Function and ontogeny of Treg subpopulations. (A) Suppression of T_con cytokine secretion by Treg subpopulations: CD4⁺CD25^hi CD127^lo CD45⁻RA^hi, CD95⁻, CCR4^lo (subpopulation A), CD4⁺CD25^hi CD127^lo CD45-RA^lo CD95⁺ CCR4^hi (subpopulation B) and CD4⁺CD25^low T_con were sorted (FacsAria) and cultured for 5 days with anti-CD3/CD28 beads (T_con:Treg:bead = 20:20:1). After 5 days of culture, the supernatant was analyzed with ProcartaPlex 6 Plex (eBioscience) according to the manufacturer’s instructions. The cytokine concentrations were corrected for the cell number. Treg B cells were able to significantly reduce both IFN-γ and TNF-α secretion by T_con (P < .05) in a 1:1 coculture. Average of 3 replicates, Student t test, *P < .05. Error bars are standard error of mean. (B) Pairwise comparison of TCR repertoire overlaps. The color shading reflects the numerical value of the (PG indices. The TCR sequences shared between T_con, Treg A, and Treg B were very low with a PG index <0.001 in all comparisons. (C-D) GEP of Treg A and Treg B subpopulations compared with T_con: We have used the published GEP profile of Tregs (Ferraro et al.) as the reference list, and all 3 T-cell populations were compared with the list, which is sorted based on highly expressed genes in human Treg. Both Treg A and Treg B subpopulations were enriched with Treg-related genes, in particular IKZF2, FCRL3, FOXP3, CTLA4, and IL-2R. However, the genes that are expressed at a lower level in human Tregs were enriched in T_con, but in none of the Treg subpopulations. (D) The frequency of common genes between Treg A, Treg B, and referenced published genes (Ferraro et al.) are demonstrated (E), which includes genes that are highly expressed by human Tregs.