Fig. 2: Interval low-dose IL-2 immunotherapy selectively expands FOXP3⁺HELIOS⁺ Tregs.

a UMAP plot depicting n = 149,202 unstimulated cells sorted from the CD127^lowCD25^hi Treg gate. Clusters were manually annotated based on the expression of key mRNA and protein markers. Clusters corresponding to: (i) naïve Tregs (defined as CD45RA⁺ FOXP3⁺HELIOS⁺); (ii) memory Tregs (defined as CD45RA^– FOXP3⁺HELIOS⁺); or (iii) CD25⁺ FOXP3^–HELIOS^– Teffs (defined as CD45RA^– FOXP3^–HELIOS^–) are annotated in green, blue and orange, respectively. All other clusters not conforming to this definition are annotated in black. b Relative frequency changes of naïve FOXP3⁺HELIOS⁺ Treg, memory FOXP3⁺HELIOS⁺ Treg, and CD25⁺ FOXP3^–HELIOS^– Teff clusters on Day 27 compared to baseline (Day 0) levels, for unstimulated cells. c, d Corresponding UMAP plot (c) and relative frequency of the identified subsets (d) for n = 115,153 stimulated CD4⁺ T cells sorted from the CD127^lowCD25^hi Treg gate. e Absolute cell numbers of naïve FOXP3⁺HELIOS⁺ Tregs, memory FOXP3⁺HELIOS⁺ Tregs, and CD25⁺ FOXP3^–HELIOS^– Teffs on Day 0, Day 27 and Day 35, for unstimulated and stimulated cells. Absolute cell numbers of the Treg subsets were calculated by multiplying the absolute number of Tregs measured by FACS by the relative frequency of the respective subset measured by single-cell multiomics. Data are presented as mean values ± SEM. f Per-cell Treg suppression efficiency for each time point, assessed in vitro by culturing CD4⁺CD25^hiCD127^low Tregs with autologous CD4⁺CD25^−/lowCD127⁺ Teffs at various ratios (x axis). n = 12 participants. In (b) and (d), P values were calculated using two-sided Wilcoxon signed-rank tests comparing the relative frequency of the subsets between Day 27 and Day 0. In (b, d and f), each dot represents cells from a single participant. Dots with the same colour represent the same participant. Each box ranges from the first quartile (Q1) to the third quartile (Q3), with a central line indicating the median. The bottom and top whiskers extend to the most extreme data points within Q1 − 1.5 × (Q3 − Q1) and Q3 + 1.5 × (Q3 − Q1), respectively. In (b, d, and e), n = 13 participants. Source data for (b, d, e, f) are provided as a Source Data file.