Figure 7. DOCK8-deficient Tregs have decreased expression of cytoskeleton- and Treg-associated genes.

(A and B) CD4⁺CD25⁺CD39⁺YFP⁺ (DOCK8-deficient) and YFP^– (DOCK8-sufficient) Tregs were sorted from Foxp3^YFP–Cre/+/Dock8^flox/flox female mice. Tregs were cultured overnight in media alone (A) or with anti-CD3+CD28 beads (B). Heatmaps of selected genes differentially expressed in YFP⁺ and YFP^– Tregs from Foxp3^YFP–Cre/+Dock8^flox/flox female mice. The cutoff for significance was P < 0.05. P values were calculated using the Wald test for differential expression analysis. P values were corrected afterward for multiple testing. Expression of genes is centered and scaled by row to highlight differences in each gene sample. Each column represents an individual mouse. (C and D) CD4⁺CD25⁺CD39⁺ Tregs were FACS sorted from Dock8^–/– and WT mice. RNA was prepared from Tregs directly after isolation (C) or after 24-hour culture with anti-CD3+CD28 beads (C and D). qPCR results are expressed as fold increase of mRNA of interest/b2microglobulin ratio relative to the unstimulated WT Tregs. (E) MFI of surface marker staining on YFP^– DOCK8-sufficient and YFP⁺ DOCK8-deficient CD4⁺CD25⁺CD39⁺ Tregs from Foxp3^YFP–Cre/+/Dock8^flox/flox female mice. Symbols represent individual mice. Bars in C–E represent the mean and SEM. t test, NS P > 0.05, *P < 0.05, ***P < 0.001.