Figure 1. Generation of CAR-Tregs.

CD4⁺ T cells were isolated from human donor peripheral blood mononuclear cells (PBMCs) and enriched for CD25⁺ cells using positive selection. (A) Sorting gates for Tregs: CD4^mid, CD25⁺⁺, and CD127^lo. Sorting gates for Tconvs: CD4⁺ and CD25^lo. Gates were drawn on cells prior to CD25 enrichment. (B) Foxp3 (clone PCH101) intracellular stain after sort. Data are representative of 7 independent sorts from different human donors. (C) Methylation status averaged across sites in the Treg-specific demethylation region (TSDR) on the X chromosome of the sorted T cell populations from female-donor leukopacks (n = 2 female donors, mean plotted). Dots within bars represent individual data points. (D) Vector maps of CD19 CAR constructs. L, leader sequence; scFv, single-chain variable fragment; TM, hinge and transmembrane domain. (E) Experimental design and preparation of CAR-Tregs. (F) Whiskers plots showing mCherry mean fluorescence intensity (MFI) of CAR T cells 12 days after lentivirus transduction at an MOI of 5 measured by flow cytometry (n = 7 human donors). **adj-P < 0.01, by paired ratio t test with Holm-Bonferroni method adjustment for 3 tests between Tregs and Tconvs. Tr, Treg; Tc, Tconv.