Fig. 3. Retinoic acid down-regulates the IL-6Rα but enhancement of Foxp3 expression by RA occurs in the absence of IL-6.

A) Expression of IL-6Rα is down-regulated in CD4⁺ T cells by retinoic acid. Splenocytes (1×10⁵) were activated with anti-CD3/CD28 beads in the absence (TGF, open histogram) or presence of RA (TGF +RA, dark grey histogram). Representative FACS plots are shown (n=6 independent experiments). B) CD4⁺ T cells from retinoic acid receptor alpha deficient (RARα KO) mice do not down regulate the IL-6R after culture with RA. CD4+ T cells (0.5 × 10⁵) from wild-type (WT) or RARαKO mice were activated with anti-CD3/CD28 beads and TGFβ (10 ng/ml) in the absence (open histogram) or presence (filled histogram) of retinoic acid (100nM). Representative FACS plots are shown (n=3 independent experiments). C) Retinoic acid prevents the inhibition of Foxp3 expression mediated by IL-6 in CD4⁺ T cells activated with TGFβ. CD4⁺ T cells (0.5 × 10⁵) were activated with anti-CD3/CD28 beads and various concentrations of RA. After 24hrs TGFβ (10 ng/ml) and IL-6 were added and cells were cultured for an additional 4 days. Representative FACS data is shown for Foxp3 expression in CD4⁺ T cells (n=3 independent experiments). D) Retinoic acid enhances Foxp3 expression in CD4⁺ T cells in the absence of IL-6. Splenocytes from wild-type or IL-6KO mice were stimulated (as in A) and analyzed 4 days later for Foxp3 expression in CD4⁺ T cells. Representative FACS plots are shown (n=3 independent experiments).