RA enhances T reg cell conversion independently of secreted cytokines and neutralizes the effect of cytokines on naive T cells. (A, left) Cytokines in supernatants of stimulated naive or CD44^hi cells. Naive CD4⁺ T cells or CD44^hi cells were activated with anti-CD3 and anti-CD28, and the supernatants were measured after 48 h by ELISA for IL-4, IFN-γ, and IL-21. In some cultures 0.6 ng/ml TGF-β was added. (right) Supernatants of CD44^hi but not naive T cells contain cytokines inhibiting T reg cell conversion. Conversion of naive T cells is enhanced by a direct effect of RA: naive CD4⁺ T cells were activated with anti-CD3 and anti-CD28 and 0.6 ng/ml TGF-β in medium or supernatant from CD44^hi or naive CD4⁺ T cells, and diluted with fresh media at a ratio of 1:1 in the presence (open histogram) or absence (shaded histogram) of 2.5 nM RA. Representative data (means ± SEM) are shown for two independent experiments. (B) RA neutralizes the adverse effect on conversion of various cytokines. 0.1 ng/ml of recombinant IL-4, IFN-γ, and IL-21 were added individually or in combination to cultures of naive CD4⁺ T cells activated with anti-CD3 and anti-CD28 and 0.3 ng/ml TGF-β in the presence (open histogram) or absence (shaded histogram) of 2.5 nM RA. Shown are representative FACS data from three individual experiments. (C) Antibodies to IL-4, IFN-γ, and IL-21 can neutralize the inhibitory effect of CD44^hi cell supernatant on T reg cell conversion. (D) RA enhances conversion of IL-4–deficient naive T cells. Naive CD4⁺ T cells were activated with anti-CD3 and anti-CD28 and TGF-β in the presence (open histogram) or absence (shaded histogram) of RA. One representative experiment out of three is shown.