Elucidation of mechanism of clinical cardiac toxicity. (A) Analysis for expression of the correctly paired MAGE-A3–engineered TCR (MAGE dextramer staining, y-axis) of input (untransduced) T cells, transduced T-cell product, and T cells recovered from PBMCs in both patients at time of death. The x-axis shows staining for specific TCR-β chain (Vβ5.1) used by the engineered MAGE-A3 TCRs. Numbers shown in each quadrant indicate percentages of the gated CD3⁺ cells. Cell populations that stain only for Vβ5.1 but not dextramer are the sum of (1) endogenous TCR that uses that β chain (ie, population shown in input T cells) and (2) mispaired MAGE-A3–engineered TCR. No expansion of T cells with mispaired TCRs was detected after infusion in the patients. (B) A sample of the T-cell product infused into Case 2, along with fresh MAGE-A3^a3a–transduced T cells and untransduced T cells, was tested for IFN-γ production by ELISPOT when cocultured with a large panel of HLA-A1⁺ cell lines, including one that was MAGE-A3⁺ (EJM) as a positive control. Bars indicate mean ± standard error of the mean (SEM) of 3 replicates. (C) Activation and cytokine production of MAGE-engineered T cells incubated in vitro with HLA-A*01⁺, titin-positive, MAGE-A3⁻ beating cardiac myocyte cells derived from iPSC-CM or iCells. The EJM plasmacytoma cell line expresses HLA-A*01 and MAGE-A3 (positive control), and the colo205 cancer cell line expresses HLA-A*01 but not MAGE-A3 or titin (negative control). Controls for the effector cells are nontransduced (ntd) T cells, or no T cells (targets only). Bars indicate mean ± SEM of 3 replicates. *P < .0001 by Student t test.