

Follicular regulatory T (Tfr) cells are a regulatory T cell subset that controls antibody production by inhibiting T follicular helper (Tfh)-mediated help to B cells. Tfh and Tfr cells possess opposing functions suggesting unique programming. Here we elucidated the transcriptional program controlling Tfr suppressive function. We found that Tfr cells have a program for suppressive function fine-tuned by tissue microenvironment. The transcription factor FoxP3 and chromatin-modifying enzyme EZH2 are essential for this transcriptional program but regulate the program in distinct ways. FoxP3 modifies the Tfh program to induce a Tfr-like functional state, demonstrating that Tfr cells coopt the Tfh program for suppression. Importantly, we identified a Tfr cell population that loses the Tfr program to become "ex-Tfr" cells with altered functionality. These dysfunctional ex-Tfr cells may have roles in modulating pathogenic antibody responses. Taken together, our studies reveal mechanisms controlling the Tfr transcriptional program and how failure of these mechanisms leads to dysfunctional Tfr cells.

PubMed DOI

Miller BC, Sen DR, Abosy RA, Bi K, Virkud YV, LaFleur MW, Yates KB, Rodig SK, Sharpe AH, Haining NW, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol. 2019;20 (3).Abstract

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.

PubMed DOI

LaFleur MW, Nguyen TH, Coxe MA, Yates KB, Trombley JD, Weiss SA, Brown FD, Gillis JE, Coxe DJ, Deonch JG, et al. A CRISPR-Cas9 delivery system for in vivo screening of genes in the immune system. Nat Commun. 2019;10 (1668).Abstract

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.

PubMed DOI

2018
Sheng W, LaFleur MW, Nguyen TH, Chen S, Chakravarthy A, Freeman GJ, Carvalho DD, He HH, Sharpe AH, Shi Y. LSD1 Ablation Stimulates Anti-tumor Immunity and Enables Checkpoint Blockade. Cell. 2018;174 (3) :549-563.Abstract

Chromatin regulators play a broad role in regulating gene expression and, when gone awry, can lead to cancer. Here, we demonstrate that ablation of the histone demethylase LSD1 in cancer cells increases repetitive element expression, including endogenous retroviral elements (ERVs), and decreases expression of RNA-induced silencing complex (RISC) components. Significantly, this leads to double-stranded RNA (dsRNA) stress and activation of type 1 interferon, which stimulates anti-tumor T cell immunity and restrains tumor growth. Furthermore, LSD1 depletion enhances tumor immunogenicity and T cell infiltration in poorly immunogenic tumors and elicits significant responses of checkpoint blockade-refractory mouse melanoma to anti-PD-1 therapy. Consistently, TCGA data analysis shows an inverse correlation between LSD1 expression and CD8+ T cell infiltration in various human cancers. Our study identifies LSD1 as a potent inhibitor of anti-tumor immunity and responsiveness to immunotherapy and suggests LSD1 inhibition combined with PD-(L)1 blockade as a novel cancer treatment strategy.

PubMed DOI

Tan CL, Peluso MJ, Drivjers JM, Mera CM, Grande SM, Brown KE, Godec J, Freeman GJ, Sharpe AH. CD160 Stimulates CD8+ T Cell Responses and Is Required for Optimal Protective Immunity to Listeria monocytogenes. ImmunoHorizons. 2018;2 (7) :238-250.Abstract

CD160 promotes NK cell cytotoxicity and IFN-γ production, but the function of CD160 on CD8+T cells remains unclear with some studies supporting a coinhibitory role and others a costimulatory role. In this study, we demonstrate that CD160 has a costimulatory role in promoting CD8+ T cell effector functions needed for optimal clearance of oral Listeria monocytogenes infection. CD160−/− mice did not clear oral L. monocytogenes as efficiently as wild type (WT) littermates. WT RAG−/− and CD160−/− RAG−/− mice similarly cleared L. monocytogenes, indicating that CD160 on NK cells does not contribute to impaired L. monocytogenes clearance. Defective L. monocytogenes clearance is due to compromised intraepithelial lymphocytes and CD8+ T cell functions. There was a reduction in the frequencies of granzyme B–expressing intraepithelial lymphocytes in L. monocytogenes–infected CD160−/−mice as compared with WT littermate controls. Similarly, the frequencies of granzyme B–expressing splenic CD8+ T cells and IFN-γ and TNF-α double-producer CD8+ T cells were significantly reduced in L. monocytogenes–infected CD160−/− mice compared with WT littermates. Adoptive transfer studies showed that RAG−/− recipients receiving CD160−/− CD8+ T cells had a higher mortality, exhibited more weight loss, and had a higher bacterial burden compared with RAG−/− recipients receiving WT CD8+ T cells. These findings demonstrate that CD160 provides costimulatory signals to CD8+ T cells needed for optimal CD8+ T cell responses and protective immunity during an acute mucosal bacterial infection.

PubMed DOI

LaFleur MW, Muroyama Y, Drake CG, Sharpe AH. Inhibitors of the PD-1 Pathway in Tumor Therapy. J Immunol. 2018;200 (2) :375-383.Abstract

The programmed death 1 (PD-1) pathway delivers inhibitory signals that function as a brake for immune responses. This pathway limits the initiation and duration of immune responses, thereby protecting tissues from immune-mediated damage and autoimmune diseases. However, the PD-1 pathway also inhibits immune responses to tumors. The critical role of PD-1 in preventing antitumor immunity is demonstrated by the transformative effects of PD-1 pathway blockade in a broad range of cancers with the hallmark of durability of response. Despite this success, most patients do not respond to PD-1 monotherapy, and some patients experience adverse events. In this review, we discuss the functions of the PD-1 pathway and its translation to cancer immunotherapy. We also consider current challenges and opportunities for PD-1 cancer immunotherapy, including mechanisms of response and resistance, identification of biomarkers of response to PD-1 therapy, characterization and treatment of PD-1 therapy-related adverse events, and development of safe and effective combination therapies.

PubMed DOI

2017
Sharpe AH, Pauken KE. The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol. 2017;18 (3) :153-167.Abstract

T cell activation is a highly regulated process involving peptide-MHC engagement of the T cell receptor and positive costimulatory signals. Upon activation, coinhibitory 'checkpoints', including programmed cell death protein 1 (PD1), become induced to regulate T cells. PD1 has an essential role in balancing protective immunity and immunopathology, homeostasis and tolerance. However, during responses to chronic pathogens and tumours, PD1 expression can limit protective immunity. Recently developed PD1 pathway inhibitors have revolutionized cancer treatment for some patients, but the majority of patients do not show complete responses, and adverse events have been noted. This Review discusses the diverse roles of the PD1 pathway in regulating immune responses and how this knowledge can improve cancer immunotherapy as well as restore and/or maintain tolerance during autoimmunity and transplantation.

PubMed DOI

Zhang R, Sage PT, Finn K, Huynh A, Blazar BR, Marangoni F, Mempel TR, Sharpe AH, Turka LA. B Cells Drive Autoimmunity in Mice with CD28-Deficient Regulatory T Cells. J Immunol. 2017;199 (12) :3972-3980.Abstract

Follicular regulatory T (TFR) cells are a newly defined regulatory T cell (Treg) subset that suppresses follicular helper T cell-mediated B cell responses in the germinal center reaction. The precise costimulatory signal requirements for proper TFR cell differentiation and function are still not known. Using conditional knockout strategies of CD28, we previously demonstrated that loss of CD28 signaling in Tregs caused autoimmunity in mice (termed CD28-ΔTreg mice), characterized by lymphadenopathy, accumulation of activated T cells, and cell-mediated inflammation of the skin and lung. In this study, we show that CD28 signaling is required for TFR cell differentiation. Treg-specific deletion of CD28 caused a reduction in TFR cell numbers and function, which resulted in increased germinal center B cells and Ab production. Moreover, residual CD28-deficient TFR cells showed a diminished suppressive capacity as assessed by their ability to inhibit Ab responses in vitro. Surprisingly, genetic deletion of B cells in CD28-ΔTreg mice prevented the development of lymphadenopathy and CD4+ T cell activation, and autoimmunity that mainly targeted skin and lung tissues. Thus, autoimmunity occurring in mice with CD28-deficient Tregs appears to be driven primarily by loss of TFR cell differentiation and function with resulting B cell-driven inflammation.

PubMed DOI

Sharpe AH. Introduction to checkpoint inhibitors and cancer immunotherapy. Immunol Rev. 2017;276 (1) :5-8.Abstract
Kamphorst AO, Wieland A, Nasti T, Yang S, Zhang R, Barber DL, Konieczny BT, Daugherty CZ, Koenig L, Yu K, et al. Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent. Science. 2017;355 (6332) :1423-1427.Abstract

Programmed cell death-1 (PD-1)-targeted therapies enhance T cell responses and show efficacy in multiple cancers, but the role of costimulatory molecules in this T cell rescue remains elusive. Here, we demonstrate that the CD28/B7 costimulatory pathway is essential for effective PD-1 therapy during chronic viral infection. Conditional gene deletion showed a cell-intrinsic requirement of CD28 for CD8 T cell proliferation after PD-1 blockade. B7-costimulation was also necessary for effective PD-1 therapy in tumor-bearing mice. In addition, we found that CD8 T cells proliferating in blood after PD-1 therapy of lung cancer patients were predominantly CD28-positive. Taken together, these data demonstrate CD28-costimulation requirement for CD8 T cell rescue and suggest an important role for the CD28/B7 pathway in PD-1 therapy of cancer patients.

PubMed DOI

Juneja VR, McGuire KA, Manguso RT, LaFleur MW, Collins N, Haining NW, Freeman GJ, Sharpe AH. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med. 2017;214 (4) :895-904.Abstract

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.

PubMed DOI

2016
Karunarathne DS, Horne-Debets JM, Huang JX, Faleiro R, Leow CY, Amante F, Watkins TS, Miles JJ, Dwyer PJ, Stacey KJ, et al. Programmed Death-1 Ligand 2-Mediated Regulation of the PD-L1 to PD-1 Axis Is Essential for Establishing CD4(+) T Cell Immunity. Immunity. 2016;45 (2) :333-45.Abstract
Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.
Im SJ, Hashimoto M, Gerner MY, Lee J, Kissick HT, Burger MC, Shan Q, Hale SJ, Lee J, Nasti TH, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature. 2016;537 (7620) :417-421.Abstract
Chronic viral infections are characterized by a state of CD8(+) T-cell dysfunction that is associated with expression of the programmed cell death 1 (PD-1) inhibitory receptor. A better understanding of the mechanisms that regulate CD8(+) T-cell responses during chronic infection is required to improve immunotherapies that restore function in exhausted CD8(+) T cells. Here we identify a population of virus-specific CD8(+) T cells that proliferate after blockade of the PD-1 inhibitory pathway in mice chronically infected with lymphocytic choriomeningitis virus (LCMV). These LCMV-specific CD8(+) T cells expressed the PD-1 inhibitory receptor, but also expressed several costimulatory molecules such as ICOS and CD28. This CD8(+) T-cell subset was characterized by a unique gene signature that was related to that of CD4(+) T follicular helper (TFH) cells, CD8(+) T cell memory precursors and haematopoietic stem cell progenitors, but that was distinct from that of CD4(+) TH1 cells and CD8(+) terminal effectors. This CD8(+) T-cell population was found only in lymphoid tissues and resided predominantly in the T-cell zones along with naive CD8(+) T cells. These PD-1(+)CD8(+) T cells resembled stem cells during chronic LCMV infection, undergoing self-renewal and also differentiating into the terminally exhausted CD8(+) T cells that were present in both lymphoid and non-lymphoid tissues. The proliferative burst after PD-1 blockade came almost exclusively from this CD8(+) T-cell subset. Notably, the transcription factor TCF1 had a cell-intrinsic and essential role in the generation of this CD8(+) T-cell subset. These findings provide a better understanding of T-cell exhaustion and have implications in the optimization of PD-1-directed immunotherapy in chronic infections and cancer.

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