In adoptive transfer EAE, antigen-specific Th17, but not Th1, cells enter into CNS independently of α4 blockade. (A) Naive CD4⁺CD44⁻CD25⁻ T cells from 2D2 MOG_35-55-specific TCR transgenic mice were polarized under Th1 or Th17 conditions in vitro. On day 3, cytokine status was checked by intracellular cytokine staining and 2 × 10⁶ cytokine-positive cells were injected i.v. into Rag1^−/− mice. Before injection, polarized cells were incubated with control Ig or blocking antibodies to α4 integrin (PS/2). According to the pretreatment of the transferred cells, host mice were administered rIgG or anti-α4 antibody every 3 d until the development of disease. Means of clinical scores +SEM, n = 4. Note that host mice that received Th17 cells under conditions of α4 blockade developed signs of atypical EAE with ataxia and hemiparesis. (B) At the peak of disease, mononuclear cells were isolated from the CNS of individual mice, stained for CD3 and CD4, and analyzed by flow cytometry. Numbers in the histograms represent percentages of CD4⁺ T cells among CNS-infiltrating mononuclear cells. Shown are representatives of six independent experiments. (C) MOG_35-55-specific TCR transgenic 2D2 mice were crossed to Ifng^−/− mice. Naive T cells were differentiated under Th17 conditions and transferred into Rag1^−/− mice, which were then treated with rIgG or blocking antibodies to α4 integrin. CNS-infiltrating mononuclear cells were isolated at the peak of disease and stained for surface and intracellular antigens as indicated. Shown are representatives of three independent experiments.