Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions

Abstract

Pancreatic cancer (PC) is an aggressive disease with dismal prognosis. Surgical resection is the recommended treatment for long-term survival, but patients with resectable PC are in the minority (with a 5-year survival rate of 20 %). Therefore, development of novel therapeutic strategies, such as anti-PC immunotherapy, is crucial. α-Enolase (ENO1) is an enzyme expressed on the surface of pancreatic cancer cells and is able to promote cell migration and cancer metastasis. The capacity of ENO1 to induce an immune response in PC patients renders it a true tumor-associated antigen. In this study, we characterized the effector functions of ENO1-specific T cells isolated from PC patients, and we specifically evaluated the successful role of intra-tumoral T helper 17 (Th17) cells and the inhibitory role of regulatory T (Tregs) cells in respectively promoting or reducing the cancer-specific immune response. In this ex vivo study, we have demonstrated, for the first time, that ENO1-specific Th17 cells have a specific anti-cancer effector function in PC patients, and that there are decreased levels of these cells in cancer compared to healthy mucosa. Conversely, there are elevated levels of ENO1-specific Tregs in PC patients which lead to inhibition of the antigen-specific effector T cells, thus highlighting a possible role in promoting PC progression. These results may be relevant for the design of novel immunotherapeutic strategies in pancreatic cancer.

Fig. 1

Repertoire and cytokine production of TIL-derived T-cell clones (Tcc) obtained from patients with pancreatic cancer. a The histogram represents the total number of T cell clones isolated from single tumor tissue sites and the ratio of CD4⁺/CD8⁺ populations. b The cytokine phenotype distribution of CD4⁺ Tcc and c CD8⁺ Tcc. The percentages of clone cytokine profiles of CD4 (Th1, Th17, Th0, Th17) and of CD8 (Tc1, Tc17, Tc0, Tc17) were calculated comparing the number of Th/Tc types with the total number of CD4/CD8 clones obtained from the same tumor tissue sites (HM, MT, CT). The production of IFN-γ, IL-17 and IL-4 was measured in culture supernatants by specific ELISA tests

Fig. 2

Evaluation of Treg/Tnull populations in the three different tumor tissue sites. For single T cell clones, we evaluated the expression of CD25, TGF-β, IL-10 and FoxP3 by flow cytometry. Tregs were defined as the T cells producing IL-10, TGF-β and FoxP3⁺, and Tnull cells were the FoxP3⁻ T blasts unable to synthesize IL-10 and TGF-β. The figure indicates the number of Treg and Tnull T clones isolated from the healthy mucosa (HM), marginal tumor (MT) and central tumor (CT). The percentage is calculated by referring to the total CD4⁺/CD8⁺ population isolated from the same tumor tissue site (HM, MT, CT)

Fig. 3

Clonality and functional profile of TIL-derived T cells reactive to ΕΝΟ1. a T cell clones were tested for proliferation in response to ENO1 in the presence of irradiated autologous APCs. Results are expressed as mean values of mitogenic index obtained in triplicate microcultures. b TCR Vβ chain collection of ENO1-specific T cell clones. The clonality of T cell clones reactive to ENO1 was analyzed by a panel of 24 monoclonal antibodies specific for human TCR Vβ chain families. T cell blasts from each clone were divided into aliquots and stained with each monoclonal antibody and the appropriate isotype controls. c Functional profile distribution of CD4⁺ ENO1-specific T cell clones

Fig. 4

Cytotoxic activity of ENO1-specific T cell clones. a ENO1-induced cytotoxicity by TIL-derived ENO1-specific CD4⁺ and CD8⁺ T cell clones. To assess their perforin-mediated cytotoxicity, T cell clones were co-cultured with ⁵¹Cr-labeled autologous EBV-B cells pulsed with ENO1 for 8 h. Results represent mean values of percentage-specific ⁵¹Cr release at an effector-to-target ratio of 10-to-1 in triplicate microcultures. The dotted line represents 3 SD over the mean ⁵¹Cr release in cultures with ⁵¹Cr-labeled autologous unpulsed EBV-B cells. b Evaluation of the specific cytolytic activity of T cell clones obtained from HM compared with those obtained from CT. We compared the specific release of ⁵¹Cr of Th17 and Th1 isolated from healthy mucosa and tumor tissue

Fig. 5

Evaluation of characteristics of ENO1-specific Treg clones. a ENO1-specific Treg clones suppress the proliferation of ENO1-specific effector T cell clones. We used a suppression test with ENO1-loaded DCs and TT-loaded DCs as controls. b Dose–response curve of proliferation of ENO1-specific T cell clones. We compared the proliferation between blasts of Treg and effector T cell clones after different doses of ENO1. The results represent mean values (±SD) of three repeated experiments