Tregs underwent clonal restriction of the TCR repertoire and activation during GVHD suppression. (A) Clonality of the TCRA and TCRB repertoire in Tregs at day 0 (light pink box and symbols) and day 8 (purple box and symbols) after HCT. (B) Representative example of overlap of the TCRA and TCRB repertoire in Tregs before transplantation and at day 8 after HCT. Scatter plots represent frequency of clones before and after HCT, and the number of unique clones (dot size). Clones that are observed at only 1 time point are colored in light gray, whereas overlapping clones are colored in dark gray. (C) Venn diagram representing the number of overlapping and nonoverlapping clones between day-0 (light pink box and symbols) and day-8 (purple box and symbols) Tregs. (D) Representative example of overlap of the TCRA and TCRB repertoire in CD4 Tcons (x-axis) and Tregs (y-axis) at day 8 after HCT in allogeneic mice receiving both Tcons and Tregs. Scatter plots represent frequencies of clones in Tcons and Tregs, and the number of unique clones (dot size). Clones that are observed in only 1 population are colored in light gray, whereas overlapping clones are colored in dark gray. Jaccard indexes are indicated. (E) Venn diagram representing the number of overlapping and nonoverlapping clones between Tregs and Tcons treated or not treated with Tregs recovered at day 8 after HCT. (F) Heatmap and hierarchical clustering based on the 500 most highly differentially expressed genes across all samples. Immune-related genes are highlighted. Expression for each gene is scaled (z scored) across single rows.