Figure 2. CD4+ T cells from Dock8pri/pri mice ameliorate EAE with reduced CNS infiltration and migration. (A and B) The encephalitogenic CD4+ T cells were purified from Dock8pri/+ or Dock8pri/pri mice to check surface expression of CD44 and CD25 by FACS (A). IL-2 concentrations were checked by ELISA in the supernatants of Dock8pri/pri or Dock8pri/+ encephalitogenic CD4+ T cells stimulation with MOG for 3 d (B). n = 6. (C–E) Adoptive transfer of encephalitogenic Dock8pri/+ and Dock8pri/priCD4+ T cells into the sublethally irradiated WT mice (n = 6) to assess clinical scores & EAE incidence (C). (D) H&E staining and Luxol blue staining of the representative tissue sections of the spinal cords. Bars, 70 μm. (E) Total number of CD4+ T cells in the CNS and in the blood by flow cytometry. n = 6. (F) The encephalitogenic CD4+ T cells were isolated from the Dock8pri/+ and Dock8pri/pri mice at day18 after EAE induction for SDF-1α– or CCL5-induced migration (left), or for a FACS assay to check the surface expression of CXCR4 and CCR5 (right). n = 4–6. (G) The percentages of IFN-γ+ cells and IL-17A+ cells in CD4+ T cells in spleen from the recipient mice presented in C. n = 5. (H–I) T8.1 cells were transfected with FLAG-tagged DOCK8 or the pri mutant and treated with or without SDF-1α, followed by immunostaining with anti-FLAG and anti-CD44 (H; bar, 5 µm; n = 50), or for a transwell assay (I; left; n = 3 per group), or for a FACS assay to check the surface expression of CXCR4 (I, right). n = 4. (J) The migration of CD4+ T cells from naive Dock8pri/+ or Dock8pri/pri mice was examined in response to SDF-1α (left). Or WT and Dock8pri/pri CD4+ T cells were reconstituted with the vector or DOCK8 to assess T cell migration in response to CCL5 (right). n = 3. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.005. Data are representative of three independent experiments (C, mean ± SEM; E–F and H–I, mean ± SD), or two experiments (A, B, G, and J, mean ± SD). Statistical significance was determined using unpaired Student’s t test.
Figure 2.

CD4+ T cells from Dock8pri/pri mice ameliorate EAE with reduced CNS infiltration and migration. (A and B) The encephalitogenic CD4+ T cells were purified from Dock8pri/+ or Dock8pri/pri mice to check surface expression of CD44 and CD25 by FACS (A). IL-2 concentrations were checked by ELISA in the supernatants of Dock8pri/pri or Dock8pri/+ encephalitogenic CD4+ T cells stimulation with MOG for 3 d (B). n = 6. (C–E) Adoptive transfer of encephalitogenic Dock8pri/+ and Dock8pri/priCD4+ T cells into the sublethally irradiated WT mice (n = 6) to assess clinical scores & EAE incidence (C). (D) H&E staining and Luxol blue staining of the representative tissue sections of the spinal cords. Bars, 70 μm. (E) Total number of CD4+ T cells in the CNS and in the blood by flow cytometry. n = 6. (F) The encephalitogenic CD4+ T cells were isolated from the Dock8pri/+ and Dock8pri/pri mice at day18 after EAE induction for SDF-1α– or CCL5-induced migration (left), or for a FACS assay to check the surface expression of CXCR4 and CCR5 (right). n = 4–6. (G) The percentages of IFN-γ+ cells and IL-17A+ cells in CD4+ T cells in spleen from the recipient mice presented in C. n = 5. (H–I) T8.1 cells were transfected with FLAG-tagged DOCK8 or the pri mutant and treated with or without SDF-1α, followed by immunostaining with anti-FLAG and anti-CD44 (H; bar, 5 µm; n = 50), or for a transwell assay (I; left; n = 3 per group), or for a FACS assay to check the surface expression of CXCR4 (I, right). n = 4. (J) The migration of CD4+ T cells from naive Dock8pri/+ or Dock8pri/pri mice was examined in response to SDF-1α (left). Or WT and Dock8pri/pri CD4+ T cells were reconstituted with the vector or DOCK8 to assess T cell migration in response to CCL5 (right). n = 3. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.005. Data are representative of three independent experiments (C, mean ± SEM; E–F and H–I, mean ± SD), or two experiments (A, B, G, and J, mean ± SD). Statistical significance was determined using unpaired Student’s t test.

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