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. 2024 Aug 4;15:6613. doi: 10.1038/s41467-024-50881-1

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 3 — a Flow cytometry charts and (b) quantification of IFNγ (spleens: Arf6^WT n = 8, Arf6^f/f n = 16; tumours: ARF6^WT n = 10, ARF6^f/f n = 17), granzyme B (GmzB) (spleens: Arf6^WT n = 8, Arf6^f/f n = 15; tumours: ARF6^WT n = 10, ARF6^f/f n = 16) and PD-1(spleens: Arf6^WT n = 10, Arf6^f/f n = 15; tumours: ARF6^WT n = 10, ARF6^f/f n = 16 in CD8+ T cells from spleens and tumours of mice bearing ARF6^WT or ARF6^f/f tumours upon T cell reactivation), two-tailed Mann Whitney test. c Flow charts and (d) quantification of the CD4+ FoxP3+ regulatory T cell fraction from spleens and tumours (spleens: Arf6^WT n = 15, Arf6^f/f n = 9; tumours: ARF6^WT n = 15, ARF6^f/f n = 19) of mice bearing ARF6^WT or ARF6^f/f tumours and Treg/CD8 ratio (tumours: ARF6^WT n = 15, ARF6^f/f n = 19), two-tailed Mann Whitney test. e Uniform manifold approximation and projection (UMAP) (scRNA-seq) showing intratumoural CD45+ cells (n = 19,367 cells from ARF6^WT, n = 28,003 cells from ARF6^f/f tumours, n = 3 tumours of each genotype) and histogram showing mean % of immune cell types among total CD45+ cells. Unpaired t-tests Two-stage step-up (Benjamini, Krieger, and Yekutieli). f UMAP showing projection of T cell clusters onto ProjecTILs reference and histogram showing mean % of CD8 T cell subtypes among total CD8+T cells. (n = 1402 cells from ARF6^WT, n = 1661 cells from ARF6^f/f, n = 3 tumours of each genotype) Likelihood ratio test, mixed effects model with fixed group effect and random effect for samples within groups. Two-way ANOVA with Sidak’s multiple comparisons tests (g) UMAP showing tumour infiltrating myeloid cells and stacked histogram showing proportion of each cell type (%) (n = 12,295 cells from ARF6^WT, n = 13,807 cells from ARF6^f/f tumours, n = 3 tumours of each genotype). Two-sided chi-square test of homogeneity. h Tumour-free survival (Two-sided Kaplan–Meier log-rank test) and rate of tumour growth (untreated n = 50, isotype n = 7, anti-CD8 n = 12), Two-tailed Welch’s t-test, Arf6^f/f mice with or without CD8 T cell depletion. Antibody treatments were initiated when mice were 5 weeks old and continued for 8 weeks. See also Supplementary Figs. 2 and 3. b, d, h Solid line within data points = mean. e–f Solid line within data points = mean. Error bars = SD. Source data are provided as a Source Data file.