(A) LNs from CD4-IL17A^ind/+ and IL-17A^ind/+ littermates were isolated and stained for CD4 and CD8 coreceptors. EGFP expression is depicted in the histograms after indicated gating. Percentages of gated cells are shown. (B) Splenocytes from naive CD4-IL17A^ind/+ mice were restimulated in the presence of Brefeldin A and subsequently stained for CD4 and IL-17A. Percentages of cells in the quadrants are indicated in the corners after gating was performed on all EGFP^– (plots on the left) or all EGFP⁺ (plots on the right) cells. Data shown are representative of 3 independent experiments. (C) FACS-sorted CD4⁺ T cells (2 × 10⁵) were cultured for 24 hours in the presence or absence of anti-CD3 and anti-CD28, after which IL-17A secretion was measured by flow cytomix assay. Error bars represent mean ± SD. n.d., not detectable. (D) Clinical scores after MOG_35–55-induced EAE are not significantly altered by increased IL-17A expression in CD4-IL17A^ind/+ and IL-17A^ind/+ littermates. Error bars represent mean ± SEM. Data shown represent 1 out of 3 independent experiments. (E) Lymphocytes isolated from the diseased EAE brain and spinal cord at day 14 from CD4-IL17A^ind/+ and IL-17A^ind/+ littermates were restimulated and surface stained for CD4 and examined for EGFP expression. Further staining for IL-17A and IFN-γ was performed. Percentages of EGFP⁺IL-17A⁺ or EGFP⁺IFN-γ⁺ are given in the quadrant corners. Plots shown are gated on CD4⁺ CNS-derived T cells. (F) At peak disease, mononuclear infiltrates were isolated from inflamed CNS extracts from either CD4-IL17A^ind/+ or IL-17A^ind/+ mice. Cellular extracts were cultured for 2 days in the presence of 20 μg/ml MOG peptide, after which cytokine secretions were measured by flow cytomix. Error bars represent mean ± SD and significance is shown where relevant. *P = 0.039, Student’s t test.