Figure 6
PGE2 enhances Tr differentiation and concomitantly inhibits Th2 differentiation. (a and b) MLN-DCs (2 × 104) preincubated with 0.2 μg ml−1 OVA peptide with or without retinoic acid and/or TGF-β for 6 h. After washing, the cells were incubated with 5 × 105 CFSE-labeled CD4+KJ1.26+ T cells in the presence of TGF-β with or without retinoic acid for 96 h. For analysis of PGE2 effects, CD4+KJ1.26+ T cells were stimulated with 16,16-dimethyl prostaglandin E2 (26 μm) 2 h before the start of the culture as well as during the 96 h culture. At 96 h, CD4+KJ1.26+ cells were analyzed for Foxp3 by flow cytometry (n=3). (b and c) CD4+ T cells were isolated from the culture by eliminating DC with mAbs and anti-rat magnetic beads and analyzed for Foxp3− (b) and IL-4− (c) mRNA expression by PCR. (d) A total of 5 × 105 CFSE-labeled CD4+KJ1.26+ T cells were stimulated with anti-CD3 (10 μg ml−1) and anti-CD28 (10 μg ml−1) in the absence of TGF-β and presence of 16,16-dimethyl prostaglandin E2 (26 μM) for 72 h. Release of IL-4 in cell culture supernatants was determined by cytometric bead array (CBA). *Statistically significant (P<0.05). CFSE, 5,6-carboxy-succinimidyl-fluoresceine ester; DCs, dendritic cells; IL, interleukin; mAb, monoclonal antibody; MLN, mesenteric lymph node; PGE2, prostaglandin E2; RA, retinoic acid; TGF-β, transforming growth factor-β.
