Fig. 6.

Tonic signaling CAR Tregs are functional in vitro but not in vivo. (A) Suppression of CPD-ef450-labeled PBMCs by untransduced (UT), non-tonic signaling (non-TS), or tonic signaling (TS-) CAR CPD-ef-670-labeled Tregs was determined after 4 d of coculture. n = 8 to 10 from four independent experiments (B) The Treg division index from cultures in (A) was determined. n = 6 to 7 from three independent experiments (A and B) Analysis were done using one-way ANOVA with Turkey’s multiple comparisons test. (C–F) Irradiated NSG mice were injected with PBS or 6 × 10⁶ PBMCs without or with 3 × 10⁶ UT or TS-CAR Tregs. Data from two independent experiments (C) Schematic diagram of the experiment set up. (D) Survival curve, log-rank (Mantel-Cox) test. (E) Absolute number of human CD45+ cells/µL of blood assessed weekly after adoptive transfer. One-way ANOVA with Dunnett’s multiple comparisons test (Left) and % Human CD45+ engraftment in the spleen upon experimental or humane endpoint (of live singlets) in the different groups (Right). (F) Absolute number of human Tregs/100 µL of blood assessed weekly after adoptive transfer. Tregs were gated as live human CD45⁺CD4⁺HLA-A2^neg. Two-way ANOVA showed no differences between groups, mean ± 95% CIs are shown. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.