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Summary/AbstractWe previously identified the adhesion molecule PSGL-1 as a T cell intrinsic immune checkpoint regulator of T cell exhaustion. Here we show that the ability of PSGL-1 to restrain TCR sginaling correlates with decreased expression of the Zap70 inhibitor Ubash3b (Sts-1) in PSGL-1-deficient T cells. PSGL-1-deficency in T cells supports antitumor responses to a PD-1 blockade resistant melanoma wherein tumor-specific CD8+ T cells sustain an enhanced metabolic state, with an elevated metabolic gene signature that promotes increased glycolysis and glucose uptake to support effector functions. In models of chronic virus infection and cancer, this outcome was associated with CD8+ T cell stemness, as PSGL-1 deficient CD8+ T cells displayed increased TCF-1 and decreased TOX expression, a phenotype shown to be crucial for responsiveness to checkpoint inhibition. Further, we demonstrate that PSGL-1 signaling promotes development of exhaustion in human CD8+ T cells. Finally, pharmacologic blockade of PSGL-1 was sufficient to curtail T cell exhaustion and enhance functionality both with melanoma tumors and chronic LCMV infection, demonstrating that PSGL-1 represents a therapeutic target for immunotherapy for PD-1/PD-L1 resistant tumors.

Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in responses to chronic viral infection and melanoma tumors. Subsequently, our studies have focused on investigating the mechanisms by which PSGL-1 regulates T cell activation and exhaustion and evaluating PSGL-1 as a target for cancer immunotherapy. Using the CD4cre system in combination with PSGL-1-floxed mice, we find that T cell intrinsic deletion of PSGL-1 is sufficient to promote enhanced tumor growth control of mesothelioma as well as melanoma tumors. We have found that in vivo PSGL-1 blockade recapitulates the enhanced melanoma tumor growth control observed with PSGL-1-/- CD8+ T cells and limits the development of T cell exhaustion during chronic LCMV Clone 13 viral infection. Conversely, ligation of PSGL-1 in vitro or in vivo drives enhanced upregulation T cell exhaustion markers, most notably PD-1 expression. Phosphoproteomic analysis of early T cell activation revealed the rapid upregulation of more than 30 phosphorylated proteins with significantly enhanced expression following PSGL-1 ligation during T cell activation than T cell activation alone. GSEA and western blot analysis confirmed the increased expression of several regulators of T cell signaling including pAkt1 and pErk in both activated PSGL-1-/- T cells and following PSGL-1 ligation during T cell activation. Single-cell RNA-sequencing of melanoma tumor-specific PSGL-1-/- CD8+ T cells identified differential modulation of genes associated with T cell metabolism and effector responses, including increased Mtor and Hif1a in tumors and Tcf7 in tumor-draining lymph nodes. The Seahorse glycolysis stress test identified that PSGL-1-/- T cells show increased glycolysis after 72 hours of in vitro activation at both sub-optimal and optimal levels of αCD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1-/- CD8+ T cells within two hours of stimulation. Further, 2-NBDG uptake was increased by PSGL-1-/- CD8+ T cells ex vivo from melanoma tumors. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. We hypothesize that PSGL-1 serves as a key regulator of early T cell activation and repeated signaling through PSGL-1 promotes differentiation into exhausted CD8+ T cells by sustaining TCR signaling. Conversely, we hypothesize that the absence of PSGL-1 allows for faster initiation and termination of TCR signaling, inhibiting differentiation into exhausted CD8+ T cells. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile which may drive their enhanced responses to tumors. Citation Format: Jennifer L. Hope, Dennis C. Otero, Eun-ah Bae, Christopher J Stairiker, Ashley B. Palete, Hannah A. Faso, Petrus de Jong, Garth Powis, Linda M. Bradley. PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO014.

Endoplasmic reticulum (ER) stress is induced by the accumulation of misfolded proteins and other cellular stresses, which lead to the unfolded protein response to restore ER homeostasis. Previous studies have shown that the ER stress response is required for T cell activation and differentiation. However, recent studies demonstrated that inhibition or deletion of ER stress mediators in T cells actually promotes anti-tumor immunity. Therefore, this incongruity as to the role of ER stress response in effector T cell function warrants further investigation. Our lab previously identified that the adhesion molecule, P-selectin glycoprotein ligand-1 (PSGL-1), acts as a T cell immune checkpoint regulator. PSGL-1-deficiency enhances T cell function, resulting in the clearance of chronic viral infection and dramatic tumor control. To better understand the contribution of ER stress to T cell function, we assessed T cell phenotype and function together with the ER stress mediators, XBP1s and ATF4, in highly-functional PSGL-1−/− T cells as well as exhausted wild-type T cells in both tumor and virus models. In melanoma models, ER stress was highly induced in activated tumor-infiltrating T cells (TILs). Effector CD8+ TILs producing IFNg displayed elevated XBP1s and ATF4 compared to IFNg− TILs. Upon chronic LCMV Cl13 viral infection in PSGL-1−/− mice, we found increased virus-specific T cells with enhanced multi-functional effector responses and increased memory/stem cell-like T cell populations characterized by the expression of the transcription factor TCF-1, as well as increased ER stress responses. Together, these data suggest that ER stress plays an essential role in effector T cell functions in settings of chronic antigen stimulation.

We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in response to chronic viral infection and tumors. Subsequent studies have focused on investigating the role of PSGL-1 signaling in T cell responses, with an emphasis on understanding the mechanisms by which PSGL-1 regulates T cell exhaustion. Single-cell RNA-sequencing of tumor infiltrating PSGL-1−/− CD8+ T cells identified the upregulation and differential modulation of several genes associated with T cell metabolism and enhanced intratumoral responses, including Mtor, Hif1a, and Mki67 in Gzmb/Ifng double-positive cells from tumors and Tcf7 in both tumor draining and non-draining inguinal lymph nodes. These data suggest an important role for PSGL-1 signaling in the development and maintenance of effective anti-tumor T cell responses to melanoma. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. In situ activation of PSGL-1−/− CD8+ T cells demonstrated that PSGL-1−/− CD8+ T cells have increased glycolysis and increased glycolytic capacity at both sub-optimal and optimal levels of α-CD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1−/− CD8+ T cells within two hours of stimulation. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile in response to melanoma tumors.