PMC

Phenotype of antigen-activated chimeric antigen receptor (CAR)–regulatory T cells (Tregs). (A–H) LNGFR+ CAR–Tregs were sorted and expanded for 10–12 days before analysis of (A) LNGFR expression and (B) dextran-binding (n = 12 for LNGFR+ sorted and n = 11 for LNGFR− sorted from four independent experiments). (C,D) FoxP3 expression in LNGFR+ sorted CAR–Tregs was analyzed ex vivo and after 21 days; (D) representative dot plot of one donor and (C) statistical summary of several donors (n = 6 from two independent experiments). (E–H) CAR–Tregs were restimulated for 6 h with bead-bound dextran and (E,G) FoxP3 expression (n = 6 from two independent experiments) and (E,F) CD137 and CD154 expression (n = 12 for from four independent experiments) were analyzed; (E) representative dot plot of one donor and (F,G) statistical summary of several donors. (H) Cytokine expression was analyzed on CD137- and CD154-expressing CAR–Tregs (n = 6 from two independent experiments). (A–C,F–H) Each dot represents one donor, and statistical significances were determined by (C) paired t-test or (H) Wilcoxon signed-rank test; lines indicate (A,B) median or (F–H) mean.

CD137+CD154− expression identifies stable regulatory T cells (Tregs) ex vivo. (A) CD137 and CD154 expression were analyzed on CD25+CD127− Tregs after 6-h stimulation with anti-CD3/anti-CD28 ex vivo (n = 68, 17 independent experiments were performed). (B) Frequencies of FoxP3+ cells were analyzed among CD137- and CD154-expressing cells within the CD25+CD127− Treg compartment (n = 30, five independent experiments were performed). (C) CD137+CD154− and CD137+CD154+ Tregs (CD25+CD127−CD45RO+) were sorted after 6-h stimulation with anti-CD3/anti-CD28, and methylation of indicated regions was analyzed (data from two independent experiments are shown, and five and six male donors were pooled for each experiment). Methylation of central memory and effector memory T cells was derived from the study by Durek et al. (). (A,B) Each dot represents one donor, lines indicate (A,B) median, (C) mean ± SEM is shown; statistical significances were determined by (B) Kruskal–Wallis test.

Phenotype of in vitro expanded regulatory T cells (Tregs). CD25+ Tregs were sorted and expanded for 14 or 28 days before analysis of (A,B) FoxP3 expression; (A) representative dot plot of one donor and (B) statistical analysis of several donors (n = 30 from nine independent experiments for d0 and d14, and n = 19 from seven different experiments for d28). (C) Cytokine expression was analyzed on d28 after 6-h restimulation with PMA/ionomycin (n = 38 from 12 different experiments for IFN-γ, n = 40 from 13 different experiments for TNF-α, n = 19 from 7 different experiments for IL-17, and n = 17 from 6 different experiments for IL-2). (D,E) CD137 and CD154 expression were analyzed on expanded Tregs on d14 after restimulation with anti-CD3/-CD28; (D) representative dot plot of one donor and (E) statistical analysis of several donors (n = 64, 21 independent experiments were performed). (B,C,E) Each dot represents one donor, and (B) statistical significances were determined by one-way analysis of variance; lines indicate (B) mean or (C,E) median.

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.

Phenotype of CD137- and CD154-expressing cells within expanded regulatory T cell (Treg) cultures. (A,B) CD25+ Tregs were sorted and expanded before analysis of FoxP3 expression; (A) representative dot plot of one donor and (B) statistical summary of several donors (n = 61, 20 independent experiments were performed). (C,D) CD25+ Tregs were sorted and expanded before 6-h restimulation with PMA/ionomycin for analysis of cytokine expression on CD137- and CD154-expressing cells; (C) representative dot plot of one donor and (D) statistical summary of several donors (n = 30 from nine different experiments for IFN-γ and TNF-α, n = 11 from four different experiments for IL-17 and IL-2). (E) Tregs were sorted from expanded CD25+ Tregs according to CD137 and CD154 expressions or left unsorted, and all populations were expanded for another 14 days before in vitro suppression of proliferation of CD4+CD25− effector T cells was analyzed (n = 4–6, two independent experiments were performed); inhibition of proliferation relative to untreated responder T cell (Tresp) is shown. (F) Tregs were sorted from expanded cultures according to CD137 and CD154 expression after 6-h restimulation with anti-CD3/anti-CD28 before Treg-specific demethylated region (TSDR) demethylation was analyzed (n = 7, two independent experiments were performed). (G,H) CD25-enriched Tregs were expanded for 14 or 28 days before analysis of TSDR demethylation; correlation of TSDR demethylation with (G) CD137+CD154− expression, and (H) FoxP3 expression is shown (n = 11, three independent experiments were performed). Statistical significances were determined by (B,F) Kruskal–Wallis test, (D) Friedman test, or (G,H) linear regression analysis. (B,D,F,G,H) Each dot represents one donor, lines indicate (B,D,F) median, (E) mean ± SEM is shown.

Comparison of regulatory T cell (Treg) activation by different chimeric antigen receptor constructs. (A–B) CD25-enriched Tregs were transduced with dextran (Dex)–CAR constructs with different spacer lengths (L = 228aa, M = 119aa, S = 45aa, XS = 12aa), and (A) binding of FITC-labeled Dex was analyzed (n = 12, four independent experiments were performed). (B) CAR–Tregs were restimulated for 6 h with 2 µg/ml FITC-labeled Dex, and CD137 expression was analyzed on Dex-binding cells; CD137 expression in unstimulated samples was subtracted, and negative values were set to 0 (n = 6–9, and two to three independent experiments were performed). (C–F) CD25-enriched Tregs were transduced with Dex–CAR constructs with different costimulatory domains combined with CD3ζ or CD3ε. (C) CD137 expression was analyzed on CAR–Tregs after 6-h restimulation with bead-bound Dex, and CD137 expression in unstimulated samples was subtracted (n = 22–26, seven to eight different experiments were performed). (D–F) CAR–Tregs with different signaling domains were pooled and expanded in the presence of anti-CD3/anti-CD28 or bead-bound Dex; (A) LNGFR expression was analyzed at different time points (n = 5–7, two to three different experiments were performed). (E,F) Expression (relative to GAPDH) of the different signaling domains with CD3ζ was determined at different time points of expansion with (E) bead-bound Dex or (F) anti-CD3/anti-CD28. Expression was quantified by qPCR and normalized to relative expression on d0 (n = 7, three different experiments were performed). Statistical significances were determined by (A) Kruskal–Wallis test, (B) one-way analysis of variance, or (C) Wilcoxon signed-rank test indicating activation above background. (A–C) Each dot represents one donor, lines indicate (A,C) median, or (B) mean; (D–F) mean ± SEM is shown.