Supplementary Figure 1: CD161⁺ T_reg cells are a discrete population of memory T_reg cells.

(a) Heat map showing signal intensity of selected markers for each node of the populations shown in Fig. 1c. (b–e) Clustering of populations of T_conv and T_reg cells by single-cell RNA-seq (scRNAseq) showing the tSNE plot of 2,636 cells from n = 3 independent donors separated into six major (clusters 0–6) and two minor (clusters 7 and 8) clusters (b), feature plots showing expression of three T_reg markers (IL2RA encoding CD25, IL7R encoding CD127 and FOXP3 encoding FOXP3), KLRB1 (encoding CD161) and two naive/memory markers (SELL encoding CD62L and CCR7 encoding CCR7) (c), a heat map showing the top 20 discriminatory genes per cluster with representative genes of each cluster indicated (d) and population clustering (e) based on expression of the transcripts shown in c. Clusters in b are color-coded and labeled according to expression of the markers shown in e. (f) Clustering of T_conv and T_reg subpopulations using protein expression data sourced from CyTOF in Fig. 1a incorporating the same markers as in e and also including CD45RA and CD45RO. Clustering of subpopulations in f is similar to that from scRNAseq (e). (g,h) viSNE plots (g) and 2D minimum spanning tree (h) of CD4⁺ T cells clustered following staining with anti-CD4, anti-CD25, anti-CD127, anti-CD45RA and anti-CD161 for flow cytometry. Shown in g are heat maps for expression of the indicated markers, with an arrow indicating CD161 expression in T_reg cells. Node size in h represents cell number and color CD161 median intensity. Grouped together are naive (circled in orange), memory (circled in yellow) and CD161⁺ (circled in purple) T_reg cells, as well as populations of naive (circled in black), memory (circled in black) and CD161⁺ (circled in red) T_conv cells. The data in g and h are representative of n = 4 experiments. *P < 0.01, **P < 0.001, ***P < 0.0001 by one-way ANOVA.