Joe-Marc Chauvin

TIGIT and PD-1 impair tumor antigen–specific CD8 + T cells in melanoma patients

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.

CD4CD8αα Lymphocytes, A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

Gut bacterium Faecalibacterium prausnitzii activates a newly identified set of human IL-10-producing Treg cells (CD4CD8αα lymphocytes), revealing a mechanism by which commensal microbes contribute to host immunity.

IL10 and PD-1 Cooperate to Limit the Activity of Tumor-Specific CD8+ T Cells

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-1(high) (PD-1(high)) tumor antigen (TA)-specific CD8(+) T cells present at periphery and at tumor sites in patients with advanced melanoma upregulate IL10 receptor (IL10R) expression. Multiple subsets of peripheral blood mononucleocytes from melanoma patients produce IL10, which acts directly on IL10R(+) TA-specific CD8(+) T cells to limit their proliferation and survival. PD-1 blockade augments expression of IL10R by TA-specific CD8(+) T cells, thereby increasing their sensitivity to the immunosuppressive effects of endogenous IL10. Conversely, IL10 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 IL10 and PD-1 to strengthen the counteraction of T-cell immunosuppression and to enhance the activity of TA-specific CD8(+) T cell in advanced melanoma patients.

HLA Anchor Optimization of the Melan-A–HLA-A2 Epitope within a Long Peptide Is Required for Efficient Cross-Priming of Human Tumor-Reactive T Cells

The uptake and long-term cross-presentation of tumor Ag long peptides (LP) by dendritic cells (DC) make them attractive cancer vaccine candidates. However, it remains to be established whether LP can prime long-lived tumor-reactive CTL and whether other cell types are able to cross-present them. Using HLA-A2 healthy donor and melanoma patient-derived PBMC, we studied the in vitro cross-priming potential of Melan-A 16–40 LP bearing the HLA-A2–restricted epitope 26–35 or its analog 26–35A27L and compared it to the priming capacity of the short analog. We then addressed LP priming capacity in vivo using HLA-A2 mice. We also studied LP cross-presentation by monocyte-derived DC, plasmacytoid DC, monocytes, and B cells. We showed that the modified LP gave rise to high and sustained cross-presentation by monocyte-derived DC. This led to cross priming in vitro and in vivo and to expansion of long-lived tumor-reactive cytotoxic T cells. In contrast, the LP containing the natural 26–35 epitope primed specific T cells poorly, despite its long-lived cross-presentation, and T cells primed against the short analog were short-lived. We further showed that LP cross-presentation is restricted to monocytes and conventional DC. These results document for the first time, to our knowledge, the strong immunogenicity of a human tumor Ag LP. Of note, they underscore that this property is critically dependent on sufficient HLA binding affinity and/or TCR ligand potency of the cross-presented epitope. We conclude that LP fulfilling this requirement should be used as tumor vaccines, together with DC maturating agents, especially the Melan-A 16–40A27L LP, for the treatment of HLA-A2+ melanoma patients.

CD226 opposes TIGIT to disrupt Tregs in melanoma

CD4+ Tregs impede T cell responses to tumors. They express multiple inhibitory receptors that support their suppressive functions, including T cell Ig and ITIM domain (TIGIT). In melanoma patients, we show that Tregs exhibit increased TIGIT expression and decreased expression of its competing costimulatory receptor CD226 as compared with CD4+ effector T cells, resulting in an increased TIGIT/CD226 ratio. Tregs failed to upregulate CD226 upon T cell activation. TIGIT+ Tregs are highly suppressive, stable, and enriched in tumors. TIGIT and CD226 oppose each other to augment or disrupt, respectively, Treg suppression and stability. A high TIGIT/CD226 ratio in Tregs correlates with increased Treg frequencies in tumors and poor clinical outcome upon immune checkpoint blockade. Altogether, our findings show that a high TIGIT/CD226 ratio in Tregs regulates their suppressive function and stability in melanoma. They provide the rationale for novel immunotherapies to activate CD226 in Tregs together with TIGIT blockade to counteract Treg suppression in cancer patients.

Abstract 4046: Interleukin-10 and programmed death-1 cooperate to regulate tumor antigen-specific CD8+ T cells in melanoma patients

With the recent success of immune checkpoint blockade in the clinic, it is expected that strategies aimed at counteracting the negative immunoregulatory networks in the tumor microenvironment will develop into potent therapy for melanoma. 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. IL-10 is produced by multiple PBMC subsets in melanoma patients and acts directly on IL-10R+ TA-specific CD8+ T cells to impede proliferation and increase apoptosis. PD-1 blockade augments IL-10R expression by TA-specific CD8+ T cells, suggesting that PD-1 blockade renders PD-1+ TA-specific CD8+ T cells more sensitive to the immunosuppressive effects of endogenous IL-10. As a result, we show that IL-10 blockade adds to PD-1 blockade to increase the expansion and functions of TA-specific CD8+ T cells. Collectively, our findings support dual IL-10 and PD-1 blockade to counteract melanoma-induced T cell dysfunction and enhance TA-specific CD8+ T cell responses in patients with advanced melanoma. Citation Format: Zhaojun Sun, Julien Fourcade, Ornella Pagliano, Joe-Marc Chauvin, Cindy Sander, John M. Kirkwood, Hassane M. Zarour. Interleukin-10 and programmed death-1 cooperate to regulate tumor antigen-specific CD8+ T cells in melanoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4046. doi:10.1158/1538-7445.AM2015-4046

Phase Ib/II Study of Pembrolizumab and Pegylated-Interferon Alfa-2b in Advanced Melanoma

PURPOSE Objective responses are reported in 34% to 37% of patients with programmed death-1 (PD-1)-naïve advanced melanoma treated with PD-1 inhibitors. Pre-existing CD8+ T-cell infiltrate and interferon (IFN) gene signature correlate with response to PD-1 blockade. Here, we report a phase Ib/II study of pembrolizumab/pegylated (PEG)-IFN combination in PD-1-naïve advanced melanoma. PATIENTS AND METHODS PEG-IFN (1, 2, and 3 μg/kg per week) was dose escalated using a modified toxicity probability interval design in three cohorts of four patients each, whereas pembrolizumab was dosed at 2 mg/kg every 3 weeks in the phase Ib portion. Thirty-one patients were enrolled in the phase II portion. Primary objectives were safety and incidence of dose-limiting toxicities. Secondary objectives included objective response rate, progression-free survival (PFS), and overall survival. RESULTS Forty-three patients with stage IV melanoma were enrolled in the phase Ib and II portions of the study and included in the analysis. At the data cutoff date (December 31, 2017), median follow-up duration was 25 months (range, 1 to 38 months). All 43 patients experienced at least one adverse event; grade 3/4 treatment-related adverse events occurred in 21 of 43 patients (48.8%). Objective responses were seen at all three dose levels among 43 evaluable patients. The objective response rate was 60.5%, with 46.5% of patients exhibiting ongoing response. Median PFS was 11.0 months in all patients and unreached in responders, whereas median overall survival remained unreached in all patients. The 2-year PFS rate was 46%. CONCLUSION Pembrolizumab/PEG-IFN demonstrated an acceptable toxicity profile with promising evidence of clinical efficacy in PD-1-naïve metastatic melanoma. These results support the rationale to further investigate this pembrolizumab/PEG-IFN combination in this disease.

Abstract 459: Targeting TIGIT and PD-1 to increase the expansion and function of tumor antigen-specific CD8+ T cells in melanoma patients

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA T-cell Immunoglobulin and ITIM Domain (TIGIT) is an inhibitory receptor expressed by activated T cells, regulatory T cells and NK cells. Here, we show that tumor antigen (TA)-specific CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) upregulate TIGIT and most often co-express TIGIT and PD-1 in melanoma patients. CD8+ TILs downregulate CD226, supporting an imbalance of TIGIT/CD226 expression in metastatic melanoma. TIGIT is a marker of early T cell activation, which is further upregulated by T cells upon PD-1 blockade and by dysfunctional PD-1+Tim-3+ TA-specific CD8+ T cells. PD-1+TIGIT+, PD-1−TIGIT+ and PD-1+ TIGIT− CD8+ TILs exhibit similar functional capacities ex vivo, suggesting that TIGIT upregulation alone or together with PD-1 is not a marker of T cell dysfunction. However, in the presence of TIGIT ligand-expressing cells, TIGIT blockade adds to PD-1 blockade to increase the proliferation, cytokine production and degranulation of TA-specific CD8+ T cells and CD8+ TILs. Collectively, our findings show that TIGIT together with PD-1 regulate the expansion and function of TA-specific CD8+ T cells and CD8+TILs in melanoma patients. They also support the use of dual TIGIT and PD-1 blockade to stimulate potent antitumor CD8+ T cells responses in patients with advanced melanoma. Citation Format: Joe-Marc Chauvin, Ornella Pagliano, Julien Fourcade, Zhaojun Sun, Cindy Sanders, John M. Kirkwood, Tseng-hui T. Chen, Mark Maurer, Alan Korman, Hassane Zarour. Targeting TIGIT and PD-1 to increase the expansion and function of tumor antigen-specific CD8+ T cells in melanoma patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 459. doi:10.1158/1538-7445.AM2015-459

Abstract 4576: Immunization with CPG in combination with MHC class I and class II peptides stimulates rapid and strong tumor antigen-specific CTL responses in melanoma patients.

There is now ample evidence that the toll-like receptor 9 ligand CPG/PF-3512676 represents a potent adjuvant for peptide/protein-based cancer vaccines capable of stimulating ex vivo detectable tumor antigen (TA)-specific CD8+ T cell responses in patients with advanced melanoma. Although CD4+ T cells appeared to promote the expansion and functions of CD8+ T cells in experimental models, melanoma-associated helper peptides have been shown to paradoxically decrease CD8+ T cell responses to a multipeptide melanoma vaccine emulsified in Montanide, possibly by inducing antigen-specific Tregs. To further investigate this question, we have performed one Phase I study of immunization where patients with advanced melanoma were immunized with CPG, Montanide and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V either alone (Arm 1, n=5) or in combination with the pan-DR CD4 helper peptide NY-ESO-1 119-143 (Arm 2, n=7). Patients in both arms exhibited a rapid increase of NY-ESO-1-specific CD8+ T cells that were detectable ex vivo by flow cytometry. Strikingly, the mean frequency of vaccine-induced NY-ESO-1-specific CD8+ T cells after 6 biweekly immunizations was five times higher in patients who also received the helper peptide. Melanoma patients immunized with the helper peptide (Arm 2) developed a rapid increase of Foxp3− NY-ESO-1-specific CD4+ T cells that produced Th-1 type cytokines and IL-21 ex vivo. As compared to Arm 1, melanoma patients included in Arm 2 developed vaccine-induced NY-ESO-1-specific CD8+ T cells that were more differentiated, produced more IFN-γ and exhibited higher cytolytic functions in ex vivo assays. Collectively, our data support the inclusion of well-defined MHC class II peptides in combination with CPG/PF-3512676 and MHC class I peptides in multipeptide melanoma vaccines to better augment the expansion, differentiation, IFN-γ production and lytic function of TA-specific CTLs in patients with advanced melanoma. This work was supported by the National Institutes of Health/National Cancer Institute grants R01CA90360 and R01CA157467 (to H.M. Zarour). Citation Format: Julien Fourcade, Zhaojun Sun, Ornella Pagliano, Joe-Marc Chauvin, Cindy Sander, Stergios Moschos, Ahmad Tarhini, Hussein Tawbi, John M. Kirkwood, Hassane M. Zarour. Immunization with CPG in combination with MHC class I and class II peptides stimulates rapid and strong tumor antigen-specific CTL responses in melanoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4576. doi:10.1158/1538-7445.AM2013-4576