Figure 1: Phosphotyrosine-dependent ShcA signalling promotes breast cancer immune suppression.

(a) Schematic diagram illustrating engagement of TK/ShcA signalling complexes to promote immune suppression. (b) MMTV/MT transgenic mice of the indicated genotypes were evaluated for mammary tumour onset. Percentage of tumour-free mice over time. Number (n) of mice analysed is indicated. (c,d) Cell lines derived from MT-driven transgenic mammary tumours that are homozygous for WT ShcA (864) or phosphotyrosine-deficient ShcA mutants (Y313F (313F-6738)) or Y239F/Y240F (2F-5372) were injected into the fourth mammary fat pad of FVB, CD8^−/− or IFNγ^−/− mice. Tumour outgrowth was measured and represented as mean tumour volume (mm³)±s.e.m. (n=8–12). (e) Immunohistochemical staining of tumour tissue (n=6–12 per group) harvested from the indicated mice using Granzyme B (GZMB)-specific antibodies. The data are represented as percentage GZMB⁺ cells relative to total cells per field±s.e.m. Representative images are shown. Scale bars, 50 μm. (f,g) Flow cytometric analysis of immune infiltrates into MT/ShcA^+/+ (864), MT/Shc^2F/2F (5372) and MT/Shc^313F/313F (6738) (f) tumour tissue or (g) matching spleen derived from FVB mice. Presence of CD8⁺ cells, CD8⁺CD69⁺ cells and CD11b⁺Gr1⁺ MDSCs (n=4–6 mice per group). The data are represented as percentage of each cell type relative to total cells analysed±s.e.m. Significance was determined by Wilcoxon’s rank-sum test for e–g, by multiple t-test with Holm–Sidak method for c,d (*statistically significant time points as indicated in the top left corner), and by two-tailed two sample t-test for a,b.