Figure 1. The CTLA4/PKCη pathway controls human Treg-suppressive activity.

(A) CD4⁺CD25⁺CD127^lo Tregs from human blood were left unstimulated (ns) or were stimulated (stim) for 5 minutes with anti-CD3 and anti-CTLA4 antibodies. CTLA4 immunoprecipitates and whole cell lysates (WCL) were immunoblotted with anti-PKCη and anti-CTLA4 antibodies. Data representative of 2 independent experiments are shown. (B–D) Human Tregs were retrovirally transduced with irrelevant shRNA (ShControl) or 2 different shRNA targeting Prkch (shPrkch-1 and shPrkch-2). PKCη expression in purified transduced Tregs was assessed by immunoblotting (B) and quantitated as the percentage of expression in the ShControl group (C). Foxp3 expression in transduced Tregs was assessed by intracellular staining (D). (E and F) Suppressive activity of the transduced Tregs was analyzed by coculture at different ratios with CTV-prelabeled PBMCs stimulated with anti-CD3. (E) Representative histograms of CTV dilution in gated CD4⁺ responder cells. (F) Cumulative data expressed as the percentage inhibition of responder CD4⁺ cell proliferation (mean ± SEM of 5 independent experiments). Statistical significance of differences between groups was determined by 1-way ANOVA and Tukey’s multiple comparisons test. ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ****P ≤ 0.0001. Statistical significance levels are shown against the + ShControl Treg group.