Fig. 4

CD4-Cre-SNX27^fl/+ splenocytes show normal growth and enhanced proliferation upon stimulation. A, B Splenocytes from SNX27^fl/fl or CD4-Cre-SNX27^fl/+ mice were stimulated with plate-bound anti-CD3 (2.5 µg/ml) and soluble anti-CD28 (1.25 µg/ml) for 1, 2, 6 or 7 days. CD19⁻ CD3⁺ CD4⁺ and CD19⁻ CD3⁺ CD8⁺ T cells were gated by flow cytometry (gating strategy shown in Suppl Fig. 4). GMFI of the forward scatter, indicative of cell size, was calculated on these populations. Data are shown as mean ± SEM; two-way ANOVA with Bonferroni post-test was used for multiple comparisons; ns p > 0.05; n = 5 mice. C-E Splenocytes from SNX27^fl/fl or CD4-Cre-SNX27^fl/+ mice were stained with CFSE. Labeled splenocytes were stimulated with plate-bound anti-CD3 (2.5 μg /mL) and soluble anti-CD28 (1.25 μg /mL) for 2 days. Cells were then stained with LIVE/DEAD violet fixable dye, and for CD19, CD3, CD4, and CD8 surface markers. CD19⁻ CD3⁺ CD4⁺ or CD19⁻ CD3⁺ CD8⁺ T cells were gated (gating strategy shown in Suppl Fig. 6). C CFSE dilution profiles of the gated populations are depicted. Representative flow cytometry plots are shown. D, E The percentage of cells by generation (D) and the proliferation index (E) are shown. Data shown as ± SEM; *p < 0.05; ***p < 0.001; ****p < 0.0001; two-way ANOVA with the Bonferroni post-test was used for multiple comparisons in (D) and unpaired t-test in (E); n = 5 mice in (C-E). F, G Total splenocytes or (H, I) isolated naïve CD4⁺ T cells from SNX27^fl/fl or CD4-Cre-SNX27^fl/+ mice were stimulated 2 days with plate-bound anti-CD3 (2.5 µg/ml) and soluble anti-CD28 (1.25 µg/ml). Afterwards, the concentration of the depicted cytokines was assessed by ELISA. Data are shown as mean ± SEM of a representative experiment run in triplicate (n = 4–5 mice); *p < 0.05; unpaired t-test. SNX Sorting nexin, GMFI Geometric mean fluorescence intensity, SEM Standard error of the mean