Figure 2.

CD4 T cell proliferation was reduced by MIS416 administration in vitro or in vivo but only when APC were present. (a) Splenocytes (1 × 10⁶/ml) were isolated from EAE mice that had been immunized 21 days previously, labelled with CSFE, and stimulated in vitro with MOG (27 μg/ml), MIS416 (20 μg/ml), or both for 72 hours. The dotted line represents proliferation in wells with medium alone. Shown are the means and SEM of proliferative indexes from individual mice (n = 4) from one experiment. ***p < 0.001 compared to MOG by one-way ANOVA with Tukey’s multiple comparison test. (b,c). Splenocytes (1 × 10⁶/ml) were isolated from healthy mice treated with MIS416 15 days previously, labelled with CSFE, and stimulated in vitro with Con A (3 μg/ml), MIS416 (20 μg/ml), or both for 48 hours. The % of CD4⁺ cells that proliferated (b) and were IFN-γ⁺ (c) was quantified by flow cytometry. Shown are the means and SEM of individual mice (n = 8/group; 4 for Con A + MIS) from 2 independent experiments. ***p < 0.001 and **p < 0.01 by two-way ANOVA with Sidak’s multiple comparison test. (d,e) CFSE-labelled 2D2 cells were transferred to congenic mice (CD45.1) one day before MIS416 treatment and EAE immunization. After 5 days, proliferation of CD45.2 2D2 CD4⁺ T cells in the draining lymph node and spleen was determined by flow cytometry (gating strategy shown in Suppl Fig. 2). Shown are the means and SEM of values from individual mice (n = 9–10/group) from two independent experiments. ****p < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test. (f,g). Purified CD4 T cells were isolated from healthy, untreated and MIS416-treated mice at 24 hour post dose 3 (day 15), labelled with CFSE, and stimulated in vitro with anti-CD3/CD28 expander beads at increasing cell:bead ratios for 48 hours. The proliferation (f) and division (g) indexes were measured by flow cytometry. Shown are the means and SEM of duplicate wells from one experiment. ***p < 0.001 and **p < 0.01 by two-way ANOVA with Sidak’s multiple comparison test.