Figure 5. GIT2 controls mouse and human Treg-suppressive activity.

(A) Rag1^−/− mice received an adoptive transfer of CD25-depleted spleen cells with or without Tregs from WT or Git2^–/– FIG mice and were implanted with B16-F10 melanoma cells, as in Figure 2. Tumor areas (mm²) were measured 3 times/week. Cumulative data of 4 independent experiments are shown (mean ± SEM). no Tregs, n = 11; + WT, Tregs, n = 14; + Prkch^−/− Tregs, n = 12). (B–F). Human Tregs were retrovirally transduced with irrelevant shRNA (ShControl) or shRNAs targeting GIT2 (shGIT2) or PKCη (ShPrkch-2). Transduced Tregs were analyzed for GIT2 protein expression by immunoblotting (B) and for the expression of Foxp3 by intracellular staining (C). Suppressive activity of transduced Tregs was evaluated by coculture with CTV-labeled PBMCs stimulated with anti-CD3 (D). Data are expressed as inhibition of gated CD4⁺ Teff cell proliferation. (E and F) Analysis of CD86 expression by CD19⁺ APCs in suppression cocultures, showing histograms of a representative mouse in each group (E) and cumulative data of 3 experiments (mean ± SEM) (F). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Statistical significance was determined by repeated-measures 2-way ANOVA (A) or 1-way ANOVA (D and F) followed by Tukey’s multiple comparisons test. Significance against the shControl Tregs group is shown (D and F). (G and H) Human Tregs were left unstimulated or stimulated using anti-CD3, anti-CTLA4, or a combination of both antibodies. Activating phosphorylation of PAK1 (Ser¹⁴⁴) and PAK2 (Ser¹⁴¹) and expression levels of PAK2 were assessed in cell lysates by immunoblotting. Representative data and quantification of signals in G are expressed as p-PAK2/PAK2 ratio (H). Cumulative data from 2 experiments (mean ± SEM). (I and J) Human Tregs transduced with ShControl or ShPrkch-2 were stimulated with an anti-CTLA4 antibody. Activating phosphorylation (Ser¹⁴¹) and total expression of PAK2 were assessed in cell lysates by immunoblotting (I). Signals in I were quantified by densitometry and expressed as p-PAK2/PAK2 ratio (J). Cumulative data from 2 experiments (mean ± SEM).