Fig. 4. Retinoic acid acts indirectly on Foxp3 expression.

A) Retinoic acid does not enhance TGFβ mediated Foxp3 expression in purified naïve CD4⁺ T cells. Naïve CD25⁻CD44^loCD62L^hi CD4⁺ T cells (0.5 × 10⁵) were activated with anti-CD3/CD28 beads and TGFβ (10 ng/ml) in the absence (left panel) or presence of RA (100 nM) (middle panel) for 4 days. Representative FACS data is shown for Foxp3 expression with the results of multiple experiments plotted in the right panel (n=6, and p-value for student’s t-test). B) Retinoic acid does enhance TGFβ mediated Foxp3 expression in CD4⁺ T cells from unfractionated splenocytes. Splenocytes (0.5 × 10⁵) were activated in vitro (as in A) in the absence (left panel) or presence of RA (100 nM) (middle panel) for 4 days. Representative FACS data is shown for Foxp3 expression in CD4⁺ T cells with the results of multiple experiments plotted in the right panel (n=6, and p value for student’s t-test). C) Retinoic acid does not enhance Foxp3 expression in purified naïve CD4⁺ T cells over a range of TGFβ concentrations. Naïve CD4⁺ T cells (purified as in A) were activated with anti-CD3/CD28 beads in the presence of 100nM RA and the indicated doses of TGFβ, then analyzed at day 4 for Foxp3 expression by FACS. Representative data is shown for 3 independent experiments. D) Foxp3 expression is not enhanced in naïve CD4⁺ T cells over a range of retinoic acid doses. Naïve CD4⁺ T cells were activated (as in A) in the presence of the indicated concentrations of RA, then analyzed for Foxp3 expression by FACS. Summarized data from 3 independent experiments is shown.