Figure 6. Overexpression of p65 and Foxp3^S418E in Stk3/4-deficient Treg cells restores their regulatory functions.

(A) Flow cytometric analysis and scatter plot representation of MFI of p65, CD25 and Foxp3 in Foxp3^YFPCre and Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells transfected with either empty vector or with p65-encoding retrovirus, as indicated (n=5 per group). (B) Survival of Foxp3^∆EGFPiCre mice injected with empty vector-transfected (n=8), p65-transfected (n=5) or Foxp3^S418E transfected Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells (n=5). (C) Flow cytometric analysis and frequencies of CD62L^loCD44^hi CD4⁺Foxp3⁻ and CD62L^hiCD44^lo CD4⁺Foxp3⁻ Teff cells from spleens of Foxp3^∆EGFPiCre mice injected with Foxp3^YFPCreStk3^∆/∆Stk4^∆/∆ Treg cells that had been transfected with either empty vector (n=7), p65 (n=7), Foxp3^S418A (n=6) or Foxp3^S418E encoding vectors (n=5). (D, E) Flow cytometric analysis and frequencies of IFNγ⁺ CD4⁺Foxp3⁻ T cells from spleens (D) and lungs (E) of the respective groups (n=7 for empty vector; n=7 for p65; n=6 for Foxp3^S418A; n=5 for Foxp3^S418E). The results represent three pooled independent experiments. Each point represents one mouse. Error bars indicate S.E.M. Statistical tests: log-rank-test (B), one-way ANOVA with post-test analysis (A,C,D,E), ns: Not significant, *P<0.05, **P<0.01, ***,P<0.005, ****P<0.0001.