TNFα and IL-6 present at the site of inflammation induce PKB/c-akt activation resulting in resistance to suppression. (A) IL-1β, IL-6, IL-17, IL-2, TNFα, and IFNγ expression in n = 4 PB plasma of HCs and n = 8 paired PB plasma and SF of JIA patients measured by Luminex. (B-C) PBMCs from HCs were untreated or incubated overnight with TNFα (50 ng/mL), IL-6 (100 ng/mL) or both TNFα and IL-6. After incubation period, cells were stained for phosphorylated PKB/c-akt expression by flow cytometry (B) or CFSE labeled and cultured in the presence or absence of recombinant human TGFβ1 (40 ng/mL) and TNFα and IL-6 to measure TGFβ-mediated suppression (C). (B) MFI of phosphorylated PKB/c-akt (p-PKB) in CD4⁺ T cells in the presence of TNFα (dark gray bars), IL-6 (light gray bars) or both (white bars) relative to cultures without cytokines added (black bars). Bars represent mean ± SEM of n = 5, *P < .05. (C) TGFβ-mediated suppression of CD4⁺ T-cell proliferation in the absence (black bars) or presence of TNFα (dark gray bars), IL-6 (light gray bars) or both (white bars). The data show the change in TGFβ-mediated suppression in the presence of cytokines compared with cultures without cytokines added. Bars represent mean ± SEM of n = 5, *P < .05.