Fig. 1: Development of a CRISPR knock-in strategy to engineer armoured T cells with tumour-restricted transgene expression.

a, Schematic depicting the CRISPR knock-in approach. b, Design of the PD-1/GFP homologous repair template. c,d, Flow cytometry plots (c) and quantification of GFP expression (d) in mock or PD-1/GFP OT-I cells stimulated with anti-CD3/CD28 antibodies or the indicated tumour cell lines for 24 h or 72 h. Data are mean ± s.d. of technical duplicates to triplicates, representative of n = 3 experiments. Red asterisks in b,c denote the location of a stop codon. e,f, Mock or PD-1/GFP OT-I cells were adoptively transferred into mice bearing AT-3-ova tumours and analysed 8 days after transfer. Experimental workflow (e) and data (f) showing flow cytometry plots (left) and quantification (right) of GFP expression in OT-I cells, shown as mean ± s.e.m. from n = 7 mice per group. g–k, OT-I cells engineered to express the indicated cytokines through the Pdcd1 locus were adoptively transferred into mice bearing AT-3-ova tumours. Data are tumour growth curves (g–i); body weight of mice treated with the indicated doses (j); and serum IL-12 concentration (k) in mice treated with 5 × 10⁶ OT-I cells at day 3 after treatment. Dashed line in j indicates 20% weight loss. Data in g–k are mean ± s.e.m. from n = 18 mice per group pooled from n = 3 experiments (g) or n = 6 mice per group (IFNγ, IFNα9 and IFNβ), or n = 4 (non-treated, PD-1/IL-2) and 5 (mock) mice per group (IL-2), representative of n = 2 (IFNα9 and IFNβ) or 3 (IFNγ and IL-2) experiments (h); n = 5 mice per group (i–k). d,g, Two-way analysis of variance (ANOVA); f, two-sided paired t-test; h, two-way ANOVA (IFNγ, IFNα9 and IFNβ) and two-sided unpaired t-test (IL-2); k, one-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001. Illustrations in a and e created using BioRender: a, Chen, A., https://BioRender.com/y6lxgqk (2025); e, Chen, A., https://BioRender.com/dzawe25 (2025).

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