Fig 1. Establishment of an in vitro CRS model using a K562/CD19 cell, 19-28z CAR T-cell, and CD14⁺ peripheral blood mononuclear cell (PBMC) co-culture system.

A) Construction of the 19-28z chimeric antigen receptor (CAR) using the anti-CD19 scFv (clone FMC63), the hinge, transmembrane, and cytoplasmic domains of CD28, and the cytoplasmic domain of the CD3-ζ chain. B) CD4 and CD8 expression in CAR T cells. Following 14 days of in vitro expansion, the percentages of CD4⁺ and CD8⁺ CAR T cells were determined by flow cytometry. C) CD19 expression in target cells (NALM-6 cells, and CD19-transduced K562 cells), determined by flow cytometry. D) Cytotoxicity of CAR T cells on NALM-6 cells and K562/CD19 cells. Target cells (NALM-6/fLucEGFP, K562/fLucEGFP, and K562/CD19/fLucEGFP) were co-cultured with CAR T cells (E/T = 0–10), and the numbers of viable cells were determined using the luciferase assay. As a control, peripheral blood T cells were cultured for 14 days in vitro. E) CRS co-culture model. K562/CD19 cells (3 × 10³), 19-28z CAR T cells (1.5 × 10⁴), and CD14⁺ PBMCs (1.5 × 10⁴) were co-cultured in a 96-well plate for up to 72 h. F) Cytotoxicity of CD4⁺, CD8⁺, and unfractionated CAR T cells. K562/CD19/fLucEGFP cells (3 × 10³) and CD4⁺, CD8⁺, or unfractionated 19-28z CAR T cells (0–3 × 10⁴) were co-cultured in a 96-well plate in the absence or presence of CD14⁺ PBMCs (1.5 × 10⁴). After 72 h of co-culture, viable cells were quantified by the luciferase assay. Values were normalized to the control well containing only K562/CD19/fLucEGFP. The error bars represent standard deviations (SDs) from three independent experiments. Difference in dose-dependent changes between two groups were assessed using a two-factor linear mixed-effect model with interaction terms. In this model, the dose variable was entered as a continuous variable after log-transformation (to the base 10), in which the zero dose was replaced by the log (minimal dose) - 1. (*p < 0.05, **p < 0.01. ***p < 0.001).