Fig. 3: CD39⁺PD-1⁺CD8⁺ T cells emerge in dormant breast cancer.

Female BALB/c mice were injected with 10⁵ 4T1 or 4T07 cells in the mammary fat pad and primary tumors were analyzed by flow cytometry 20 d later. a t-SNE visualization of markers after gating on single, live, CD45⁺ TCRβ⁺ CD44⁺ cells. b Upper panel: t-SNE plots of the main T cell subsets. Lower left panel: frequency of the main T cell subsets in both groups. Each symbol represents an individual mouse, *p = 0.019 (CD4⁺ Tconv), *p = 0.011 (CD8⁺ T cells). Lower right panel: Heat map with marker expression of the main T cell subsets. c t-SNE visualization of markers on CD8⁺ TCRβ⁺ cells. d Upper panel: t-SNE plots of the main CD8⁺ T cell subsets identified by FlowSOM algorithm. Lower panel: frequency of the main CD8⁺ T subsets identified by FlowSOM algorithm in both groups, *p = 0.028, **p = 0.0069. e Scaled histograms of arcsinh-transformed marker expression showing the relative marker distribution of the population identified by CellCnn (red) among all CD8⁺ T cells (blue). KS indicates the Kolmogorov–Smirnov two-sample test between the whole-cell population and the selected cell subsets. f Heat map with marker expression of the main CD8⁺ T cell subsets identified by FlowSOM algorithm. g Representative FACS plot of 4T1 and 4T07 primary tumors after gating on CD8⁺ TCRβ⁺. h Frequency of the population of total CD8⁺ identified by CellCnn in both models, ****p < 0.0001. i Number of CD39⁺PD-1⁺CD8⁺ T cells in the lungs, *p = 0.0317. j Representative CD103-staining of live CD39⁺PD-1⁺CD8⁺ T cells from the lungs. Each symbol represents an individual mouse. n = 4 per group for all panels, except panel h (n = 3) and i (n = 5). Two-tailed Student’s t-test with Welch’s correction. The bar represents the mean ± SD.