Fig 1: Knock-out of IL6RA in human Tregs impairs IL-6-mediated signaling. (A) Tregs were incubated without (control) or with IL-6 for 24 hours and subsequently cultured in suppression assays with CFSE-labeled PBMCs. FACS plots of one representative experiment (left panel) and data combining 4 independent donors (right panel). Error bars represent mean ± SD. (B–F) Human Tregs were cultured in vitro for 7 days prior gene editing (following protocol described in Figure 1A). (B) FOXP3 and CD126 expression were analyzed in mock and IL6RA-KO Tregs that were harvested 5 days after RNP nucleofection. FOXP3 plots are pre-gated on living cells and CD126 plots are pre-gated on living FOXP3+ cells. Data are from one representative experiment. (C) CD126 expression was studied in mock and IL6RA-KO Tregs 4 to 7 days after nucleofection in n = 7 independent donors. Error bars represent mean ± SD. (D) RNP-transfected cells were FACS-sorted for CD126 expression and as living (L/D nearIR-) CD4+CD25+CD127- cells. Left panel represents pre-sorted cells and right panel shows re-analysis post sorting. (E) FOXP3 expression in CD126- sorted cells (n = 3). Error bars represent mean ± SD. (F) STAT3 phosphorylation was analyzed in CRISPR-edited Tregs. FACS plot of one representative experiment (left panel) and data from 3 independent donors (right panel). Error bars represent mean ± SD. MFI is normalized over IL-6-stimulated mock. Statistical significance was calculated by two-tailed t test. **p = 0.01, ****p = 0.0001.