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. 2024 Nov 18;15:9917. doi: 10.1038/s41467-024-53996-7

Fig. 2. Both hematopoietic and non-hematopoietic expression of CD40 contribute to on-target off-tumor toxicity.

Fig. 2

a Production of IL-6 or IL-1β after co-culture of CD40 CAR-T cells with A20 or peritoneal macrophages (Mφ) for 24 h, measured in triplicate. Results are representative of three independent experiments. b–f Balb/C mice were irradiated (2.5 Gy) for lymphodepletion on day -1 and injected with 5 × 106 (b, d, f) or 1 × 106 (c, e) control T cells or CD40 CAR-T cells on day 0. IL-6 or IL-1β neutralization (b, c) and phagocyte depletion (d–f) were performed as described in “Methods”. Body weight change (b–e, n = 5) and serum levels of IL-6 (f, n = 5) were measured. Each dot in (f) represents the value of a single mouse. *: p = 0.0211 on day 5, p = 0.0187 on day 8 (c), and p = 0.0029 on day 8 (e), compared to CD40 CAR‐T group. g, h BM chimeras were established (e.g., CD40-/-»WT denotes the transfer of donor BMs from CD40-knockout mice to B6 wild-type recipients). After 8 weeks, chimeric mice were irradiated (2.5 Gy) on day -1 and injected with CD40 CAR-T cells (5 × 106) on day 0. Body weight change (g, n = 5) and survival (h, n = 10) were measured. p for WT»CD40-/- versus WT»WT (h). i Balb/C mice were irradiated (2.5 Gy) on day -1 and injected with GFP-efflux T (control-Luc-T) or CD40 CAR-efflux T cells (CD40 CAR-Luc-T) (5 or 1 × 106) on day 0. In vivo, the distribution of injected T cells was visualized by bioluminescence imaging. j Histology of lung and liver was examined by H&E staining 3 and 10 days after CAR-T (1 × 106) injection (100× magnification, scale bar: 200 μm). Measurements of body weight were halted when the mice began to die (red asterisks in b, d, g). Data in (h) are pooled from two replicate experiments. All other data are representative of at least two independent experiments. Data in a–g are presented as mean ± SEM. Statistical significance was determined by 1-way ANOVA (c, e), unpaired two-tailed t test (f), or log-rank (Mantel-Cox) test (h). p: p-value, n.s.: not significant. Source data are provided in the Source Data file.