Eomes expression delineates a population of highly activated CD4⁺ T cells that accumulate in the CNS during neuroinflammation. (A) Representative dot plots of CD4 versus GFP expression of naïve 2D2-Eomes-GFP cells unstimulated (left) or stimulated for 72 h in vitro with APCs loaded with 10 μg/ml of MOG_35–55 peptide (right). (B) Percentages of GFP-expressing cells of naïve 2D2-Eomes-GFP cells stimulated in vitro with APCs loaded with MOG_35–55 (1 or 10 μg/ml), NFM (1 μg/ml), or OVA (10 μg/ml) and was measured after 48 and 72 h of stimulation (n = 4 mice). (C) Representative dot plots of CTV staining according to GFP expression in 2D2 CD4⁺ T cells stimulated in vitro for 72 h with MOG_35–55- or NFM-loaded APCs. (D) GFP expression compared with activation marker expression against the number of cell divisions in 2D2 CD4⁺ T cells (n = 4 mice). (E) Naïve 2D2-Eomes-GFP CD45.1⁺ cells were injected into Eomes-GFP CD45.2 recipient mice prior to immunization with MOG_35–55 peptide in CFA. (F) Representative dot plots of CD4 versus GFP (left) and percentages of GFP-expressing cells (right) gated on CD44^high CD45.1⁺ and CD44^hi CD45.2⁺ CD4⁺ T cells, in dLN and spleen (SPL) of immunized mice at 8 dpi (n = 5 mice). (G and H) Expression of (G) Ki67, activation markers, and (H) migratory markers in CD44^high CD45.1⁺ and CD44^hi CD45.2⁺ CD4⁺ T cells in the spleen at 8 dpi (n = 5 mice). (I) Percentages of GFP⁺ cells were analyzed in CD44^hi CD45.1⁺ and CD44^hi CD45.2⁺ CD4⁺ T cells at 14 dpi in dLN, spleen, brain, and spinal cord (n = 6 mice per group, and for SC, cells were pooled to have three independent samples). (J) Percentage of GFP⁺ cells was analyzed in CD44^high CD4⁺ T cells in a model of active EAE at 14 dpi in dLN, spleen, brain, and SC (n = 8 mice). (K) Intracellular expression of IFN-γ, IL-10, IL-17, and IFN-γ/IL-17 was assessed in Eomes-GFP⁻ and Eomes-GFP⁺ cells from the brain after an overnight restimulation with 10 µg/ml of MOG_35–55 peptide (n = 6 mice). Data are representative of at least two independent experiments. Error bars = SEM; P values for B and F–J (two-way ANOVA with Bonferroni correction), P values in K (Mann–Whitney U test)—****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. See also Fig. S2.