The paradoxical coexistence of spontaneous tumor antigen–specific immune responses with progressive disease in cancer patients furthers the need to dissect the molecular pathways involved in tumor-induced T cell dysfunction. In patients with advanced melanoma, we have previously shown that the cancer-germline antigen NY-ESO-1 stimulates spontaneous NY-ESO-1–specific CD8+ T cells that up-regulate PD-1 expression. We also observed that PD-1 regulates NY-ESO-1–specific CD8+ T cell expansion upon chronic antigen stimulation. In the present study, we show that a fraction of PD-1+ NY-ESO-1–specific CD8+ T cells in patients with advanced melanoma up-regulates Tim-3 expression and that Tim-3+PD-1+ NY-ESO-1–specific CD8+ T cells are more dysfunctional than Tim-3−PD-1+ and Tim-3−PD-1− NY-ESO-1–specific CD8+ T cells, producing less IFN-γ, TNF, and IL-2. Tim-3–Tim-3L blockade enhanced cytokine production by NY-ESO-1–specific CD8+ T cells upon short ex vivo stimulation with cognate peptide, thus enhancing their functional capacity. In addition, Tim-3–Tim-3L blockade enhanced cytokine production and proliferation of NY-ESO-1–specific CD8+ T cells upon prolonged antigen stimulation and acted in synergy with PD-1–PD-L1 blockade. Collectively, our findings support the use of Tim-3–Tim-3L blockade together with PD-1–PD-L1 blockade to reverse tumor-induced T cell exhaustion/dysfunction in patients with advanced melanoma.

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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 melanoma patients. These cells were partially dysfunctional, producing less IFN-γ than BTLA− T cells, but more IFN-γ, TNF and IL-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.

Although melanoma vaccines stimulate tumor antigen (TA)-specific CD8+ T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8+ T cells with regard to the inhibitory T cell co-receptors PD-1 and Tim-3 in metastatic melanoma patients who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant (IFA), CpG oligodeoxynucleotide (CpG) and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid TA-specific CD8+ T-cell responses detected ex vivo, however, TA-specific CD8+ T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8+ T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8+ T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8+ T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T cell responses and increase the likelihood of clinical responses in advanced melanoma patients.

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Immune checkpoint inhibitors show great promise as therapy for advanced melanoma, heightening the need to determine the most effective use of these agents. Here, we report that programmed death-1high (PD-1high) tumor antigen (TA)-specific CD8+ T cells present at periphery and at tumor sites in patients with advanced melanoma upregulate IL-10 receptor (IL-10R) expression. Multiple subsets of peripheral blood mononucleocytes from melanoma patients produce IL-10, which acts directly on IL-10R+ TA-specific CD8+ T cells to limit their proliferation and survival. PD-1 blockade augments expression of IL-10R by TA-specific CD8+ T cells, thereby increasing their sensitivity to the immunosuppressive effects of endogenous IL-10. Conversely, IL-10 blockade strengthened the effects of PD-1 blockade in expanding TA-specific CD8+ T cells and reinforcing their function. Collectively, our findings offer a rationale to block both IL-10 and PD-1 to strengthen the counteraction of T cell immunosuppression and enhance the activity of TA-specific CD8+ T cell in advanced melanoma patients.

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.

Analogue peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific CTLs. Here, we report the results of a pilot trial designed to study the immunogenicity of the analogue peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1-expressing melanoma. Eight Patients were immunized either with Montanide and CpG (arm 1, three patients), Montanide and peptide NY-ESO-1 157-165V (arm 2, two patients) or with Montanide, CpG and peptide NY-ESO-1 157-165V (arm 3, three patients). Only the three patients immunized with Montanide, CpG and peptide NY-ESO-1 157-165V in arm 3, developed a rapid increase of effector-memory NY-ESO-1-specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28−). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1-specific CD8+ T cells in two patients with advanced disease but failed to prime tumor-reactive NY-ESO-1-specific T cells in one patient with no spontaneously tumor-induced CD8+ T cell responses to NY-ESO-1. Collectively, our data support the capability of the analogue peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1-specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1-specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1-specific CD8+ T cells following peptide-based vaccine strategies.