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 tumorassociated 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.tumor-infiltrating lymphocytes | IL-6 | IL-10 | T cell receptor T he presence of tumor-infiltrating lymphocytes within the tumor microenvironment is considered to be an indication of the host immune response to tumor antigens and is thought to reflect the dynamic process of "cancer immunoediting" (1). In epithelial ovarian cancer (EOC), support for the role of immune surveillance of tumors comes from observations indicating that the presence of intraepithelial CD8 + -infiltrating T lymphocytes in tumors is associated with improved survival of patients with the disease (2). Although several lines of evidence have shown that spontaneous or vaccine-induced tumor-antigen-specific CD8 + T cells can recognize EOC targets (3), prolongation of survival in patients treated with immunization has only rarely been observed. This is probably a reflection of several in vivo immunosuppressive mechanisms in EOC-bearing hosts (4). Therefore, understanding factors that regulate the function(s) of tumor-antigen-specific CD8 + T cells is critical for effective control of tumor recurrence.The NY-ESO-1 tumor antigen is a major target of CD8 + T cell recognition in EOC, eliciting both cellular and humoral immune responses in a proportion of patients with advanced NY-ESO-1-expressing tumors (5). However, similar to infectious disease models, chronic antigenic stimulation may result in exhaustion of antigen-specific CD8 + T cells (6) and loss of ability to produce key cytokines that are critical for the maintenance of CD8 + T ce...

T cell trafficking at vascular sites has emerged as a key step in antitumor immunity. Chemokines are credited with guiding the multistep recruitment of CD8+ T cells across tumor vessels. However, the multiplicity of chemokines within tumors has obscured the contributions of individual chemokine receptor/chemokine pairs to this process. Moreover, recent studies have challenged whether T cells require chemokine receptor signaling at effector sites. Here, we investigate the hierarchy of chemokine receptor requirements during T cell trafficking to murine and human melanoma. These studies reveal a non-redundant role for GαI-coupled CXCR3 in stabilizing intravascular adhesion and extravasation of adoptively transferred CD8+ effectors that is indispensable for therapeutic efficacy. In contrast, functional CCR2 and CCR5 on CD8+ effectors fail to support trafficking despite the presence of intratumoral cognate chemokines. Taken together, these studies identify CXCR3-mediated trafficking at the tumor vascular interface as a critical checkpoint to effective T cell-based cancer immunotherapy.

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.

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NY-ESO-1 is a ''cancer-testis'' antigen expressed in epithelial ovarianHLA-DP4 ͉ peptide epitope ͉ tumor recognition ͉ vaccine T here is increasing evidence that the immune system has the ability to recognize tumor-associated antigens expressed in human malignancies and to induce antigen-specific humoral and cellular immune responses to these targets. In epithelial ovarian cancer (EOC), support for the role of immune surveillance of tumors comes from our recent observation indicating that the presence of intraepithelial CD8 ϩ -infiltrating T lymphocytes in tumors is associated with improved survival of patients with the disease (1). Although the majority of women with advanced-stage ovarian cancer respond to first-line chemotherapy, most of these responses are not durable, and Ͼ70% of patients die of recurrent disease within 5 years of diagnosis. Therefore, the development of strategies to enhance the potential of tumor antigen-specific CD8 ϩ T and CD4 ϩ T cells is urgently needed for extending remission rates in this disease. In this regard, cancer-testis antigens, a unique class of antigens that demonstrate high levels of expression in adult male germ cells but generally not in other normal adult tissues and aberrant expression in a variable proportion of a wide range of different cancer types, are promising candidates for immunotherapy. Among cancer-testis antigens, NY-ESO-1 (2) is one of the most spontaneously immunogenic tumor antigens described so far. Previously, we reported that NY-ESO-1 is a promising target for specific immunotherapy of EOC (3).Although the majority of cancer vaccine trials have focused on eliciting antigen-specific CD8 ϩ T cells, a growing body of evidence indicates that CD4 ϩ T cells play a pivotal role in orchestrating these responses. The multiple roles of antigen-specific CD4 ϩ T cells include the provision of help to CD8 ϩ T cells during the primary and secondary immune responses, direct cytolysis, and activation of B cells for production of tumor antigen-specific Abs. Therefore, we have focused on the NY-ESO-1 epitope, ESO 157-170 , a naturally processed helper epitope that is recognized by CD4 ϩ T cells in the context of HLA-DPB1*0401 and *0402 (4), prevalent MHC class II alleles present in Ϸ43-70% of Caucasians. Moreover, the NY-ESO-1 HLA-DP4 epitope has HLA-A2 (ESO 157-165 ) (5) and HLA-A24 (ESO 158-166 ) (6) motifs embedded in its natural sequence. In this study, we evaluated whether active immunization with ESO 157-170 would elicit NY-ESO-1-specific CD4 ϩ and CD8 ϩ T cell responses in ovarian cancer patients with minimal disease burden. In addition, we characterized NY-ESO-1-specific CD8 ϩ and CD4 ϩ T cell receptor (TCR) repertoires in conjunction with functional analysis of vaccine-elicited T cell clones.