Suppression activity of Tr17 cells
To provide an answer to the question of whether Tr17 cells can suppress effector CD4+ T cells, we tried to compare the ability of RORγt-positive and -negative Treg cells to inhibit the proliferation of effecter CD4+ T cells in vitro. Because it is difficult to isolate RORγt-positive cells by their expression, we tried to enrich them using CCR6 expression. FACS analysis revealed that about 30% of CCR6+ Foxp3-positive Treg cells expressed RORγt (Supplementary Figure 3) and, thus, we used CCR6+ Treg cells as Tr17-enriched Treg cells (Figure 5a). To check the equivalence between Tr17 cells and CCR6+Treg cells, we examined the expression of the cell surface molecules typically highly expressed in Tr17 cells (as mentioned above; Figure 2). FACS analysis revealed nearly identical expression levels of CD25, CTLA-4, and GITR between Tr17 cells and CCR6+Treg cells (Supplementary Figure 4) and, thus, it was thought to be reasonable to use CCR6+Treg cell for evaluation of suppressive function as enriched-Tr17 cells.
CD4+GFP+ Treg cells and CD4+GFP-conventional T cells were isolated from LN in C57BL/6-Foxp3GFP reporter mice 10 days after 1st CII-immunization, and cocultured with stimulation of T cell receptor in vitro. CCR6+Tr17-enriched Treg cells suppressed the proliferation of conventional T cells (Figure 5b, c). Interestingly, compared to CCR6-Treg cells, the suppressive capacity of CCR6+Tr17-enriched Treg cells was significantly enhanced (Figure 5b, c). These results showed the possibility that Tr17 cells have enhanced suppressive capability compared with RORγt-negative Treg cells.