Extended Data Fig. 3: Nnmt−/− mice display increased numbers of CD8+ T cells in the tumour.
From: NNMT inhibition in cancer-associated fibroblasts restores antitumour immunity
a, Nnmt−/− mice: Exon 2 of Nnmt and flanking splicing regions were constitutively deleted using CRISPR-Cas9 gene editing technology in mouse zygotes. Knockout was verified by PCR, which showed a 475 base pair (bp) gene product due to exon 2 deletion. P = primer. b, Omental tumour weights 16 days after intraperitoneal (i.p.) injection of ID8 ovarian cancer cells into Nnmt+/+ and Nnmt−/− mice (n = 8 per group) (two-tailed unpaired Student’s t-test). c, ID8 omental tumours from (b) were formalin-fixed and H&E stained. The left panel shows a healthy omentum from a tumour-naïve female C57BL/6 mouse with a milky spot containing immune cells. d, Flow cytometry gating strategy to identify CD4+/CD8+ T cells, F4/80+ macrophages, Ly6G+ neutrophils, and Ly6Chigh monocytes and their PD-L1, C5aR and cytokine expression. e, Splenocytes from tumour-naïve Nnmt+/+ (n = 9) and Nnmt−/− (n = 7) mice were stimulated with PMA/Ionomycin and flow cytometry was performed (two-tailed unpaired Student’s t-test). f, Blood samples were collected from tumour-naïve Nnmt+/+ and Nnmt−/− mice and flow cytometry was performed (n = 6 per group) (two-tailed unpaired Student’s t-test). g, Flow cytometry of subcutaneous (s.c.) MC38 colon cancers from Nnmt+/+ and Nnmt−/− mice. CD8+ T cell cytokine production was quantified after PMA/ionomycin stimulation (n = 6 per group) (two-tailed Mann-Whitney test). h, E0771-LMB cells were administered via retro-orbital intravenous (i.v.) injection into Nnmt+/+ (n = 5) and Nnmt−/− (n = 6). After 12 days, lungs were formalin-fixed, and metastases size was assessed by H&E staining. Mean metastases size represents the average size of the five largest metastases per mouse (two-tailed unpaired Student’s t-test). i, E0771-LMB cells were administered via retro-orbital i.v. injection into Nnmt+/+ and Nnmt−/− mice (n = 9 per group). After 10 days, lungs were stimulated with PMA/Ionomycin and CD8+ T cell cytokine production was quantified using flow cytometry (two-tailed unpaired Student’s t-test). Data are shown as mean ± SEM. One experiment representative of at least two independent experiments is shown for panels f, g, and h.
