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. Author manuscript; available in PMC: 2019 Jul 15.
Published in final edited form as: J Immunol. 2018 May 23;201(2):440–450. doi: 10.4049/jimmunol.1701654

Figure 5. PRMT1 overcomes IL-2-mediated inhibition of Th17 differentiation by controlling reciprocal STAT3 and STAT5 recruitments.

Figure 5

A) Flow cytometric analysis of the percentage of IL-17+ cells among naïve CD4+ T cells differentiated under Th17 priming conditions in the presence of increasing concentrations of exogenous IL-2 (0, 10 or 100 IU/ml). Right panel is the quantification results of the left panels. B) Flow cytometric analysis of the percentage of IL-17+ cells among naïve CD4+ T cells differentiated under Th17 priming conditions in the presence of IL-2 (100 IU/ml of exogenous rhIL-2), rested for 24hrs and re-stimulated with increasing concentrations of IL-6 (0, 2.5, 25 ng/ml). Right panel is the quantification results of the left panels. C and D) Flow cytometric analysis of the percentage of IL-17+ cells among naïve CD4+ T cells transduced with retrovirus expressing GFP and WT PRMT1 (C) or inactive PRMT1-E153Q (D), and differentiated under Th17 priming conditions in the presence of IL-2 (100 IU/ml), rested for 24 hrs and re-stimulated with increasing concentrations of IL-6 (0, 2.5, 25 ng/ml). Right panels are the quantification results of the left panels. E) Flow cytometric analysis of the percentage of IL-17+ cells among naïve CD4+ T cells differentiated under Th17 priming conditions in the presence of IL-2 (100 IU/ml), increasing concentrations of IL-6 (0, 2.5, 25 ng/ml) and PRMT1 inhibitor (3μM). Right panel is the quantification results of the left panel. F-J) ChIP analysis of the enrichment of H4R3me2a, STAT3 and STAT5 at four different sites at IL-17 gene locus in CD4+ T cells differentiated under Th17 priming conditions (Th17) (F), Th17 conditions + IL-2 (100 IU/ml) (G), Th17 conditions + IL-2 (100 IU/ml) + IL-6 (25ng/ml) + retroviral expression of WT PRMT1 (H), or inactive PRMT1-E153Q (I), or Th17 conditions + IL-2 (100 IU/ml) + IL-6 (25ng/ml) + PRMT1 inhibitor (3μM) (J). IgG is a negative control. K) Immunoblot analysis of indicated proteins from immune-complexes eluted from chromatin precipitates with anti-RORγt antibody from CD4+ T cells treated as described in F-J. L) Immunoblot analysis of phosphorylated or whole STAT3 and STAT5 in naïve CD4+ T cells differentiated under indicated conditions as described in F-J. K and L are the representative results of three independent experiments. *P<0.05, (A-E, One-way ANOVA, with Tukey's post-analysis multiple comparison), NS: non-significant.