Impact of Fut1 KO splenocytes and THBS1 on T cell proliferation and differentiation
Next, to investigate the effects of Fut1 KO splenocytes and their reduced levels of THBS1 on the proliferation and differentiation of CD4 T cells, we cocultured CD4+ cells isolated from WT mice with either Fut1 KO splenocytes or WT splenocytes (Fig. 2A ). The CFSE dilution assay revealed that the proliferation of CD4+ cells cocultured withFut1 KO splenocytes was significantly higher than those cocultured with WT splenocytes (Fig. 2B ). Remarkably, the addition of THBS1 to the coculture reversed the effects of Fut1KO splenocytes on CD4+ cell proliferation (Fig. 2A ).
Similar findings were observed with CD4 T cell differentiation. The coculture of CD4+ cells with Fut1 KO splenocytes significantly induced the differentiation of CD4+ cells into IFN-γ+CD4+ Th1 cells and IL-17+CD4+ Th17 cells, compared to the coculture with WT splenocytes (Fig. 2C ). The replenishment of THBS1 in CD4+ cell-Fut1 KO splenocyte cocultures effectively suppressed the differentiation of CD4+ cells into Th1 and Th17 subsets (Fig. 2C ). However, neither Fut1 KO splenocytes nor THBS1 affected the differentiation of CD4+ cells into Foxp3+ regulatory T cells (Tregs) (Fig. 2D ).
Furthermore, we examined the levels of inflammatory cytokines in the coculture supernatants. The secretion of IL-2, IFN-γ and IL-17 were significantly increased, while IL-10 was reduced, in CD4+ cells cocultured with Fut1 KO splenocytes compared to those cocultured with WT splenocytes (Fig. 2E ). THBS1 was also effective in reducing the levels of IL-2, IFN-γ and IL-17, as well as enhancing IL-10, in the coculture of CD4+ cells and Fut1 KO splenocytes (Fig. 2E ).
These results suggest that Fut1 KO splenocytes promote CD4 T cell proliferation and the differentiation towards Th1 and Th17 cells through reduced levels of THBS1 at least in part.