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. 2021 Aug 2;12:655122. doi: 10.3389/fimmu.2021.655122

Figure 4.

Figure 4

Knock-out of IL2RA in human Tregs impairs suppressive capacity and IL-2-mediated signaling. Human Tregs were cultured in vitro for 7 days prior gene editing (following protocol described in Figure 1A). (A) FOXP3 and CD25 expression were analyzed in mock and IL2RA-KO Tregs that were harvested 4 days after RNP nucleofection. FOXP3 plots are pre-gated on living cells and CD25 plots are pre-gated on living FOXP3+ cells. Data are from one representative experiment. (B) CD25 expression was studied in mock and IL2RA-KO Tregs 4 to 7 days after nucleofection. n = 11 independent donors. Error bars represent mean ± SD. (C) RNP-transfected cells were FACS-sorted for CD25 expression and as living (PI-) CD4+CD127- cells. Top panel displays pre-sorted cells and bottom panels represent re-analysis post sorting. (D) FOXP3 expression after CD25-sorting in mock (n = 3), CD25+ (n = 6) and IL2RA-KO (n = 6) Tregs. Error bars represent mean ± SD. (E) STAT5 phosphorylation was studied in CRISPR-edited Tregs. FACS plot of one representative experiment (left panel) and combined data of three independent donors (right panel). Error bars represent mean ± SD. MFI is normalized over IL-2-stimulated mock. (F) Suppressive capacity of IL2RA-KO Tregs was measured by their ability to suppress T cell proliferation in vitro (ratio 1:2). Left panels show FACS plots of one representative donor. Cells are pre-gated for viability (all columns) and CD4 or CD8 expression (middle and right column, respectively). Right panels show CD4+ and CD8+ T cell proliferation, displayed by dilution of the cell proliferation dye CFSE. Proliferation is normalized over T cell proliferation in the absence of Tregs (n = 3-6 independent donors). Error bars represent mean ± SD. Significance was calculated by two-tailed paired t test (B), Kruskal-Wallis test (D, F) or one-way ANOVA (E). *p ≤ 0.05, **p ≤ 0.01. ****p ≤ 0.0001.