(A) Schematic diagram of the first- and second-generation CARs used. Tregs expressing the indicated CARs were stained with CPDe450 and cocultured with HLA-A2⁺ K562 cells for 3 days. (B) The percentage of CAR-Tregs that divided, determined by CPDeF450 dilution (left); n = 12–20 replicates from at least 5 independent experiments; and IL-10 secretion (right), measured in culture supernatants; n = 5–7 replicates from at least 3 independent experiments. (C) CAR-Tregs were cocultured with OTII CD4⁺ T cells at varying ratios in the presence of irradiated HLA-A2⁺ splenocytes and OVA peptide. CAR-Tregs mediated suppression of the OTII CD4⁺ T cell proliferation, as determined by Ki67 expression; n = 3–6 replicates from at least 2 independent experiments. UT, untransduced. (D–H) BL/6 mice were transplanted with skin grafts from syngeneic or HLA-A2⁺ BL/6 mice and administered 1 × 10⁶ CAR-Tregs i.v. (D) Skin graft survival curves and (E) levels of anti–HLA-A2 IgG Abs from mice infused with Tregs expressing first- and second-generation CARs. (F) Persistence of CAR-Tregs measured as the percentage of Thy1.1⁺ CAR-Tregs of total CD45⁺ T cells in peripheral blood over time. (G and H) CAR-Treg expression of (G) CAR (c-Myc+) and (H) FoxP3 and FoxP3 and Helios. In vivo data pooled from 3 individual experiments with n = 3–13 mice per group. Data are reported as mean ± SEM. Data from the A2.28z, HER2.28z, and UT conditions are also shown in Figure 2, C and E, and Figure 3, A, B, and E–G. Statistical significance was determined using (B) 1-way or (C and E–H) 2-way ANOVA with a (C and E) Holm-Šidak posttest or (D) log-rank Mantel-Cox test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Co-stim, costimulatory; UT, untransduced.