Fig. 1. Murine CD40 CAR-T cells show lethal on-target off-tumor toxicity in a syngeneic lymphoma model.

a Schematic diagram of the murine CD40 CAR construct. EC, extracellular; TM, transmembrane; Cyt, cytoplasmic domain. b Representative flow cytometry plot of CD40 CAR expression on mouse T cells 4 days after transduction. c Cytotoxicity of CD40 CAR-T cells against A20 cells. d IFN-γ production after co-culture of control T cells or CD40 CAR-T cells with A20 cells for 24 h, measured in triplicate. Results are representative of three independent experiments (c, d). e–g Balb/C mice were injected i.v. with A20-Luc cells (1 × 10⁶) on day 0, irradiated (2.5 Gy) for lymphodepletion on day 6, and injected i.v. with control T or CD40 CAR-T cells (5 × 10⁶) on day 7. Body weight change (e, n = 5) and survival (f, n = 10) were measured. Serum levels of IL-6 were measured three days after T cell injection (g, n = 9). Each dot represents the value of a single mouse. h–j The same experiments as in e–g were performed including the groups not injected with A20-Luc cells. Body weight change (h, n = 5), survival (i, n = 5), and serum levels of IL-6 (j, n = 7) were measured. k–m Wild-type and CD40 knockout B6 mice were irradiated (3 Gy) on day 6 and injected i.v. with CD40 CAR-T cells (5 × 10⁶) on day 7. Body weight change (k, n = 5), survival (l, n = 10), and serum levels of IL-6 (m, n = 9) were measured. Measurements of body weight were halted when the mice began to die (red asterisks in e, h, k). Data in (e, h, i, and k) are representative of at least two independent experiments. Data in (f, g, j, l, and m) are pooled from two replicate experiments. Data in (d, e, g, h, j, k, and m) are presented as mean ± SEM. Statistical significance was determined by either the log-rank (Mantel-Cox) test (f, i, l) or the unpaired two-tailed t test (d, g, j, m). p: p-value, n.s.: not significant. Source data are provided in the Source Data file.