Novel effector phenotype of TIM-3^þ Regulatory T cells leads to enhanced suppressive function in head and neck cancer patients

Purpose: Regulatory T (Treg) cells are important suppressive cells among tumor-infiltrating lymphocytes (TIL). Treg cells express the well-known immune checkpoint receptor PD-1, which is reported to mark "exhausted" Treg with lower suppressive function. T-cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3^þ Tregs remains unclear. Experimental Design: CD4^þCTLA-4^þCD25^high Treg cells were sorted from freshly excised head and neck squamous cell carcinoma (HNSCC) TIL based on Tim-3 expression. Functional and phenotypic features of these Tim-3^þ and Tim-3 TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene-expression profiling and NanoString analysis of Tim-3^þ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3^þ Treg. Results: Despite high PD-1 expression, Tim-3^þ TIL Treg displayed a greater capacity to inhibit na€ve T-cell proliferation than Tim-3 Treg. Tim-3^þ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more robust expression of CTLA-4, PD-1, CD39, and IFN-g receptor. Exogenous IFN-g treatment could partially reverse the suppressive function of Tim-3^þ TIL Treg. Anti-PD-1 immunotherapy downregulated Tim-3 expression on Tregs isolated from murine HNSCC tumors, and this treatment reversed the suppressive function of HNSCC TIL Tregs. Conclusions: Tim-3^þ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T-cell proliferation despite high PD-1 expression. IFN-g induced by anti–PD-1 immunotherapy may be beneficial by reversing Tim-3^þ Treg suppression.