PKB/c-akt hyperactivation causes resistance of effector cells to suppression. (A-B) PBMCs and SFMCs were stained for phosphorylated PKB/c-akt expression by flow cytometry. (A) Histogram showing phosphorylated PKB/c-akt (p-PKB) fluorescence intensity in CD4⁺ T cells from paired PBMCs (solid gray) and SFMCs (black line), 1 representative of n = 3. (B) MFI of phosphorylated PKB/c-akt (p-PKB) in CD4⁺ T cells from paired PBMCs (gray bar) and SFMCs (white bar) of JIA patients relative to PBMC from HC (black bar). Bars represent mean ± SEM of n = 3, *P < .05. (C-D) CFSE-labeled PBMCs and SFMCs were cultured in the presence or absence of recombinant human TGFβ1 (40 ng/mL) and increasing concentrations of PKB/c-akt inhibitor VIII (PKBinh VIII; 0, 0.01, 0.1, 1μM). At day 5, proliferation of CD4⁺ T cells was analyzed. (C) TGFβ-mediated suppression of CD4⁺ T-cell proliferation for PBMCs from HCs (black bars) and PBMCs (gray bars) and SFMCs (white bars) from JIA patients. The results show percentage of suppression in the presence of TGFβ relative to cells cultured without TGFβ. Bars represent mean ± SEM of n = 3 PBMC HC, n = 4 PBMC JIA, and n = 5 SFMC JIA, *P < .05. (D) TGFβ-mediated suppression of CD4⁺ T cell proliferation for paired PBMCs (gray bars) and SFMCs (white bars) in the presence of increasing concentrations of PKB/c-akt inhibitor VIII. The data show the change in TGFβ-mediated suppression for each concentration of PKB/c-akt inhibitor relative to cultures without PKB/c-akt inhibitor. Bars represent mean ± SEM of n = 3, *P < .05 compared with 0μM PKB/c-akt inhibitor VIII. (E) CD4⁺CD25⁺CD127^low Tregs were sorted from SFMCs by flow cytometry and cocultured with CFSE-labeled PBMCs (squares) or SFMCs (triangles) at a 1:4 ratio in the presence or absence of PKB/c-akt inhibitor VIII (PKBinh VIII) (0.1μM). At day 4, Treg-mediated suppression of CD4⁺ T-cell proliferation was analyzed. The data show the change in Treg- mediated suppression in the presence PKB/c-akt inhibitor relative to cultures without PKB/c-akt inhibitor.