Zhaojun Sun

Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen–specific CD8+ T cell dysfunction in melanoma patients

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.

CD8+ T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1

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.

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.

PD-1 Is a Regulator of NY-ESO-1-Specific CD8+ T Cell Expansion in 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.

PD-1 and Tim-3 Regulate the Expansion of Tumor Antigen–Specific CD8+ T Cells Induced by Melanoma Vaccines

Although melanoma vaccines stimulate tumor antigen-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 coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freunds adjuvant, 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 tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-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 patients with advanced melanoma.

Human Tumor Antigen-Specific Helper and Regulatory T Cells Share Common Epitope Specificity but Exhibit Distinct T Cell Repertoire

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-β. 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 Vβ 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.

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.

Epitope Hierarchy of Spontaneous CD4+ T Cell Responses to LAGE-1

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.

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