Fig. 4.

Mreg coculture enriches CD4⁺ T cells for GARP⁺ FoxP3⁺ iTregs. Marker screening experiments were performed in order to establish whether FoxP3⁺ and FoxP3⁻ T cells arising through Mreg coculture represented truly distinct populations. a Human Mregs were cultured at a 1:1 ratio with allogeneic human CD3⁺ T cells for 5 days before expression of Treg-associated markers was investigated by flow cytometry (representative of n = 6). b Correlation between marker expression in FoxP3⁺ miTregs and CD25⁺ FoxP3⁻ non-Tregs. Background (isotype)-subtracted MFI values were log₂-transformed and median values for n = 6 donors were plotted. GARP, CD357 (GITR), intracellular CD152 (CTLA4), CD137 (4-1BB) and CD39 were more highly expressed by miTregs. CD6, CD127 and KLRG1 were more highly expressed by non-Tregs. c GARP-selected miTregs stimulated with αCD3/αCD28 beads expanded exponentially for 21 days after isolation (n = 3). d Over this 21-day re-stimulation with αCD3/αCD28 beads, frequencies of FoxP3⁺ miTregs decayed to 36.6 ± 5.2% (n = 3; mean ± sd). e No relevant increase in TSDR demethylation was detected during re-stimulation (n = 3; mean ± sd). f To better understand the relationship between miTreg and other in vitro-derived iTregs, a phenotypic comparison was made between nine CD4⁺ T-cell subtypes from n = 6 donors considering 29 markers measured by flow cytometry (Supplementary Figure 4D). Principal component (PC) analysis readily distinguished miTregs from other in vitro-derived iTregs, activated T cells and activated nTregs. This approach revealed miTregs were more similar to naive T cells and peripherally-induced Tregs (pTregs) than any other CD4⁺ T-cell subset