Naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 for 4 days under indicated conditions in the absence or presence of RA or Vehicle (DMSO). Aliquots of cells cultured with TGF-β (A) or different combinations of cytokines (B) were fixed, permeabilized, and stained intracellularly with PEFoxp3 mAb and analyzed by flow cytometry. Cells were also reactivated with PMA and ionomycin for 4 h in the presence of GolgiStop, and then stained with APC-IFN-g and PE-IL-17 mAb and analyzed by flow cytometry. Numbers in lower right quadrants indicate the percentage of IL-17-producing cells (B). Data are representative of 3-5 experiments.

Naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 for 24 h under indicated conditions with or without RA. (A) Total RNA was isolated and expression of Smad2 and Smad3 mRNA was determined by Taqman quantitative PCR. (B) Cell lysates from indicated conditions were separated by SDS-PAGE gel, and total Smad and p-Smad were detected by immunoblotting. For quantification, the ratio of total Smad/β-actin and the ratio of p-Smad/total Smad were determined. Data are representative of three experiments. *, P <; 0.05 when RA treatment was compared to vehicle treatment.

Naïve CD4⁺ T cells were activated under indicated conditions with or without RA. After 48 h, cells were collected for the determination of gp130 and IL-6Rα mRNA by Taqman quantitative PCR (A) and surface expression of IL-6Ra protein by flow cytometry (B). Data are representative of three experiments. *, P < 0.05 when RA treatment compared to vehicle treatment.

Naïve CD4⁺ T cells were activated under indicated conditions with or without RA. Cells were collected for the determination of STAT3 phosphorylation by immunoblotting at various time points, and the ratio of p-STAT3/total STAT3 was analyzed (A). IL-21 mRNA expression at 24 h by Taqman quantitative PCR and IL-21 production in 96-h culture supernatants by ELISA (B). Expression of IL-23R mRNA at 24 h and 48 h by Taqman quantitative PCR (C). Data are representative of 3-5 experiments. *, P < 0.05 when RA treatment was compared to vehicle treatment.

Naïve CD4⁺ T cells were activated under indicated conditions with or without RA. Cells were collected after 24 h or 48 h, lyzed, and detected for IRF4 protein expression by immunoblotting. Data are representative of three experiments.

C57BL/6 mice were immunized with MOG_35-55/CFA, and RA was intraperitoneally injected every other day from day 0 to day 8. On day 10, (A) draining lymph node cells were isolated and restimulated with MOG_35-55. Proliferation and cytokine production in 48-h culture supernatants were measured by ³[H]-thymidine incorporation and ELISA assays, respectively; (B) isolated spleen cells were cultured with 5 μg/ml of MOG_35-55 for 5 days. Cytokine production from CD4⁺ cells was determined by intracellular cytokine staining following 4-h activation with PMA and ionomycin; (C) expression of IL-23R and IL-21 mRNA was measured in CD4⁺ T cells purified from spleen cells by Taqman quantitative PCR. *, P < 0.05 when RA treatment was compared to vehicle treatment. Data are the average or representative of 4-6 mice in each group.

Foxp3gfp.KI mice were immunized with MOG_35-55/CFA, and RA was intraperitoneally injected every other day from day 0 to day 8. On day 10, (A) the frequency of GFP⁺ Treg cells was determined in different lymphoid compartments by flow cytometry. “Spl”, spleen; “cer/ax”, cervical/axillary; “in”, inguinal; “m”, mesenteric; “pp”, peyer's patch; (B) spleen cells were restimulated with 2 μg/ml of MOG_35-55 for 5 days, and then stained with PE-conjugated IA^b tetramer, APC-anti-CD4, and 7-AAD. The MOG_35-55/IA^b tetramer-positive cells were determined in the live CD4⁺ cell population (CD4⁺7-AAD^-). TMEV_70-86/IA^S tetramers were used as a negative control. (C) Inflammatory cytokines IL-6, TNF-α, and IL-1β inhibited RA-enhanced Foxp3 expression but could not rescue RA-inhibited IL-17 production. Naïve CD4⁺ T cells (CD4⁺ CD62L^hi Foxp3/GFP^-) purified from Foxp3gfp.KI mice were activated with anti-CD3 and anti-CD28 under indicated conditions in the absence or presence of RA for 5 days. Cells were reactivated with PMA and ionomycin for 4 h in the presence of GolgiStop. Then, cells were stained with APC-anti-CD4, PE-anti-IL-17 and 7-AAD, and analyzed by flow cytometry for IL-17 production and Foxp3/GFP expression in gated CD4⁺7-AAD^- cells. (D) Isolated spleen cells from immunized mice on day 10 were cultured with 5 μmg/ml of MOG_35-55 peptide or 500ng/ml LPS. IL-6 production was determined in 48-h cultures by ELISA. None, no treatment.

C57BL/6 mice were immunized with MOG_35-55/CFA, and RA was intraperitoneally injected every other day starting from day 0 (RA-induction) or after all mice in the group showed signs of clinical EAE (RA-effector). Mice were evaluated daily for the signs of EAE. EAE was significantly (P < 0.05) reduced in mice in the “RA-induction” group after day 10 and in the “RA-effector” group after day12 compared to the vehicle treated mice.