CD19 CAR T-cell–expressing PD-1 and LAG-3 are functional and produce EVs. (A) Flow cytometry analysis of markers, known to be involved in T-cell exhaustion, expressed on CD19 CAR T cells, residual from the infusion bags. The 3 graphs indicate the percentage of PD-1⁺ cells, LAG-3⁺ cells, and TIM-3⁺ cells gated on total CD3⁺ CAR T cells (gray circles), CD4⁺ CAR T cells (blue circles), and CD8⁺ CAR T cells (blue circles). Data are presented as mean ± SEM (1-way ANOVA; CD4⁺PD-1⁺ CAR T cells vs CD8⁺PD-1⁺ CAR T cells, P = .0100; CD4⁺LAG-3⁺ CAR T cells vs CD8⁺LAG-3⁺ CAR T cells, P = .0386). (B) Cytotoxic function of total CD3⁺ CAR T cells, CD4⁺PD-1⁺ CAR T cells, CD4⁺PD-1^– CAR T cells, CD8⁺LAG-3⁺ CAR T cells, and CD8⁺LAG-3^– CAR T cells, isolated by FACS from the infusion bag, against a CD19-expressing cell line, Raji. After 24 hours of culture, the percentage of target living cells with CAR T cells (target-to-CAR T-cell ratio, 1:10) was assessed by flow cytometry. The bars represent the mean and SEM of killing of CAR T cells derived from at least 4 different donors. (Student t test; CD3⁺ CAR T cells vs CD8⁺LAG-3⁺ CAR T cells, P = .0115; CD4⁺PD-1⁺ CAR T cells vs CD8⁺LAG-3⁺ CAR T cells, P = .0160; CD4⁺PD-1^– CAR T cells vs CD8⁺LAG-3⁺ CAR T cells, P = .0137). (C) Comparison of the percentage of CD4⁺PD-1⁺ CAR T cells and CD8⁺LAG-3⁺ CAR T cells, assessed by flow cytometry. The cells analyzed were obtained from the residual bags or from the PB of patients, 14 days after CD19 CAR T-cell infusion (2-way ANOVA, not significant). (D) The protein functional analysis calculated by ingenuity pathway analysis with all identified proteins is shown for CD8⁺LAG3⁺ EVs paralleled with the CD8⁺LAG-3^– EV compartment.