Figure 6. In vivo and in vitro APC suppression by first- and second-generation CAR-Tregs.

(A–D and G) For in vitro assays, CAR-Tregs were cocultured with splenic HLA-A2⁺ CD11c⁺ DCs at a ratio of 1:2 or 1:5 DCs to Tregs for 1 or 2 days. (A) Schematic of in vitro DC suppression assay. (B) Representative histograms of at least 5 independent experiments showing CD80 expression on CD11c⁺ DCs after 2-days of culture with the indicated types of Tregs. (C) Expression of CD80 (left) and CD86 (right) in HLA-A2⁺ CD11c⁺ DCs relative to DCs cultured with untransduced Tregs (dotted line); n = 8–15 replicates from at least 5 independent experiments. (D) In vitro DC suppression assays performed with or without 10 μg/mL CTLA-4–Ig; n = 3 replicates from 2 independent experiments. (E and F) For in vivo assays, BL/6 mice were transplanted with skin grafts from HLA-A2⁺ BL/6 mice and treated or not with 1 × 10⁶ CAR-Tregs. dLN and spleen tissues were collected at day 7 after CAR-Treg infusion; n = 6–7 mice per group from 2 independent experiments. (E) Expression of CD80, CD86, and MHC-II in DCs from dLNs of mice treated with CAR-Tregs relative to untreated mice (dotted lines; average of 3–4 untreated mice per each experiment). (F) Expression of CD80, CD86, and MHC-II in DCs from spleens of mice treated with CAR-Tregs relative to untreated mice (dotted lines; average of 3–4 untreated mice per each experiment). (G) Expression of CD80 at day 1 (left), CD86 at day 2 (center), and MHC-II at day 1 (right) on HLA-A2⁺CD11c⁺ DCs treated with different types of CAR-Tregs in vitro; n = 6–16 replicates from 4 (CD80/MHC-II) or 5 (CD86) independent experiments. (C, D, and G) Data are shown relative to DCs cultured with untransduced Tregs, which were normalized to 100% (dotted lines). (E and F) Data are shown relative to the expression of CD80, CD86, and MHC-II in DCs from nontreated mice, which were normalized to 100% (dotted lines). Data are reported as mean ± SEM. Statistical significance was determined using (C, D, and G) 1-way ANOVA with a Holm-Šidak posttest and (E and F) Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.