T cell differentiation and antigen-specific proliferation is not impaired in CD4 Cre × α4^flox/flox mice. We generated T cell conditional α4 integrin knockout mice (α4^−/−) by crossing CD4 Cre mice with α4^flox/flox mice. (A) Naive T cells (CD4⁺CD44⁻CD25⁻) from α4^−/− mice were isolated by FACS sorting and differentiated in vitro by stimulation with anti-CD3/anti-CD28 under Th1 or Th17 polarizing conditions. Surface staining for α4 integrin (top row: tinted line, isotype control; black line, anti–α4 integrin) and intracellular cytokine staining for IL-17 and IFN-γ (bottom row) are depicted. Numbers indicate percentages of cytokine-positive cells. Shown are representatives of three independent experiments. (B and C) α4^−/− mice and wild-type littermates were immunized with MOG_35-55 in CFA. On day 8, draining lymph nodes were dissected and restimulated with MOG_35-55 in vitro. After 48 h, the antigen-specific proliferative response was determined by ³H-thymidine incorporation (B, means + SD, n = 3). (C) Fractions of antigen-specific cytokine-producing CD4⁺ T cells in draining lymph nodes of MOG_35-55-immunized wild-type and conditional α4^−/− mice were determined by intracellular CD40L (CD154) and cytokine staining after restimulation with MOG_35-55. Numbers indicate percentages of cytokine/CD40L double-positive cells (C, means ± SEM, n = 3).