Anti–programmed cell death protein 1 (PD-1) therapy provides long-term clinical benefits to patients with advanced melanoma. The composition of the gut microbiota correlates with anti–PD-1 efficacy in preclinical models and cancer patients. To investigate whether resistance to anti–PD-1 can be overcome by changing the gut microbiota, this clinical trial evaluated the safety and efficacy of responder-derived fecal microbiota transplantation (FMT) together with anti–PD-1 in patients with PD-1–refractory melanoma. This combination was well tolerated, provided clinical benefit in 6 of 15 patients, and induced rapid and durable microbiota perturbation. Responders exhibited increased abundance of taxa that were previously shown to be associated with response to anti–PD-1, increased CD8+ T cell activation, and decreased frequency of interleukin-8–expressing myeloid cells. Responders had distinct proteomic and metabolomic signatures, and transkingdom network analyses confirmed that the gut microbiome regulated these changes. Collectively, our findings show that FMT and anti–PD-1 changed the gut microbiome and reprogrammed the tumor microenvironment to overcome resistance to anti–PD-1 in a subset of PD-1 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.

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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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◥ Purpose: Natural killer (NK) cells play a critical role in tumor immunosurveillance. Multiple activating and inhibitory receptors (IR) regulate NK-cell-mediated tumor control. The IR T-cell immunoglobulin and ITIM domain (TIGIT) and its counterreceptor CD226 exert opposite effects on NK-cell-mediated tumor reactivity.Experimental Design: We evaluated the frequency, phenotype, and functions of NK cells freshly isolated from healthy donors and patients with melanoma with multiparameter flow cytometry. We assessed TIGIT and CD226 cell surface expression and internalization upon binding to CD155. We evaluated the role of IL15 and TIGIT blockade in increasing NK-cell-mediated cytotoxicity in vitro and in two mouse models.Results: NK cells are present at low frequencies in metastatic melanoma, are dysfunctional, and downregulate both TIGIT and CD226 expression. As compared with TIGIT À NK cells, TIGIT þ NK cells exhibit higher cytotoxic capacity and maturation, but paradoxically lower cytotoxicity against CD155 þ MHC class Ideficient melanoma cells. Membrane bound CD155 triggers CD226 internalization and degradation, resulting in decreased NK-cell-mediated tumor reactivity. IL15 increases TIGIT and CD226 gene expression by tumor-infiltrating NK cells (TiNKs) and, together with TIGIT blockade, increases NK-cell-mediated melanoma cytotoxicity in vitro and decreases tumor metastasis in two mouse melanoma models. Specific deletion of TIGIT on transferred NK cells enhances the antimetastatic activity of IL15, while CD226 blockade decreases the effects of IL15 and TIGIT blockade. Conclusions: Our findings support the development of novel combinatorial immunotherapy with IL15 and TIGIT blockade to promote NK-cell-mediated destruction of MHC class I-deficient melanoma, which are refractory to CD8 þ T-cell-mediated immunity.

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.