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T cell Ig and ITIM domain (TIGIT) is an inhibitory receptor expressed by activated T cells, Tregs, and NK cells. Here, we determined that TIGIT is upregulated on tumor antigen-specific (TA-specific) CD8⁺ T cells and CD8⁺ tumor-infiltrating lymphocytes (TILs) from patients with melanoma, and these TIGIT-expressing CD8⁺ T cells often coexpress the inhibitory receptor PD-1. Moreover, CD8⁺ TILs from patients exhibited downregulation of the costimulatory molecule CD226, which competes with TIGIT for the same ligand, supporting a TIGIT/CD226 imbalance in metastatic melanoma. TIGIT marked early T cell activation and was further upregulated by T cells upon PD-1 blockade and in dysfunctional PD-1⁺TIM-3⁺ TA-specific CD8⁺ T cells. PD-1⁺TIGIT⁺, PD-1⁻TIGIT⁺, and PD-1⁺TIGIT⁻ CD8⁺ TILs had similar functional capacities ex vivo, suggesting that TIGIT alone, or together with PD-1, is not indicative of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT and PD-1 blockade additively increased proliferation, cytokine production, and degranulation of both TA-specific CD8⁺ T cells and CD8⁺ TILs. Collectively, our results show that TIGIT and PD-1 regulate the expansion and function of TA-specific CD8⁺ T cells and CD8⁺ TILs in melanoma patients and suggest that dual TIGIT and PD-1 blockade should be further explored to elicit potent antitumor CD8⁺ T cell responses in patients with advanced melanoma.

Patients with advanced melanoma can develop spontaneous cellular and humoral responses to tumor antigens. Understanding the failure of spontaneous or vaccine-induced tumor antigen-specific T-cell responses to promote the immunologic clearance of melanomas is critical. Multiple mechanisms of melanoma-induced immune escape, which are likely to cause the failure of the spontaneous or vaccine-induced immune responses to promote tumor regression in humans, have been elucidated. In addition, a number of negative factors in the tumor microenvironment dampen antitumor immune responses, including cytokines (like transforming growth factor-β or interleukin-10), suppressive cells (regulatory T cells and myelosuppressive dendritic cells), defective antigen presentation by tumor cells (human leukocyte antigen or T antigen expression loss, antigen processing machinery defects), amino acid catabolizing enzymes (indoleamine-2-3 dioxygenase, arginase), and immune inhibitory pathways (like cytotoxic T-lymphocyte antigen 4/cluster of differentiation 28, programmed death 1/programmed death 1 ligand 1). This information has been used to develop a number of therapies to specifically target these negative regulators of antimelanoma immune responses to enhance tumor antigen-specific immune responses and to increase the likelihood of clinical benefits in patients with advanced melanoma., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

NY-ESO-1 and LAGE-1 represent highly homologous cancer-germline Ags frequently coexpressed by many human cancers, but not by normal tissues, except testis. In contrast to NY-ESO-1, little is known about spontaneous immune responses to LAGE-1. In the current study, we report on spontaneous LAGE-1-specific CD4(+) T cells isolated from PBLs of patients with advanced LAGE-1(+)/NY-ESO-1(+) melanoma and directed against three promiscuous and immunodominant epitopes. Strikingly, although the three LAGE-1-derived epitopes are highly homologous to NY-ESO-1-derived epitopes, LAGE-1-specific CD4(+) T cells did not cross-react with NY-ESO-1. LAGE-1-specific CD4(+) T cells produced Th1-type and/or Th2-type cytokines and did not exert inhibitory effects on allogenic T cells. We observed that most patients with spontaneous NY-ESO-1-specific responses exhibited spontaneous CD4(+) T cell responses to at least one of the three immunodominant LAGE-1 epitopes. Additionally, nearly half of the patients with spontaneous LAGE-1-specific CD4(+) T cell responses had circulating LAGE-1-specific Abs that recognized epitopes located in the C-terminal portion of LAGE-1, which is distinct from NY-ESO-1. Collectively, our findings define the hierarchy of immunodominance of spontaneous LAGE-1-specific CD4(+) T cell responses in patients with advanced melanoma. These findings demonstrate the capability of LAGE-1 to stimulate integrated cellular and humoral immune responses that do not cross-react with NY-ESO-1. Therefore, they provide a strong rationale for the inclusion of LAGE-1 peptides or protein in vaccine trials for patients with NY-ESO-1(+)/LAGE-1(+) tumors.

CD4(+) regulatory T cells (Tregs) accumulate at tumor sites and play a critical role in the suppression of immune responses against tumor cells. In this study, we show that two immunodominant epitopes derived from the tumor Ags (TAs) NY-ESO-1 and TRAG-3 stimulate both CD4+ Th cells and Tregs. TA-specific Tregs inhibit the proliferation of allogenic T cells, act in a cell-to-cell contact dependent fashion and require activation to suppress IL-2 secretion by T cells. TRAG-3 and NY-ESO-1-specific Tregs exhibit either a Th1-, a Th2-, or a Th0-type cytokine profile and dot not produce IL-10 or TGF-beta. The Foxp3 levels vary from one Treg clone to another and are significantly lower than those of CD4+CD25high Tregs. In contrast to NY-ESO-1-specific Th cells, the NY-ESO-1-specific and TRAG-3-specific Treg clonotypes share a common TCR CDR3 Vbeta usage with Foxp3+CD4+CD25high and CD4+CD25- T cells and were not detectable in PBLs of other melanoma patients and of healthy donors, suggesting that their recruitment occurs through the peripheral conversion of CD4+CD25- T cells upon chronic Ag exposure. Collectively, our findings demonstrate that the same epitopes spontaneously stimulate both Th cells and Tregs in patients with advanced melanoma. They also suggest that TA-specific Treg expansion may be better impaired by therapies aimed at depleting CD4+CD25high Tregs and preventing the peripheral conversion of CD4+CD25- T cells.

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8(+) T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8(+) T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8(+) T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8(+) T cells, NY-ESO-1-specific CD8(+) T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8(+) T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1(+) APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8(+) T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8(+) T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8(+) T cell numbers and functions, increasing the likelihood of tumor regression.

TCRs exhibit a high degree of specificity but may also recognize multiple and distinct peptide-MHC complexes, illustrating the so-called cross-reactivity of TCR-peptide-MHC recognition. In this study, we report the first evidence of CD4(+) T cells recognizing the same tumor peptide-epitope from NY-ESO-1, in the context of multiple HLA-DR and HLA-DP molecules. These cross-reactive CD4(+) T cells recognized not only autologous but also allogenic dendritic cells previously loaded with the relevant protein (i.e., the normally processed and presented epitope). Using clonotypic real-time RT-PCR, we have detected low frequencies of CD4(+) T cells expressing one cross-reactive TCR from circulating CD4(+) T cells of patients with stage IV melanoma either spontaneously or after immunization but not in normal donors. The maintenance of cross-reactive tumor Ag-specific CD4(+) T cells in PBLs of cancer patients required the presence of tumor Ag/epitope in the context of the MHC molecule used to prime the Ag-specific CD4(+) T cells. Our findings have significant implications for the optimization of TCR gene transfer immunotherapies widely applicable to cancer patients.