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Recent studies have demonstrated that β-catenin in DCs serves as a key mediator in promoting both CD4⁺ and CD8⁺ T-cell tolerance, although how β-catenin exerts its functions remains incompletely understood. Here we report that activation of β-catenin in DCs inhibits cross-priming of CD8⁺ T cells by up-regulating mTOR-dependent IL-10, suggesting blocking β-catenin/mTOR/IL-10 signaling as a viable approach to augment CD8⁺ T-cell immunity. However, vaccination of DC–β-catenin−/−(CD11c-specific deletion of β-catenin) mice surprisingly failed to protect them against tumor challenge. Further studies revealed that DC–β-catenin−/−mice were deficient in generating CD8⁺ T-cell immunity despite normal clonal expansion, likely due to impaired IL-10 production by β-catenin−/−DCs. Deletion of β-catenin in DCs or blocking IL-10 after clonal expansion similarly led to reduced CD8⁺ T cells, suggesting that β-catenin in DCs plays a positive role in CD8⁺ T-cell maintenance postclonal expansion through IL-10. Thus, our study has not only identified mTOR/IL-10 as a previously unidentified mechanism for β-catenin–dependent inhibition of cross-priming, but also uncovered an unexpected positive role that β-catenin plays in maintenance of CD8⁺ T cells. Despite β-catenin’s opposite functions in regulating CD8⁺ T-cell responses, selectively blocking β-catenin with a pharmacological inhibitor during priming phase augmented DC vaccine-induced CD8⁺ T-cell immunity and improved antitumor efficacy, suggesting manipulating β-catenin signaling as a feasible therapeutic strategy to improve DC vaccine efficacy.

NY-ESO-1 is a \"cancer-testis\" antigen frequently expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8⁺ T cells derived from peripheral blood lymphocytes (PBLs), tumor-infiltrating lymphocytes (TILs), and tumor-associated lymphocytes (TALs) of EOC patients with NY-ESO-1-expressing tumors, with or without humoral immunity to NY-ESO-1. Whereas NY-ESO-1-specific CD8⁺ T cells were readily detectable ex vivo with tetramers in TILs and TALs of seropositive patients, they were only detectable in PBLs following in vitro stimulation. Compared with PBLs, tumor-derived NY-ESO-1-specific CD8⁺ T cells demonstrated impaired effector function, preferential usage of dominant T-cell receptor, and enriched coexpression of inhibitory molecules LAG-3 and PD-1. Expression of LAG-3 and PD-1 on CD8⁺ T cells was up-regulated by IL-10, IL-6 (cytokines found in tumor ascites), and tumor-derived antigen-presenting cells. Functionally, CD8⁺LAG-3⁺PD-1⁺ T cells were more impaired in IFN-γ/TNF-α production compared with LAG-3⁺PD-1⁻ or LAG-3⁻PD-1⁻ subsets. Dual blockade of LAG-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8⁺ T cells, indicating that antitumor function of NY-ESO-1-specific CD8⁺ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

Immunosuppressive factors such as regulatory T cells (Tregs) limit the efficacy of immunotherapies. Histone deacetylase (HDAC) inhibitors have been reported to have antitumor activity in different malignancies and immunomodulatory effects. Herein, we report the Tregs-targeting and immune-promoting effect of a class I specific HDAC inhibitor, entinostat, in combination with either IL-2 in a murine renal cell carcinoma (RENCA) model or a survivin-based vaccine therapy (SurVaxM) in a castration resistant prostate cancer (CR Myc-CaP) model. RENCA or CR Myc-CaP tumors were implanted orthotopically or subcutaneously, respectively. Inoculated mice were randomized into four treatment groups: vehicle, entinostat, cytokine or vaccine, and combination. Tregs in the blood were assessed by FACS analysis. Real time quantitative PCR and Western blot analysis of isolated T cell subpopulations from spleen were performed to determine Foxp3 gene and protein expression. The suppressive function of Tregs was tested by T cell proliferation assay. Low dose (5 mg/kg) entinostat reduced Foxp3 levels in Tregs and this was associated with enhanced tumor growth inhibition in combination with either IL-2 or a SurVaxM vaccine. Entinostat down-regulated Foxp3 expression transcriptionally and blocked Tregs suppressive function without affecting T effector cells (Teffs). In vitro low dose entinostat (0.5 µM) induced STAT3 acetylation and a specific inhibitor of STAT3 partially rescued entinostat-induced down-regulation of Foxp3, suggesting that STAT3 signaling is involved in Foxp3 down-regulation by entinostat. These results demonstrate a novel immunomodulatory effect of class I HDAC inhibition and provide a rationale for the clinical testing of entinostat to enhance cancer immunotherapy.

Adoptive cell therapy (ACT) employing ex vivo-generated tumor antigen-specific CD8+ T cells shows tumor efficacy when the transferred cells possess both effector and memory functions. New strategies based on understanding of mechanisms that balance CD8+ T cell differentiation toward effector and memory responses are highly desirable. Emerging information confirms a central role for antigen-induced metabolic reprogramming in CD8+ T cell differentiation and clonal expansion. The mitochondrial protein uncoupling protein 2 (UCP2) is induced by antigen stimulation of CD8+ T cells; however, its role in metabolic reprogramming underlying differentiation and clonal expansion has not been reported. Employing genetic (siRNA) and pharmacologic (Genipin) approaches, we note that antigen-induced UCP2 expression reduces glycolysis, fatty acid synthesis and production of reactive oxygen species to balance differentiation with survival of effector CD8+ T cells. Inhibition of UCP2 promotes CD8+ T cell terminal differentiation into short-lived effector cells (CD62L lo KLRG1 Hi IFNγ Hi ) that undergo clonal contraction. These findings are the first to reveal a role for antigen-induced UCP2 expression in balancing CD8+ T cell differentiation and survival. Targeting UCP2 to regulate metabolic reprogramming of CD8+ T cells is an attractive new approach to augment efficacy of tumor therapy by ACT.

Recombinant poxviruses (vaccinia and fowlpox) expressing tumor-associated antigens are currently being evaluated in clinical trials as cancer vaccines to induce tumor-specific immune responses that will improve clinical outcome. To test whether a diversified prime and boost regimen targeting NY-ESO-1 will result in clinical benefit, we conducted two parallel phase II clinical trials of recombinant vaccinia-NY-ESO-1 (rV-NY-ESO-1), followed by booster vaccinations with recombinant fowlpox-NY-ESO-1 (rF-NY-ESO-1) in 25 melanoma and 22 epithelial ovarian cancer (EOC) patients with advanced disease who were at high risk for recurrence/progression. Integrated NY-ESO-1-specific antibody and CD4+ and CD8+ T cells were induced in a high proportion of melanoma and EOC patients. In melanoma patients, objective response rate [complete and partial response (CR+PR)] was 14%, mixed response was 5%, and disease stabilization was 52%, amounting to a clinical benefit rate (CBR) of 72% in melanoma patients. The median PFS in the melanoma patients was 9 mo (range, 0–84 mo) and the median OS was 48 mo (range, 3–106 mo). In EOC patients, the median PFS was 21 mo (95% CI, 16–29 mo), and median OS was 48 mo (CI, not estimable). CD8+ T cells derived from vaccinated patients were shown to lyse NY-ESO-1-expressing tumor targets. These data provide preliminary evidence of clinically meaningful benefit for diversified prime and boost recombinant pox-viral-based vaccines in melanoma and ovarian cancer and support further evaluation of this approach in these patient populations.

PurposeResident memory CD8 T cells, owing to their ability to reside and persist in peripheral tissues, impart adaptive sentinel activity and amplify local immune response, and have beneficial implications for tumor surveillance and control. The current study aimed to clarify the less known chemotactic mechanisms that govern the localization, retention, and residency of memory CD8 T cells in the ovarian tumor microenvironment.Experimental designRNA and protein expressions of chemokine receptors in CD8+ resident memory T cells in human ovarian tumor-infiltrating CD8+ T cells and their association with survival were analyzed. The role of CXCR6 on antitumor T cells was investigated using prophylactic vaccine models in murine ovarian cancer.ResultsChemokine receptor profiling of CD8+CD103+ resident memory tumor-infiltrating lymphocytes in patients with ovarian cancer revealed high expression of CXCR6. Analysis of The Cancer Genome Atlas (TCGA) (ovarian cancer database revealed CXCR6 to be associated with CD103 and increased patient survival. Functional studies in mouse models of ovarian cancer revealed that CXCR6 is a marker of resident, but not circulatory, tumor-specific memory CD8+ T cells. CXCR6-deficient tumor-specific CD8+ T cells showed reduced retention in tumor tissues, leading to diminished resident memory responses and poor control of ovarian cancer.ConclusionsCXCR6, by promoting retention in tumor tissues, serves a critical role in resident memory T cell-mediated immunosurveillance and control of ovarian cancer. Future studies warrant exploiting CXCR6 to promote resident memory responses in cancers.

Tumor antigen-specific CD4 + T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4 + T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize anti-tumor effects of a unique human CD4 + helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4 + T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8 + T cells by enhancing cytotoxic activity and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8 + T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TR-CD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients.

NY-ESO-1 is a \"cancer-testis\" antigen expressed in epithelial ovarian cancer (EOC) and is among the most immunogenic tumor antigens defined to date. The NY-ESO-1 peptide epitope, ESO₁₅₇₋₁₇₀, is recognized by HLA-DP4-restricted CD4⁺ T cells and HLA-A2- and A24-restricted CD8⁺ T cells. To test whether providing cognate helper CD4⁺ T cells would enhance the antitumor immune response, we conducted a phase I clinical trial of immunization with ESO₁₅₇₋₁₇₀ mixed with incomplete Freund's adjuvant (Montanide ISA51) in 18 HLA-DP4⁺ EOC patients with minimal disease burden. NY-ESO-1-specific Ab responses and/or specific HLA-A2-restricted CD8⁺ and HLA-DP4-restricted CD4⁺ T cell responses were induced by a course of at least five vaccinations at three weekly intervals in a high proportion of patients. There were no serious vaccine-related adverse events. Vaccine-induced CD8⁺ and CD4⁺ T cell clones were shown to recognize NY-ESO-1-expressing tumor targets. T cell receptor analysis indicated that tumor-recognizing CD4⁺ T cell clones were structurally distinct from non-tumor-recognizing clones. Long-lived and functional vaccine-elicited CD8⁺ and CD4⁺ T cells were detectable in some patients up to 12 months after immunization. These results confirm the paradigm that the provision of cognate CD4⁺ T cell help is important for cancer vaccine design and provides the rationale for a phase II study design using ESO₁₅₇₋₁₇₀ epitope or the full-length NY-ESO-1 protein for immunotherapy in patients with EOC.

BackgroundPerturbation of the mechanistic target of rapamycin (mTOR) pathway can instruct effector versus memory cell fate of tumor antigen-specific T cells in preclinical models. In this study, we sought to understand the impact of rapamycin (sirolimus), an mTOR inhibitor, on reprogramming vaccine-induced T cells to enhance memory responses in patients with solid tumors following completion of their standard therapy.MethodsWe conducted three phase I clinical trials employing New York esophageal squamous cell carcinoma-1 (NY-ESO-1) vaccination approaches, with or without schedule-varied rapamycin. T cell phenotypes, functions, and Vβ usage in peripheral blood were analyzed to ask whether rapamycin influenced the generation of vaccine-induced T cells with memory attributes.ResultsThe addition of rapamycin to all vaccination approaches was safe and well tolerated. Immediate (days 1–14 postvaccination) or delayed (days 15–28 postvaccination) administration of rapamycin led to a significant increase in the generation of vaccine-induced NY-ESO-1-specific T cells exhibiting central memory phenotypes (CD45RO+CD45RA− CCR7+). Moreover, delayed administration resulted in a greater than threefold (p=0.025) and eightfold (p=0.005) increase in the frequency of NY-ESO-1-specific CD4+ T and CD8+ T cells respectively at the time of long-term follow-up, compared with its immediate usage.ConclusionOur novel finding is that delayed administration of rapamycin to patients during the contraction phase of vaccine-induced antitumor immune responses was particularly effective in increasing the frequency of memory T cells up to 1 year postvaccination in patients with solid tumors. Further studies are warranted to identify the impact of this approach on the durability of clinical remission.Trial registration number NCT00803569, NCT01536054, NCT01522820.

The attributes of specificity and memory enable CD8 + T cells to provide long-lasting protection against a variety of challenges. Although, the importance of CD8 + T cells for protection against intracellular infections and transformation is well-established, the functional type; effector phenotypes (Tc1, Tc2, Tc17 and/or Tcreg) and/or memory (effector or central), of CD8 + T cells most desirable for tumor immunity is not established. To determine the tumor efficacy of various effector types and/or memory CD8 T cells, it is imperative to better understand intrinsic and extrinsic factors that regulate CD8 + T cell differentiation and use this information to generate and test distinct functional cell types in tumor models. The focus of our laboratory investigations is to identify the extrinsic factors such as antigen strength, co-stimulatory molecules, cytokines, and small molecule modifiers that regulate intrinsic programs for various effector and/or memory cell fate in antigen specific CD8 T cells. The use of this information to generate immunity in murine tumor models has facilitated development of new adoptive cell transfer (ACT) as well as immunization strategies for cancer treatment.