Supplementary Materials1: Supplementary Fig. cells from HNSCC TIL, they were treated with IL-2 (200 IU/ml) with or without IFN- (200ng/ml) for 48 hours. Viability factor (A), Foxp3 (A) and ki-67 expression (B) were tested by flow cytometry. NIHMS964603-supplement-2.pptx (62K) GUID:?B92DD055-99AC-41A5-A49C-84908AB6762B Abstract Purpose Regulatory T (Treg) cells are important suppressive cells among tumor infiltrating lymphocytes (TIL). Treg express the well-known immune checkpoint receptor PD-1, which is usually reported to mark exhausted Treg with lower suppressive function. T cell immunoglobulin mucin (Tim)-3, a negative regulator of Th1 immunity, is usually expressed by a sizeable fraction of TIL Tregs, but the functional status of Tim-3+ Tregs remains unclear. Experimental design Taxol enzyme inhibitor CD4+CTLA-4+CD25high Treg 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+ and Tim-3? TIL Tregs were tested by in vitro suppression assays and multi-color flow cytometry. Gene expression profiling and NanoString Taxol enzyme inhibitor analysis of Tim-3+ TIL Treg were performed. A murine HNSCC tumor model was used to test the effect of anti-PD-1 immunotherapy on Tim-3+ Treg. Results Despite high PD-1 expression, Tim-3+ TIL Treg displayed a greater capacity to inhibit na?ve T cell proliferation than Tim-3? Treg. Tim-3+ Treg from human HNSCC TIL also displayed an effector-like phenotype, with more strong expression of CTLA-4, PD-1, CD39 and IFN- receptor. Exogenous IFN- treatment could partially reverse the suppressive function of Tim-3+ 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. Conclusion Tim-3+ Treg are functionally and phenotypically distinct in HNSCC TIL, and are highly effective at inhibiting T cell proliferation despite high PD-1 expression. IFN- induced by anti-PD-1 immunotherapy may be beneficial by reversing Tim-3+ Treg suppression. of Tim-3 expression on TIL Treg after the asminitration of anti-PD-1, a result that was reversersed with the co-administration of anti-IFN- (Physique 5C). Furthermore, we observed a significant decrease in Nrp-1 when mice were treated with anti-PD-1 alone, suggesting that anti-PD-1 monotherapy increases the fragiligt of TIL Treg. This effect was also partially reversed by the co-administration of Col18a1 IFN- Taxol enzyme inhibitor capture antibody. While it remains clear that Treg fragility is required for response to PD-1 blockade and it has been reported that IFN- drives Treg fragility to promote anti-tumor immunity through regulation of the expression of Nrp-1 (19), it remains unclear the source of IFN- that regulates expression. We have presented in vitro evidence that the source of IFN- may come from CD8+ T cells following anti-PD-1 monotherapy (36), although a deeper analysis into this complex issue is usually ongoing. It is possible that Nrp-1 marks Treg that can be destabilized, whereas Tim-3 expression is usually unassociated with this phenotype. Ultimately, genetically designed mice with selective deletion of Nrp-1, Tim-3 or IFN-, currently being generated, will be useful to definitively characterize the differential functions of Tim-3 vs Nrp-1 in TIL Treg. Tim-3 was first identified as a cell surface molecule selectively expressed on IFN–producing Th1 and Tc1 cells (7). Here, Tim-3 was shown to play an important role in the induction of autoimmune diseases by regulating macrophage activation and function and Tim-3 blockade enhanced the clinical and pathological severity of Th1-dependent autoimmune disease and increases the number of activated macrophages in mice. Furthermore, transgenic overexpression of Tim-3 on T cells resulted in an increased in granulocytic MDSC and inhibition of immune responses (37). In accordance with prior studies, we could only detect appreciable Tim-3 expression in HNSCC TIL Treg, with little expression on circulating Treg (14, 17). This localized expression within tumors makes it an attractive therapeutic target, directly or indirectly. Similarly, CD4+CD25hiFoxp3+ Tregs express more Tim-3 than CD4+CD25?Foxp3? T cells in HNSCC TIL. Taken together, Tim-3 is usually highly expressed in TIL Tregs, which appear to play an important role in antitumor immune responses. Interestingly, the suppressive effects of Tim-3+ TIL Treg appeared to be reversible during anti-PD-1 based immunotherapy in a murine model, and the suppressive function of HNSCC TIL Treg could also be reversed by anti-PD-1 Ab. Increased suppression by Tim-3+ Treg cells compared to Tim-3? Treg cells indicates that Tim-3+ Treg cells from HNSCC patients are more potent in the microenvironment. Previous studies reported that PD-1+Tim-3+ CD8+ T cells are dysfunctional in melanoma patients (38). PD-1hi TIL Treg are dysfunctional also, losing their suppressive function in malignant gliomas as compared to PD-1low TIL Treg (29). We observed that Tim-3+ TIL Treg express greater PD-1 than Tim-3? Treg. Indeed, PD-1+ Treg cells, Tim-3+ Treg have.