A negative role for the interleukin-2-inducible T-cell kinase (ITK) in human Foxp3+ TREG differentiation
Fig 1
ITK knock-down promotes increased Foxp3 expression in human CD4 T cells activated under different T cell polarizing conditions.
(A) ITK mRNA expression in sdRNA-treated cells was normalized by the ΔΔCt method to the mean of endogenous control genes (TBP, IPO8, and HPRT1). CD4 T cells were activated in the presence of a non-targeting control (NTC) sdRNA or two ITK-targeting sdRNA constructs. sd-ITK49 (henceforth sd-ITK) showed higher knockdown efficiency compared to sd-ITK47 and was therefore exclusively used in most subsequent experiments. N = 5 donors. (B and C) Human naïve CD4+ T cells were treated with NTC or ITK targeting sdRNAs and then activated under Th17, Th1, or TREG polarizing conditions. Four days later, CD4 T cells were analyzed for Foxp3 expression by flow cytometry. N = 3–5 donors per condition. (B) Foxp3 expression in CD4 T cells activated under TREG-polarizing conditions in the presence of NTC or two ITK-targeting sdRNA constructs. Right panels show representative flow cytometry plots of T cells treated with NTC or sd-ITK. (C) Foxp3 expression in CD4 T cells activated under different polarizing conditions in the presence of NTC or sd-ITK. Median ± IQR, *p<0.05, **p<0.01 (paired one-way ANOVA with Dunnett’s post-hoc test vs. NTC).