Fig. 2: B16F10 melanoma tumours with CRISPR-Cas9 knockout of IFNγR1 are efficiently controlled by the endogenous anti-tumour response.
A–D B16-OVA WT (red) or IFNγRKO (blue) cells were engrafted subcutaneously into the flanks of C57Bl/6 WT mice and tumours were harvested after 11–16 days. A Diagram of the experimental setup. Graphics created with BioRender. Surface expression of IFNγR1 (B n[WT] = 3, n[IFNγRKO] = 6 for from two independent experiments), MHC-I H2-Db (C n[WT] = 17, n[IFNγRKO] = 10 from four independent experiments), and MHC-II I-A/I-E (D n[WT] = 17 for WT, n[IFNγRKO] = 10 from four independent experiments) expression on mCherry+ CD45− cells were analysed by flow cytometry. E–I WT and IFNγRKO (KO) tumours expressing mCherry-OVA or ZsGreen-OVA transgenes were admixed 1:1 prior to engraftment in WT (F, G), IFNγKO (H) or CD8ɑKO mice (I) mice. E Experimental design. Graphics created with BioRender. F Tumour volumes of WT, IFNγRKO, or admixed tumours taken at endpoint on days 12–14 post-engraftment from three independent experiments for WT:WT (red; n = 11) and KO:KO (blue; n = 10), and admixed WT:KO tumours (purple; n = 37). G–I Tumours were harvested and analysed by flow cytometry. Outgrowth of KO tumour cells relative to WT, expressed as percent selection of mCherry+ cells in control WT:WT (red; n = 6 (G, H), 7 (I)), KO:KO (blue; n = 7 (G, H), 8 (I)), or WT:KO (purple; n = 12 (G), 15 (H), 14 (I)) tumours at days 14–17 in WT (G), IFNγ KO (H) and CD8ɑKO (I) mice. Cells were gated on live CD45− mCherry+. J, K WT and IFNγRKO tumours expressing mCherry-OVA or ZsGreen-OVA transgenes were admixed 1:1 in vitro and treated with 10 ng/ml IFNγ when indicated. Ki67 staining (n = 16) (J) and the ratio between ZsGreen and mCherry (KO/WT) (n[Ctrl] = 3; n[IFNγ] = 17) (K) were assessed by flow cytometry after 2 days. Data are from three independent experiments, each n corresponds to a well (sample). L WT and IFNγRKO tumours expressing mCherry-OVA or ZsGreen-OVA transgenes were admixed 1:1 in vitro and treated with 10 ng/ml IFNγ for 10 h. Activated OTI T cells were added to tumour cells for 5 h (OTI:tumours = 2:1). The percentage of apoptotic cells was quantified by flow cytometry using Annexin V staining (n = 17). Data are from two independent experiments. M WT and IFNγRKO tumours expressing mCherry-OVA or ZsGreen-OVA transgenes were admixed 1:1 prior to engraftment in WT or CD8ɑKO mice. Tumour volumes of admixed tumours from WT (n[WT:WT] = 9, n[KO:KO] = 7, n[admix] = 13) or CD8ɑKO (n[WT:WT] = 7, n[KO:KO] = 8, n[admix] = 14) mice was measured on day 12/13 post-engraftment. Data are from two independent experiments (N) WT and IFNγRKO (KO) tumours expressing mCherry-OVA or ZsGreen-OVA transgenes were admixed 1:1 (WT:WT = red; KO:KO = blue, WT:KO = purple) prior to engraftment in WT mice and harvested at days 12–14 post-engraftment. Infiltration of lymphocyte populations as a percent of total CD45+ cells from admixed tumours in WT mice (n[WT:WT] = 11, n[KO:KO] = 10, n[admix] = 18) was analysed by flow cytometry. Each n is a tumour. Data are pooled from two or more independent experiments unless otherwise indicated. All data show mean ± SEM with p values by non-parametric two-sided Mann–Whitney t tests for comparisons between two groups, two-sided paired t tests for paired values, Kruskal–Wallis tests between three groups with multiple comparisons correction using Dunn’s method, and two-way ANOVA using Šídák’s test for multiple comparisons between multiple two or more groups of data. A, E Created in BioRender. Gerard (2024) https://BioRender.com/k40f729.
