CXCR6 by increasing retention of memory CD8+ T cells in the ovarian tumor microenvironment promotes immunosurveillance and control of ovarian cancer.

PURPOSE: Resident 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 DESIGN: RNA 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. RESULTS: Chemokine 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. CONCLUSIONS: CXCR6, 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.

Uncoupling protein 2 regulates metabolic reprogramming and fate of antigen-stimulated CD8+ T cells.

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.

Direct tumor recognition by a human CD4(+) T-cell subset potently mediates tumor growth inhibition and orchestrates anti-tumor immune responses.

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.

LAG3 and PD1 co-inhibitory molecules collaborate to limit CD8+ T cell signaling and dampen antitumor immunity in a murine ovarian cancer model.

The immune co-inhibitory receptors lymphocyte activation gene-3 (LAG3) and programmed cell death 1 (PD1) synergistically contribute to autoimmunity and tumor evasion. Here we demonstrate how they collaborate and interact to regulate T cell function. We first show that LAG3 and PD1 are co-expressed on both OVA-specific and non-specific T cells infiltrating murine ovarian tumors. Dual antibody blockade or genetic knockout of LAG3 and PD1 significantly enhanced T effector function and delayed tumor growth. LAG3 and PD1 co-localized in activated CD8+ T cells in vitro at the trans-Golgi vesicles, early/recycling endosomal compartments, lysosomes, and microtubule organizing center. Importantly, LAG3 and PD1 cluster with pLck at the immunological synapse. Reciprocal immunoprecipitation of T cell extracts revealed physical interaction between LAG3 and PD1. Mutational analyses indicate that the cytoplasmic domain of LAG3 is not absolutely required for its association with PD1, while the ITIM and ITSM of PD1 are necessary for its association with LAG3. Finally, LAG3 protein also associates with the Src-homology-2 domain-containing phosphatases (SHP1/2) which are known to be recruited by PD1 during T cell signaling. Our data indicate that the association of LAG3 with PD1 contributes to their rapid trafficking to the immunological synapse, leading to a synergistic inhibitory effect on T cell signaling.

ß-Catenin in dendritic cells exerts opposite functions in cross-priming and maintenance of CD8+ T cells through regulation of IL-10.

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

Nonclassical antigen-processing pathways are required for MHC class II-restricted direct tumor recognition by NY-ESO-1-specific CD4(+) T cells.

Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.

ß-catenin mediates tumor-induced immunosuppression by inhibiting cross-priming of CD8⁺ T cells.

Whereas CD8⁺ T cells are essential for anti-tumor immunity, tumors often evade CD8⁺ T cell surveillance by immunosuppression. As the initiators of antigen-specific immune responses, DCs are likely to play a central role in regulating the balance between immunity and tolerance to tumor antigens and are specialized in their ability to cross-present exogenous tumor antigens on MHC class I molecules to initiate CD8⁺ T cell immunity. However, it remains unclear whether and how tumors modulate DC functions to suppress CD8⁺ T cell responses. We have shown previously that ß-catenin signaling in DCs promotes DC-mediated CD8⁺ T cell tolerance. Here, we tested the hypothesis that ß-catenin in DCs mediates tumor-induced suppression of CD8⁺ T cell immunity by inhibiting the ability of DCs in cross-priming. ß-Catenin was activated in DCs by multiple tumors in vivo and in vitro. B16 melanoma-bearing mice, when vaccinated with DC-targeting anti-DEC-205 mAb fused with tumor antigens, exhibited dampened CD8⁺ immunity, similar to DC-ß-catenin(active) mice. DCs from DC-ß-catenin(active) and tumor-bearing mice were deficient in cross-priming, and antigen-specific CD8⁺ T cells primed in these mice resulted in dampened CD8⁺ memory responses. Importantly, DC-ß-catenin⁻/⁻ mice completely abrogate tumor-mediated inhibition of cross-priming, suggesting that tumor-induced inhibition of cross-priming is dependent on ß-catenin. Finally, enhancing cross-priming at the priming or recall phase rescued ß-catenin-suppressed CD8⁺ immunity in DC-ß-catenin(active) and tumor-bearing mice. Thus, ß-catenin-mediated inhibition of cross-priming represents a new and potentially general mechanism that tumors employ to achieve immunosuppression.

A unique form of haptoglobin produced by murine hematopoietic cells supports B-cell survival, differentiation and immune response.

Haptoglobin (Hp), an acute phase reactant and major hemoglobin-binding protein, has a unique role in host immunity. Previously, we demonstrated that Hp-deficient C57BL/6J mice exhibit stunted development of mature T- and B-cells resulting in markedly lower levels of antigen-specific IgG. The current study identified leukocyte-derived pro-Hp as a relevant mediator of an optimal immune response. Reconstitution of Hp-/- mice with Hp+/+ bone marrow restored normal immune response to ovalbumin. Furthermore, transplanting a mixture of bone marrow-derived from B-cell-deficient and Hp-deficient mice into Rag1-/-/Hp+/+ recipients resulted in mice with a defective immune response similar to Hp-/- mice. This suggests that Hp generated by the B-cell compartment, rather than by the liver, is functionally contributing to a normal immune response. Leukocytes isolated from the spleen express Hp and release a non-proteolytically processed pro-Hp that uniquely differed from liver-derived Hp by not binding to hemoglobin. While addition of purified plasma Hp to cultured B-cells did not alter responses, pro-Hp isolated from splenocytes enhanced cellular proliferation and production of IgG. Collectively, the comparison of wild-type and Hp-deficient mice suggests a novel regulatory activity for lymphocyte-derived Hp, including Hp produced by B-cells themselves, that supports in vivo survival and functional differentiation of the B-cells to ensure an optimal immune response.

Prostate derived Ets transcription factor and Carcinoembryonic antigen related cell adhesion molecule 6 constitute a highly active oncogenic axis in breast cancer.

We previously reported overexpression of Prostate derived Ets transcription factor (PDEF) in breast cancer and its role in breast cancer progression, supporting PDEF as an attractive target in this cancer. The goal of this research was to identify specific PDEF induced molecules that, like PDEF, show overexpression in breast tumors and a role in breast tumor progression. PDEF expression was down regulated by shRNA in MCF-7 human breast tumor cell line, and probes from PDEF down-regulated and control MCF-7 cells were used to screen the HG-U133A human gene chips. These analyses identified 1318 genes that were induced two-fold or higher by PDEF in MCF-7 cells. Further analysis of three of these genes, namely CEACAM6, S100A7 and B7-H4, in relation to PDEF in primary breast tumors showed that in 82% of ER+, 67% of Her2 overexpressing and 24% of triple-negative breast tumors both PDEF and CEACAM6 expression was elevated 10-fold or higher in comparison to normal breast tissue. Overall, 72% (94 of 131) of the primary breast tumors showed 10-fold or higher expression of both PDEF and CEACAM6. In contrast, S100A7 and B7-H4 failed to show concordant elevated expression with PDEF in primary tumors. To determine the significance of elevated PDEF and CEACAM6 expression to tumor phenotype, their expression was down regulated by specific siRNAs in human breast tumor cell lines. This resulted in the loss of viability of tumor cells in vitro, supporting an oncogenic role for both PDEF and CEACAM6 in breast cancer. Together, these findings show that PDEF-CEACAM6 is a highly active oncogenic axis in breast cancer and suggest that targeting of these molecules should provide novel treatments for most breast cancer patients.

Rapamycin: A rheostat for CD8(+) T-cell-mediated tumor therapy.

Vaccines that generate Ag-specific CD8(+) T-cell responses of appropriate quality, magnitude and duration are highly desirable. The ability of mTOR to regulate CD8(+) T-cell functional differentiation must be exploited for clinical benefit. In a recent paper, we report that varying the regimen of rapamycin administration regulates viral vaccine-induced CD8(+) T-cell responses for tumor immunity. These observations validate the use of rapamycin in vaccination strategies and demonstrate the efficacy of memory CD8(+) T-cell responses for tumor immunity.

Effects of 1-methyltryptophan stereoisomers on IDO2 enzyme activity and IDO2-mediated arrest of human T cell proliferation.

IDO2 is a newly discovered enzyme with 43 % similarity to classical IDO (IDO1) protein and shares the same critical catalytic residues. IDO1 catalyzes the initial and rate-limiting step in the degradation of tryptophan and is a key enzyme in mediating tumor immune tolerance via arrest of T cell proliferation. The role of IDO2 in human T cell immunity remains controversial. Here, we demonstrate that similar to IDO1, IDO2 also degrades tryptophan into kynurenine and is inhibited more efficiently by Levo-1-methyl tryptophan (L-1MT), an IDO1 competitive inhibitor, than by dextro-methyl tryptophan (D-1MT). Although IDO2 enzyme activity is weaker than IDO1, it is less sensitive to 1-MT inhibition than IDO1. Moreover, our results indicate that human CD4(+) and CD8(+) T cell proliferation was inhibited by IDO2, but both L-1MT and D-1MT could not reverse IDO2-mediated arrest of cell proliferation, even at high concentrations. These data indicate that IDO2 is an inhibitory mechanism in human T cell proliferation and support efforts to develop more effective IDO1 and IDO2 inhibitors in order to overcome IDO-mediated immune tolerance.

Efficacy of vaccination with recombinant vaccinia and fowlpox vectors expressing NY-ESO-1 antigen in ovarian cancer and melanoma patients.

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.

Transcription factor Foxo1 represses T-bet-mediated effector functions and promotes memory CD8(+) T cell differentiation.

The evolutionary conserved Foxo transcription factors are important regulators of quiescence and longevity. Although, Foxo1 is known to be important in regulating CD8(+) T cell trafficking and homeostasis, its role in functional differentiation of antigen-stimulated CD8(+) T cells is unclear. Herein, we demonstrate that inactivation of Foxo1 was essential for instructing T-bet transcription factor-mediated effector differentiation of CD8(+) T cells. The Foxo1 inactivation was dependent on mTORC1 kinase, given that blockade of mTORC1 abrogated mTORC2-mediated Akt (Ser473) kinase phosphorylation, resulting in Foxo1-dependent switch from T-bet to Eomesodermin transcription factor activation and increase in memory precursors. Silencing Foxo1 ablated interleukin-12- and rapamycin-enhanced CD8(+) T cell memory responses and restored T-bet-mediated effector functions. These results demonstrate an essential role of Foxo1 in actively repressing effector or terminal differentiation processes to promote memory CD8(+) T cell development and identify the functionally diverse mechanisms utilized by Foxo1 to promote quiescence and longevity.

Regulating mammalian target of rapamycin to tune vaccination-induced CD8(+) T cell responses for tumor immunity.

Vaccine strategies aimed at generating CD8(+) T cell memory responses are likely to show augmented efficacy against chronic challenges like tumor. The abundance in variety of memory CD8(+) T cells behooves development of vaccine strategies that generate distinct memory responses and evaluate them for tumor efficacy. In this study, we demonstrate the ability of a variety of rapamycin treatment regimens to regulate virus vaccination-induced CD8(+) T cell memory responses and tumor efficacy. Strikingly, a short course of high-dose, but not low-dose, rapamycin treatment transiently blocks viral vaccination-induced mammalian target of rapamycin activity in CD8(+) T cells favoring persistence and Ag-recall responses over type 1 effector maturation; however, prolonged high-dose rapamycin administration abrogated memory responses. Furthermore, a short course of high-dose rapamycin treatment generated CD8(+) T cell memory responses that were independent of IL-15 and IL-7 and were programmed early for sustenance and greater tumor efficacy. These results demonstrate the impact a regimen of rapamycin treatment has on vaccine-induced CD8(+) T cell responses and indicates that judicious application of rapamycin can augment vaccine efficacy for chronic challenges.

Class I histone deacetylase inhibitor entinostat suppresses regulatory T cells and enhances immunotherapies in renal and prostate cancer models.

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor immunity.

The cell-intrinsic mechanisms guiding naive CD8+ T cells for clonal expansion and memory generation via homeostatic proliferation (HP) are unclear. Here, we have shown that HP of naive CD8+ T cells requires IL-7-, but not IL-15-induced mTOR kinase activation. HP-induced mTOR enhances transcription factor T-bet for functional maturation and CD122 expression, which sensitizes for an IL-15-dependent memory transition by favoring transcription factor Eomesodermin over T-bet. Inhibition of mTOR blocks T-bet and CD122 expression but preserves memory in an IL-15-independent manner by promoting Eomesodermin expression. The ability of rapamycin to augment HP-induced memory was cell-intrinsic given that silencing mTOR in CD8+ T cells generated identical outcomes. Strikingly, HP-induced CD8+ T cell memory generated by IL-15-dependent or -independent mechanisms demonstrated identical tumor efficacy. These results indicate a central role for mTOR in HP-induced CD8+ T cell responses and demonstrate the importance for CD8+ memory in HP-induced tumor efficacy.

Fine-tuning CD8(+) T cell functional responses: mTOR acts as a rheostat for regulating CD8(+) T cell proliferation, survival and differentiation?

Naïve CD8(+) T cells are instructed by antigen, co-stimulatory molecules and cytokines to undergo proliferation, clonal expansion and differentiation for effector and memory functions. The integration of extracellular instructions induces coordinated signaling cascades and transcriptional programs to determine CD8(+) T cell functional fate. Although, an important role for the energy sensitive kinase-mammalian target of rapamycin (mTOR) in regulating T cell proliferation and cell survival instep with cellular metabolic status has been demonstrated, emerging information indicates that mTOR also acts as a critical regulator of effector and memory functional fates in CD8(+) T cells. Herein, we discuss the pathways by which cellular metabolism regulates proliferation, survival and functional differentiation of CD8(+) T cells and focus on the role of mTOR as the rheostat that strikes a balance between effector and memory differentiation of CD8(+) T cells for enabling adaptive host immunity.

NY-ESO-1 protein glycosylated by yeast induces enhanced immune responses.

Vaccine strategies that target dendritic cells to elicit potent cellular immunity are the subject of intense research. Here we report that the genetically engineered yeast Saccharomyces cerevisiae, expressing the full-length tumour-associated antigen NY-ESO-1, is a versatile host for protein production. Exposing dendritic cells (DCs) to soluble NY-ESO-1 protein linked to the yeast a-agglutinin 2 protein (Aga2p) protein resulted in protein uptake, processing and MHC class I cross-presentation of NY-ESO-1-derived peptides. The process of antigen uptake and cross-presentation was dependent on the glycosylation pattern of NY-ESO-1-Aga2p protein and the presence of accessible mannose receptors. In addition, NY-ESO-1-Aga2p protein uptake by dendritic cells resulted in recognition by HLA-DP4 NY-ESO-1-specific CD4(+) T cells, indicating MHC class II presentation. Finally, vaccination of mice with yeast-derived NY-ESO-1-Aga2p protein led to an enhanced humoral and cellular immune response, when compared to the bacterially expressed NY-ESO-1 protein. Together, these data demonstrate that yeast-derived full-length NY-ESO-1-Aga2p protein is processed and presented efficiently by MHC class I and II complexes and warrants clinical trials to determine the potential value of S. cerevisiae as a host for cancer vaccine development.

Tc17 cells are capable of mediating immunity to vaccinia virus by acquisition of a cytotoxic phenotype.

CD8 T cells can acquire cytokine-secreting phenotypes paralleling cytokine production from Th cells. IL-17-secreting CD8 T cells, termed Tc17 cells, were shown to promote inflammation and mediate immunity to influenza. However, most reports observed a lack of cytotoxic activity by Tc17 cells. In this study, we explored the anti-viral activity of Tc17 cells using a vaccinia virus (VV) infection model. Tc17 cells expanded during VV infection, and TCR transgenic Tc17 cells were capable of clearing recombinant VV infection. In vivo, adoptively transferred Tc17 cells lost the IL-17-secreting phenotype, even in the absence of stimulation, but they did not acquire IFN-gamma-secreting potential unless stimulated with a virus-encoded Ag. However, examination of cells following infection demonstrated that these cells acquired cytotoxic potential in vivo, even in the absence of IFN-gamma. Cytotoxic potential correlated with Fasl expression, and the cytotoxic activity of postinfection Tc17 cells was partially blocked by the addition of anti-FasL. Thus, Tc17 cells mediate VV clearance through expression of specific molecules associated with cytotoxicity but independent of an acquired Tc1 phenotype.

Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer.

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-gamma/TNF-alpha 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.