T-cell characterization of in vivo–expanded CAR19 and CAR19–TIM-3–Fc T cells by spectral flow cytometry. (A) Cartoon of the experimental design for the identification of overexpanded T-cell subpopulations by spectral flow cytometry in mice treated with CAR19 and CAR19–TIM-3–Fc T cells. (B) UMAP visualization shows a total of 1 917 663 T cells, including both transduced (GFP⁺) and nontransduced (GFP^–), identified in BM and spleen from all mice (n = 10) treated with CAR19–TIM-3–Fc T cells (n = 5) and CAR19 T cells (n = 5). Color-coded UMAPs identifying major T-cell subsets, innate T cells, and functional and maturation-associated T-cell subsets. (C) Volcano plots identifying T-cell subpopulations differentially expanded in vivo between CAR19TIM3Fc and CAR19 groups in spleen (left) and BM (right) for transduced (GFP⁺) and nontransduced (GFP^–) T cells. T-cell populations significantly expanded in vivo in both spleens and BM from mice treated with CAR19–TIM-3–Fc T cells are represented in bold. T cells used in in vivo experiments across figures are from 2 different donors. iNKT, invariant natural killer T cells; TCR, T-cell receptor.; TFH, T-follicular helper cells; Th, T-helper cells; Treg, regulatory T cells.