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Immunosuppressive microenvironments induced by regulatory T cells (Treg) present a major barrier for successful anti-tumor immunotherapy. Understanding the mechanisms for the accumulation of different subtypes of Treg cells in the immunosuppressive tumor microenvironment is essential to improving cancer treatment. Enriched gammadelta1 T cell populations in tumor-infiltrating lymphocytes (TILs) suppress T cell responses and dendritic cell maturation in breast cancer, where their presence is correlated negatively with clinical outcomes. However, mechanism(s) that explain the increase in this class of gammadelta Treg cells in breast cancer patients have yet to be elucidated. In this study, we showed that IP-10 secreted by breast cancer cells attracted gammadelta Treg cells. Using neutralizing antibodies against chemokines secreted by breast cancer cells, we found that IP-10 was the only functional chemokine that causes gammadelta Treg cells to migrate toward breast cancer cells. In a humanized NSG mouse model, human breast cancer cells attracted gammadelta Treg cells as revealed by a live cell imaging system. IP-10 neutralization in vivo inhibited migration and trafficking of gammadelta Treg cells into breast tumor sites, enhancing tumor immunity mediated by tumor-specific T cells. Together, our studies show how gammadelta Treg cells accumulate in breast tumors, providing a rationale for their immunological targeting to relieve immunosuppression in the tumor microenvironment.

Immunotherapy is a promising approach for treating patients with malignant tumor, but immunosuppressive microenvironments induced by regulatory T cells (Tregs) present a major barrier to successful anti-tumor immunotherapy. A better understanding of the suppressive mechanisms utilized by Tregs is essential for the development of novel strategies to treat human cancer. Here we report that human Tregs can induce senescence in responder naïve and effector T cells in vitro and in vivo. Senescent responder T cells induced by human Tregs changed their phenotypes and cytokine profiles, and possessed potent suppressive function. Furthermore, Treg-mediated molecular control of senescence in responder T cells was associated with selective modulation of p38 and ERK1/2 signaling and cell cycle regulatory molecules p16, p21 and p53. We further revealed that human Treg-induced senescence and suppressor function could be blocked by TLR8 signaling and/or by specific ERK1/2 and p38 inhibition in vitro and in vivo in animal models. Our studies identify a novel mechanism of human Treg cell suppression that induces targeted responder T cell senescence, and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression for tumor immunotherapy.

Accumulating evidence suggests that T-helper 17 (Th17) cells and regulatory T (Treg) cells may exhibit development plasticity, and that CD4+ Tregs can differentiate into IL-17-producing T cells; however, whether Th17 cells can reciprocally convert into Tregs has not been described. In this study, we generated Th17 clones from tumor-infiltrating T lymphocytes (TILs). We showed that Th17 clones generated from TILs can differentiate into IFN-gamma-producing and FOXP3+ cells after in vitro stimulation with OKT3 and allogeneic peripheral blood mononuclear cells (PBMCs). We further demonstrated that T-cell receptor (TCR) engagement was responsible for this conversion, and that this differentiation was due to the epigenetic modification and reprogramming of gene expression profiles, including lineage-specific transcriptional factor and cytokine genes. In addition to expressing IFN-gamma and FOXP3, we showed that these differentiated Th17 clones mediated potent suppressive function after repetitive stimulation with OKT3, suggesting that these Th17 clones had differentiated into functional Tregs. We further demonstrated that the Th17-derived Tregs, unlike naturally occurring CD4+CD25+ Tregs, did not reconvert back into Th17 cells even under Th17-biasing cytokine conditions. These results provide the critical evidence that human tumor-infiltrating Th17 cells can differentiate into Tregs and indicate a substantial developmental plasticity of Th17 cells.

Although Th17 cells play critical roles in the pathogenesis of many inflammatory and autoimmune diseases, their prevalence among tumor-infiltrating lymphocytes (TILs) and function in human tumor immunity remains largely unknown. We have recently demonstrated high percentages of Th17 cells in TILs from ovarian cancer patients, but the mechanisms of accumulation of these Th17 cells in the tumor microenvironment are still unclear. In this study, we further showed elevated Th17 cell populations in the TILs obtained from melanoma, breast and colon cancers, suggesting that development of tumor-infiltrating CD4+ Th17 cells may be a general feature in cancer patients. We then demonstrated that tumor microenvironmental RANTES and MCP-1 secreted by tumor cells and tumor-derived fibroblasts mediate the recruitment of Th17 cells. In addition, we found that tumor cells and tumor-derived fibroblasts produce a proinflammatory cytokine milieu as well as provide cell-to-cell contact engagement that facilitates the generation and expansion of Th17 cells. We also showed that inflammatory TLR and NOD2 signaling promote the attraction and generation of Th17 cells induced by tumor cells and tumor-derived fibroblasts. These results identify Th17 cells as an important component of human TILs, demonstrate mechanisms involved in the recruitment and regulation of Th17 cells in tumor microenvironments, and provide new insights relevant for the development of novel cancer immunotherapeutic approaches.