Figure 3.

Immunosuppressant treatment mitigates in vivo rejection of allogeneic HLA-I⁺ CAR-T cells

(A) Five HIS mouse cohorts (1–5) were allocated into groups receiving vehicle (VEH) (n = 25), rapamycin (RPM) (n = 14), or tacrolimus (TAC) (n = 13) daily for 2 weeks. At 1 day post-treatment, 5 × 10⁶ allogeneic, TCR^KO (HLA⁺) and TCR^KOB2M^KOCIITA^KO (HLA-deficient) 4CAR-T cells were mixed and infused into HLA-mismatched mice. Mice in cohorts 3 and 4 also received an equal amount of syngeneic HIS mouse-derived 4CAR-T cells. (B) FACS plots indicate longitudinal frequency of peripheral allogeneic HLA⁺ and HLA-deficient 4CAR-T cells in VEH-, RPM-, or TAC-treated mice at 1, 7, and 13 days post-infusion. (C) Aggregate peripheral allogeneic HLA⁺ 4CAR-T cell persistence relative to HLA-deficient 4CAR-T cells from individual mice in cohorts 1–5 during the drug treatment interval. (D) Peripheral allogeneic HLA⁺ 4CAR-T cell persistence relative syngeneic HIS mouse-derived 4CAR-T cells from individual mice in cohorts 3–4 during the drug treatment interval. (E and F) Correlation between percentage change in allogeneic HLA⁺ 4CAR-T cells from 1 to 7 days post-infusion and contemporaneous trough plasma concentration of RPM in cohorts 1–4 (E) and TAC in cohorts 3–4 (F) at 7 days post-infusion. (G) Total splenic allogeneic HLA⁺ and HLA-deficient 4CAR-T cells from individual mice treated with VEH (n = 5) and TAC (n = 5) in cohort 5, 1 day post-drug withdrawal. (H and I) FACS plots indicate frequency (H) and summarized data show total (I) splenic allogeneic HLA⁺ and HLA-deficient 4CAR-T cells from individual mice treated with VEH (n = 4), RPM (n = 6), and TAC (n = 6) in cohorts 3 and 4, 30 days post-drug cessation. (J and K) Cumulative persistence of peripheral syngeneic 4CAR-T cells and allogeneic HLA⁺ 4CAR-T cells during the drug treatment interval (1–13 days post-infusion) (J) and post-drug cessation (22–42 days post-infusion) (K). For all data, symbols and sample sizes indicate biologically independent animals. Bars and lines represent mean and error bars show ±SEM. Statistical significance was calculated by Kruskall-Wallace test with Dunn’s test for multiple comparisons (C and D), Spearman correlation (E and F), Wilcoxon matched pairs signed rank test (G and I), and Wilcoxon rank-sum test (J and K). AUC, area under the curve; r, coefficient of correlation.