LAT recruitment to the immune synapse and late T cell activation are deficient in Rab6 cKO CD4⁺ T lymphocytes. (A) Immunoblot analysis of Rab6 and gp96 (loading control) expression in CD4⁺ T cells (left) or total kidney cells (right) isolated from control (WT) and Rab6 cKO mice. Molecular mass is indicated in kilodaltons. (B and C) Quantification by TIRFM of the number of LAT (B) or ZAP70 (C) microclusters recruited to the immune synapse in CD4⁺ T cells incubated on poly-l-lysine alone or anti-CD3+anti-CD28 mAb (1 or 10 µg/ml). Bars, 5 µm. (D–F) Flow cytometry analysis of the proliferation of CD4⁺ T cells isolated from control (Ctrl) or Rab6 cKO mice labeled with CTV proliferation dye and stimulated for 5 d by different numbers of irradiated allogeneic Balb/C splenocytes (stimulatory cells) in a mixed lymphocyte reaction. (D) Representative experiment. (E) Percentages of proliferating CD4⁺ T cells from five Rab6 cKO or control mice. (F) Rab6-deficient CD4⁺ T cells labeled with the CFSE proliferation dye were either left alone (dark colors) or mixed with (1:1) control CD4⁺ T cells labeled with CTV (light colors). Analysis of the percentages of proliferating CD4⁺ T cells exposed for 5 d to different numbers of irradiated allogeneic Balb/C splenocytes was performed as in D. (G) Percentages of proliferating CD4⁺ T cells isolated from control or Rab6 cKO mice left untreated (left) or stimulated for 5 d with low concentrations of PMA/ionomycin (7.5 × 10⁻⁷ M and 2 × 10⁻⁸ M; right). *, P < 0.05; **, P < 0.01; ****, P < 0.0001; one-way ANOVA in B and C; two-way ANOVA in E; mean and SEM in D. Horizontal lines represent median in B and C. Data from three experiments in B, from two experiments in C, and from four experiments in E. Data representative from three experiments in A, four in D, two in F, and three in G.