Integration of anti-CD19 CAR T cells early after PTCy (day +5). (A) Donor (B6C3F1), E2a-PBX leukemia (B6), and recipient (B6D2F1) cells have differing MHC haplotypes, allowing their delineation based on expression of H2k^b, H2k^k, and H2k^d. (B) Treatment schema. Mice in treatment groups not receiving leukemia, CAR T cells, NT T cells, or PTCy received vehicle on the indicated day (D). (C) Histopathologic scores of GVHD severity at day +21 post transplant. Statistical testing was performed with one-way ANOVA followed by the Holm-Sidak post hoc test on natural logarithmically transformed data using the Allo BM/Splen, PTCy, CAR D5 group as the reference. ∗P < .05. (D-E) Survival, weights, and clinical scores for (D) syngeneic (B6C3F1→B6C3F1) or (E) allogeneic (B6C3F1→B6D2F1) HCT. (F-G) Total numbers at day +21 of (F) CD4⁺ or (G) CD8⁺ T cells that were CAR⁺ as measured by Protein-L staining and gated on viable donor T-cell subsets. (H) Percentages at day +21 of viable cells that were E2a-PBX leukemia. (I-J) All mice in D-E surviving to day +120 were assessed for the frequency of (I) CD4⁺ and (J) CD8⁺ CAR T cells, (K) E2a-PBX leukemia, and (L) donor (H2k^k+H2k^b+) CD19⁺ B cells, revealing persistence of CAR T cells, clearance of leukemia, and persistent donor B-cell aplasia. Combined results from 4 independent experiments are shown for panels D, E, and I to L (numbers/group shown in supplemental Table 2) and 2 independent experiments (n = 3 per group per experiment) for panels C, F, G, and H. BM, bone marrow; D, day; IP, intraperitoneal; IV, intravenous; ns, not statistically significant (P ≥ .05); Splen, splenocytes; TBI, total body irradiation; TCD, T-cell–depleted.