Figure S2.

Eomes expression is induced in a TCR/CD28-dependent manner proportionally to signal strength and duration and Eomes⁺ CD4⁺ T cells accumulate in the CNS over the course of inflammation. (A) GFP expression in CTV-labeled Eomes-GFP CD4⁺ T cells stimulated for 48 h with 1 µg/ml of anti-CD28 and increasing doses of anti-CD3 mAb. (B) Same as A but with 0.5 µg/ml of anti-CD3 and increasing doses of anti-CD28 mAb (n = 5 mice). (C) GFP expression in Eomes-GFP CD4⁺ T cells stimulated for 24, 48, and 72 h with 2 µg/ml of anti-CD3 and 1 µg/ml of anti-CD28 mAbs (n = 3 mice). (D) GFP expression compared to activation marker expression against number of cell divisions in CD4⁺ T cells was assessed in Eomes-GFP CD4⁺ T cells stimulated in vitro for 48 h (n = 5 mice). (E) Representative gating strategy showing GFP expression in CD45.1⁺ and CD45.2⁺ compartment in the brain of WT C57BL/6J recipients transferred with 2D2-Eomes-TWT cells. (F) Ki67 and activation marker expressions were analyzed in GFP⁻ and GFP⁺ CD45.1⁺ or CD45.2⁺ CD44^high CD4⁺ T cell compartments in the brain at 14 dpi (n = 6 mice). (G) CD107a expression was assessed in Eomes-GFP⁻ and Eomes-GFP⁺ CD44^high CD4⁺ T cells from the brain at 14 dpi upon active immunization of Eomes-GFP mice and restimulation ex vivo overnight with 10 µg/ml of MOG_35–55 peptide (n = 6 mice). (H) Percentage of injected cells in NI mice in blood, spleen, dLN, and cLN 8 dpi (n = 4 mice). (I and J) Percentage and absolute number of 2D2 injected cells were analyzed at 5, 8, and 14 dpi (n = 5, 6, and 6 mice, respectively) in (I) cLN and (J) blood. (K and L) Proliferation rates were assessed at (K) 5 dpi or (L) 8 and 14 dpi in spleen (SPL), dLN, brain, cLN, and blood of immunized mice. Data are representative of two independent experiments. Error bars = SEM; P values in G (Mann–Whitney U test) and other P values (two-way ANOVA with Bonferroni correction)—****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.