Figure 3.

Splenic Treg number and function was enhanced by in vivo MIS416 administration but did not appear to correlate with IFN-γ-dependent protection from EAE. (a) Treg were isolated from healthy mice and MIS416-treated mice one day post-dose 3 and cultured with CFSE-labelled, anti-CD3/CD28 stimulated CD4+ T cells at increasing Treg ratios. After 72 hours, the CD4 T cell proliferative index was assessed by flow cytometry. Shown are the means and SEM of Treg preparations from individual mice (n = 5–6/group) from 2 independent experiments. ***p < 0.001 and *p < 0.05 by two-way ANOVA with Sidak’s multiple comparison test. (b) MIS416 increased the total number of Treg in healthy WT and IFN-γ^−/− mice 15 days post treatment initiation. Treg were identified as CD25⁺FoxP3⁺CD4⁺ cells by flow cytometry. Shown are the means and SEM of individual mice (n = 7/WT group and n = 12/IFN-γ^−/− group) from 3 experiments. *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (c) Splenocytes were isolated from WT and IFN-γ^−/− mice one day post the third dose of MIS416 (day15), labelled with CSFE, and cultured in vitro (1 × 10⁶/ml) with Con A (3 μg/ml) for 48 hours. The % of CD4⁺ cells that proliferated was quantified by flow cytometry. Shown are the means and SEM of individual mice (n = 12/group) from 3 independent experiments. ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test.