Effect of ECP therapy on viability, short-term cytotoxicity, component, and cytokine-producing capacity of CAR-T cells in 2 different in vitro models. (A) Proportion of CAR-T cells in the peripheral blood of patients after CAR T-cell therapy (n = 4). (B) Schematic diagram of in vitro dilution CAR T-cell therapy models. (C) Cell loss between the ND and the D models in different dilution ratios. Cell loss was calculated by the following formula: (the absolute number of CAR-T cells without ECP therapy cultured 24 hours) – (the absolute number of CAR-T cells with ECP therapy cultured 24 hours). (D) Representative dot plots of apoptotic CAR-T cells in different dilution ECP therapy models. Apoptotic cells were defined as Apotracker⁺ cells. (E) Viability of CAR-T cells shown for the ND and the D models in different dilution ratios. Nonliving cells were defined as Apotracker⁺cells and 7AAD⁺cells. (F) Representative dot plots of specific killing efficiency of CAR-T cells and unspecific killing capacity of PBMCs with or without ECP in a ratio of 1:1 to Raji tumor cells. (G) Summarized killing efficiency of CAR-T cells with or without ECP therapy in the ND and the D models. The killing efficiency was calculated by the following formula: ([initial number of tumor cells – residual number of tumor cells] × 100)/(initial number of tumor cells). (H) Absolute residual number of tumor cells after 24 hour coculture with ECP-treated CAR-T cells or ECP-treated PBMCs in the ND and the D models. (I) Contribution of unspecific killing capacity of PBMCs in the dilution model. The killing contribution of PBMCs was estimated by using the identical D model without CAR-T cells. (J) Component of CAR-T cells with respect to CD4/CD8 ratio before and after ECP treatment in the ND and the D models. (K) Influence of ECP treatment on TNF-α, IFN-γ, and multifunctional (TNF-α⁺IFN-γ⁺) cytokine production of CAR-T cells was determined after 4 hour tumor cell stimulation in an effector/tumor (E/T) ratio of 1:1 by an intracellular cytokine staining. A 2-tailed t test was performed for statistical analysis. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ND, nondilution; D, dilution