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. 2018 Feb 7;9:199. doi: 10.3389/fimmu.2018.00199

Figure 4.

Figure 4

CD137 expression identifies antigen-activated chimeric antigen receptor (CAR)–regulatory T cells (Tregs). (A) Schematic diagram of the dextran-specific CAR construct [transmembrane [TM], 2A peptide (P2A)]. (B–D) CAR–Tregs were generated by lentiviral transduction, and efficiencies were analyzed by (B–C) LNGFR expression; (B) representative dot plot of one donor and (C) statistical analysis of several donors is shown (n = 50 from 16 independent experiments of CAR–Tregs and n = 12 from 4 different experiments of untransduced Tregs are shown). (D) CAR surface expression was analyzed on LNGFR+ and LNGFR− cells by incubation with FITC-labeled dextran (n = 3–9 from 1 to 3 independent experiments are shown). (E,F) Tregs were restimulated for 6 h with anti-CD3/anti-CD28, bead-bound dextran or 2 µg/ml soluble FITC dextran, and expression of CD137 was analyzed. (E) Representative dot plots of one donor and (F) statistical analysis of bead-bound stimulation are shown (n = 21, seven different experiments were performed). (C,F) Each dot represents one donor, and (C) lines indicate mean; (D) mean ± SEM is shown. (F) Statistical significance was determined by paired t-test.