Fig. 5: The combination of rapamycin and vitamin C abates overactivation due to 4-1BB tonic signaling and improves CAR-Treg stability.

a Histograms of phosphoS6 staining on day 16 of culture (left panel, representative of seven donors from seven independent experiments) and frequency comparison between 4-1BB and 4-1BB+ rapamycin/vitamin C (p = 0.0006) (right panel). b Fold expansion of CD28 or 4-1BB CAR-Tregs, cultured with or without rapamycin/vitamin C cocktail from the second anti-CD3/CD28 bead activation on day 11 (arrow) to day 21. N = 5 donors from five independent experiments. Statistical significance was found when comparing 4-1BB CAR-Tregs cultured with or without Rapamycin/vitamin C, at day 18 (p = 0.0159) and day 21 (p = 0.0079). c Frequency of double-negative (HELIOS and FOXP3) cells on days 10, 16, and 21 of culture by flow cytometry. Significant difference was found between 4-1BB CAR-Tregs cultured with (n = 9) or without (n = 8) Rapamycin/vitamin C cocktail, at day 21 (p = 0.0499). d Percentage of 4-1BB CAR-Tregs with Treg-specific demethylated region (TSDR) demethylation on day 16 of culture with or without rapamycin and vitamin C starting on day 11, from four different donors. Blue circle, red and yellows triangle refer to CD28, 4-1 BB, and 4-1BB + rapamycin/vitamin C populations, respectively (panels a–c). Data are depicted as mean +/− SEM (panels a and c) or median +/− interquartile range (IQR) (panel b) and compared using two-tailed Mann–Whitney test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).