04.15 Cancer-Associated Plastic IL17⁺Foxp3⁺ and ex-Th17 Foxp3⁺ Cells are Immunosuppressive

S. E. Berkey¹, D. L. Bartlett¹, N. Obermajer^{1  1}University Of Pittsburgh,Department Of Surgery,Pittsburgh, PA, USA

Introduction: Regulatory T (T_reg) cells are integral in maintaining immune homeostasis and are associated with the inflammatory immunosuppression observed in cancer. Expansion of T_reg cells within the tumor is a known barrier to successful cancer immunotherapy. In previous experiments, we have demonstrated that Th17 cells are a novel source of tumor-induced forkhead box P3 (Foxp3+) cells in addition to the natural (n)Treg and induced (i)Treg cells, which develop from naïve precursors. During tumor growth, Th17 cells progressively transdifferentiate into IL17⁺Foxp3⁺ and ex-Th17 IL17^negFoxp3⁺ T cells. Here, we aimed to characterize the immunosuppressive ability of the ex-Th17 IL17^negFoxp3⁺ T cells.

Methods: Using novel Th17^eYFP-Foxp3^mRFP reporter mice, CD4⁺ cells were isolated from splenocytes. The cells were cultured under Th17-driving conditions (IL6, IL23, and TGF-β) for 3-4 days, followed by T_reg– driving conditions (TGF-β) for an additional 3 days, and then sorted into Th17-T_reg subsets (eYFP⁺IL17⁺Foxp3^neg, eYFP⁺IL17⁺Foxp3⁺, eYFP⁺IL17^negFoxp3^neg, eYFP⁺IL17^negFoxp3⁺, eYFP^negIL17^negFoxp3^neg and eYFP^negIL17^negFoxp3⁺ (inducible (i)T^reg cells)). CD4⁺ cells were then isolated from CD45.1 mice and stained with the proliferation dye, carboxyfluorescein succinimidyl ester (CFSE). These CD4⁺ cells were activated with anti-CD3e Ab in the presence of CD4^neg fraction and Th17-T_reg subsets (2:1 ratio of CD4⁺ target cells: Th17-T_reg subset). The cells were incubated for 72 hours and then analyzed by flow cytometry. The percentage of proliferating cells (CFSE^neg, gated on live/CD45.1⁺CD4⁺ cells) was calculated as a fraction of proliferating cells compared to control condition where CD4⁺ cells were activated with anti-CD3e Ab in the absence of Th17-T_reg subsets.

Results: We demonstrate that IL17⁺Foxp3⁺ and ex-Th17 IL17^negFoxp3⁺ T cells have greater suppressive effects on CD4⁺ T cell proliferation compared to Th17 cells and have similar suppressive effects as (i)Treg cells.

Conclusions: The conversion of Th17 into Foxp3⁺ T cells is an alternative route of IL17⁺Foxp3⁺ and IL17^negFoxp3⁺ T cell development in tumors. Due to their immunosuppressive properties, transdifferentiated T_reg cells represent a novel target and warrant new therapeutic approaches aimed at eliminating Foxp3⁺ T cells in tumor immunotherapy.