Extended Data Fig. 2: Validation of CRISPR KI strategy with PD-1/TNF OT-I and murine anti-hHer2 CAR T cells.
From: Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery
a-e. Assessment of PD-1/TNF OT-I cells in vitro (a-c) and in vivo (d-e). a. TNF concentration in supernatants of Mock, PD-1 KO or PD-1/TNF OT-I cells stimulated with anti-CD3/28 antibodies or the indicated tumor cell lines for 24 or 72 h. b. TNF-conditioned supernatants from a 24 and 72 h coculture with MC38-ova tumor cells and indicated OT-I cells were added to 51Cr-labelled parental MC38 tumor cells and incubated for 16 h before quantifying 51Cr release. c. OT-I cells were cocultured with a 1:1 mix of mCherry+ parental MC38 and GFP+ MC38-ova tumor cells and imaged using the Incucyte Live-Cell Analysis System. Tumor cells were quantified based on mCherry or GFP expression. (a-c) Data represent mean ± SD of technical triplicates, representative of n = 3 experiments. d-e. Mock, PD-1 KO or PD-1/TNF OT-I cells were adoptively transferred into mice bearing orthotopic AT-3-ova tumors. d. 8 days post transfer, tumors were harvested, processed and cocultured with fresh AT-3-ova tumor cells for 16 h prior to analysis of TNF expression in OT-I cells. Flow cytometry plots from concatenated samples of n = 6 mice/group (left) and quantification of TNF expression represented as mean ± SEM from n = 18 (Mock) and 19 (PD-1 KO, PD-1/TNF) mice/group pooled from n = 3 experiments (right). e. Body weight of mice, represented as mean ± SEM from n = 6 mice/group. f-g. Assessment of PD-1/TNF murine anti-hHer2 CAR T cells. f. TNF concentration in supernatants of Mock, PD-1/GFP or PD-1/TNF murine anti-hHer2 CAR T cells cocultured with the indicated hHer2-expressing tumor cell lines for 24 h. Data represent mean ± SD of technical triplicates, representative of n = 3 experiments. g. Mock, PD-1 KO or PD-1/TNF murine anti-hHer2 CAR T cells were adoptively transferred into mice bearing subcutaneous MC38-hHer2 tumors. Tumor growth curve represented as mean ± SEM from n = 5 (Non-treated, Mock, PD-1/TNF) and 6 (PD-1 KO) mice/group. (a-c, f-g) Two-way ANOVA. (d) One-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001. Illustrations in b and c created using BioRender: b, Chen, A., https://BioRender.com/q0g8j7b (2025); c, Chen, A., https://BioRender.com/q0g8j7b (2025).
