Fig 1. PGE2 inhibits Tr1 cell differentiation in splenocyte cultures.

(A) il10^gfp splenocytes were stimulated with 3 μg/ml anti-CD3 and 1 μg LPS in the presence of neutralizing IL-27 antibodies (5 and 10 μg/ml) or IgG control (20 μg/ml). Cells were collected on day 3 and CD4⁺CD49b⁺LAG-3⁺ Tr1 cells were identified by FACS. Data are cumulative from two independent experiments. (B) Splenocytes were stimulated with anti-CD3 and LPS in the presence of PGE2 (10^-6M). Supernatant was collected on day 3 and IL-27 levels analyzed by ELISA. (C-D) Splenocytes were stimulated with anti-CD3 and LPS in the presence or absence of IL-27 (50 ng/ml) and PGE2 (10^-6M). Cells were collected on day 3 and CD4⁺CD49b⁺LAG-3⁺ Tr1 cells were identified by FACS. (C) Representative sample shows gating strategy to determine percentage of CD49b⁺LAG-3⁺ within CD4⁺ T cells and (D) graph presents representative data from three independent experiments. Each sample was tested in duplicate and results represent means ± SD. Significance was evaluated by one-way ANOVA; **P<0.01.