Cytokine production and transcription factor expression in Tr17 cells
To determine the cytokine production with T cell subsets in CIA, we analyzed draining LN cells harvested from C57BL/6-Foxp3GFP mice at day 10 post-1st-CII-immunization. CD4+GFP+Treg cells and CD4+GFP- T cells were isolated and cultured with PMA/ionomycin for 8 h. IL-10 and IL-17 production from these cells were analyzed using flow cytometry, and its relationship to the expression of Foxp3 and RORγt was assessed. IL-10 produced was much higher in Tr17 cells compared to RORγt-negative Treg cells (Figure 4a), whereas IL-17 production was lower in Tr17 cells than RORγt-positive non-Treg cells (Figure 4a). To clarify why IL-10 was increased in Tr17 cells compared to RORγt-negative Treg cells, we checked the expression of Blimp1 and Foxp3. Blimp-1 is essential for the production of IL-10 by Foxp3+Treg cells20 and preserves Treg cell stability at sites of inflammation21. The frequency of Blimp-1 positive cells was increased in Tr17 cells compared to RORγt-negative Treg cells (Figure 4b). Moreover, expression level of Foxp3 was significantly higher in Tr17 cells compared to RORγt-negative Treg cells in spite of the findings that RORγt expression was significantly enhanced in Tr17 cells (Figure 4b). These results indicated that enhanced expression of Blimp-1 might induce increased IL-10 production and maintain expression of Foxp3 in Tr17 cells under inflammatory conditions.