(A and B) Proliferation measured by Cell Trace Violet dilution (A) and IL-2 secretion in culture supernatants (B) by CD4⁺CD25^– Teffs isolated from the spleens of Dock8^–/– and WT mice cultured for 3 days with anti-CD3+anti-CD28–coated beads. (C) Percentage of CD25⁺FOXP3⁺ Tregs among CD4⁺ cells in the thymuses, spleens, and LNs of Dock8^–/– and WT mice. n = 17 mice from each group for the thymus, n = 31 mice from each group for the spleen, n = 7 mice from each group for the LN. (D) Percentages of CD44^–CD62L^hi rTregs and CD44⁺CD62L^lo aTregs of total CD4⁺FOXP3⁺ cells in the spleens of Dock8^–/– and WT mice. n = 5 mice from each group. (E) Representative FACS plots of intracellular FOXP3 and CTLA-4 and surface CD25 expression gating on CD4⁺FOXP3⁺ splenocytes (left). Quantitative analysis of surface CD25 expression by splenic CD4⁺FOXP3⁺ cells from Dock8^–/– mice and WT controls. n = 29 mice from each group. (F) qPCR analysis of Foxp3 and Il2ra mRNA levels in FACS-sorted CD4⁺CD25⁺CD39⁺ Tregs from Dock8^–/– and WT mice. Results are expressed as fold increase relative to the WT control ratio of the mRNA of interest/b2microglobulin. (G) Suppression of the proliferation of CD4⁺CD25^– Teffs by CD4⁺CD25⁺CD39⁺ Tregs from Dock8^–/– mice and WT controls. Teff proliferation was measured by FACS analysis of Cell Trace Violet dilution. The left panel is a representative experiment; the right panel shows the pooled results. (H) qPCR analysis of Tgfb and Il10 mRNA expression by sorted CD4⁺CD25⁺CD39⁺ Tregs from Dock8^–/– mice and WT controls. Symbols represent individual mice, and error bars represent mean and SEM. Results in A, B, and F–H are representative of 3 independent experiments. t test, NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.