Persistence, phenotype, and function of TCR-T after infusion. (A) HA-1 TCR-T by MFC in a representative patient 18 days after infusion indicating CD34 tag and HA-1 TCR dextramer staining. (B) Peripheral blood CD3⁺CD34⁺ HA-1 TCR-T (cells per μL) over time across all patients (n = 9) at dose level (DL) 1 (purple), DL2 (blue), and DL3 (green). Note: Infusion 3.3 was delivered at the time of ∼80% BM blasts, and the massive and rapidly increasing tumor burden likely induced T-cell death. (C) TCR-T phenotype by MFC on gated CD34⁺ cells for CD4 and CD8 infusion products and recipient blood. (D) Activation/inhibition markers on gated CD34⁺ cells and bulk CD4⁺ and CD8⁺ T cells on days 11 (purple) and 18 (green) after infusion. Error bars show mean and standard deviation. Statistics determined by paired t test (2-sided P value ∗P < .05, ∗∗P < .01; ∗∗∗∗P < .0001). (E-F) Lysis in a chromium release assay of HA-1 peptide–pulsed (1nM) T2 cells (E) or HA-1⁺ primary AML (F) at an effector:target ratio of 20:1 by HA-1 TCR-T recovered from recipient blood after infusion. CD4 (light blue bars) and CD8 (dark blue bars) TCR-T were sorted from patient blood at approximately day 32 after infusion and expanded with anti-CD3 monoclonal antibody and IL-2 and sorted by MFC before the functional assay. The patient and infusion numbers are indicated by 1.1, 1.2, etc. Absence of a bar indicates that data were not available (eg, no CD8⁺ HA-1 TCR-T were successfully isolated and expanded from patient 3).