IL-2 responsiveness and in vitro expandability of Treg-A and Treg-B. (A-B) Western blot analysis of STAT5 and pSTAT5 protein expression in Treg-A and Treg-B after treatment with 1, 40, 60, or 80 IU/mL of human IL-2 for 15 or 30 minutes. β-ACTIN protein level is used as a loading control, and numbers represent the densitometric quantification of STAT5 and pSTAT5 protein expression levels normalized to β-ACTIN. (C) The expansion rate of Treg-A and Treg-B from HD (n = 6) and AA patients (n = 3). Treg-A and Treg-B were stimulated with anti-CD3/CD28 beads (1 cell:1 bead ratio) and 1000 IU/mL IL-2 for 4 weeks with 2 μM ATRA and 100 nM rapamycin in Prime XV T-cell expansion XSFM medium. Error bars represent mean ± SD. *P ≤ .05; ns, not significant. (D) GSEA enrichment plot shows the significant enrichment of gene set for cell proliferation in AA-expanded Treg-A compared with Treg-A before expansion. However, gene set of cell proliferation was not enriched in expanded HD Treg-A as compared with preexpansion Treg-A. This finding suggests the higher “expansion potential” of AA Treg-A compared with HD Treg-A. ES, enrichment score; NES, normalized enrichment score. (E) PCA analysis on transcriptional profiles in AA Treg-A as well as Treg-B before and after expansion. (F) Chart shows the median expression of 29 markers measured by CyTOF analysis in HD-expanded Treg-A (n = 3) and Treg-B (n = 3). Error bars represent mean ± SD. (G) Heat map shows the median expression of Treg-B–specific markers (CD45RA, CD45RO, CD95, and CCR4) in HD Treg-A (n = 3) and Treg-B (n = 3) before and after expansion.