Fig. 1.

Gating strategy for the delineation of IL-6R^hi CD127^lowCD25⁺ T cells (Tregs). (A) Gating strategy for the delineation of IL-6R^lo and IL-6R^hi CD127^lowCD25⁺ Tregs, defined as the lower and upper 20th percentile, respectively, of the IL-6R mean fluorescence intensity (MFI) distribution in total CD127^lowCD25⁺ Tregs. (B) Histograms depict the distribution of IL-6R expression and the respective frequency of IL-6R^lo and IL-6R^hi cells among: (i) CD45RA⁺ naïve (depicted in blue); (ii) total (depicted in black); and (iii) CD45RA⁻ memory (depicted in red) CD127^lowCD25⁺ Tregs. Frequencies shown in the figure represent the average frequency of the IL-6R^lo and IL-6R^hi cells in the three assessed Treg subsets from 33 healthy donors. (C) Data shown depict the expression of TIGIT versus IL-6R in one illustrative donor and the frequency (GeoMean +/− 95% CI) of IL-6R^hi cells in the TIGIT⁺ (depicted in green) and TIGIT⁻ (depicted in red) subsets of CD127^lowCD25⁺CD45RA⁻ mTregs in 33 healthy donors. P value was calculated using a two-tailed paired non-parametric Wilcoxon signed rank test, comparing the frequency of IL-6R^hi cells in the TIGIT⁺ and TIGIT⁻ subsets. (D) Histograms illustrate the distribution of the two sorting markers TIGIT and IL-6R following flow-cytometric sorting.