Fig. 4: Tumor-associated CD39⁺PD-1⁺CD8⁺ T cells prevent metastatic outgrowth.

a Experimental design. 4T07-mCh cells (10⁵) were injected into the mammary fat pad of female BALB/c mice. The breast tumor was resected on d 20 and histological analysis of lungs was performed on d 32. Mice received an i.p. injection with 250 µg anti-PD-1 or isotype control on d 15, 18, 23, 26, and 29. b Enumeration of disseminated 4T07 cells by quantitative pathology (right panel). Each symbol represents an individual mouse. Isotype, n = 6; anti-PD-1, n = 7, *p = 0.0152, (two-tailed Student’s t-test with Welch’s correction). The bar represents the mean ± SD. c Gating used for sorting of CD44⁺CD39⁺PD-1⁺CD8⁺ and the rest of the CD44⁺CD8⁺ population (termed other CD8⁺ T cells) from established 4T07 orthotopic breast tumors. d Experimental design. Two-hundred-thousand sorted CD44⁺CD39⁺PD-1⁺CD8⁺ or other CD44⁺CD8⁺ T cells were transferred after injection of 10⁵ 4T07-LZ cells into female BALB/c mice. All injections were given intravenously. Lung metastatic load was determined by bioluminescence 14 d later. e Bioluminescence of lungs at endpoint. f Quantification of lung metastatic load by bioluminescence, **p = 0.0058, ***p = 0.0002. Each symbol represents an individual mouse. Five mice per group. **p < 0.01, ***p < 0.001 (ANOVA with Bonferroni’s correction). The bar represents the mean ± SD. g Experimental design. 4T07-mCh cells (10⁵) were injected into the mammary fat pad of female BALB/c mice. Mice received an i.p. injection with 500 µg anti-IFNγ plus 500 µg anti-TNFα every 3rd day. Control mice received isotype control antibody. On d 20, 4T07-mCh cells were visualized in lungs by immunofluorescence. h Two representative examples showing clusters of proliferating 4T07-mCh cells in the lungs of mice treated with anti-IFNγ plus anti-TNFα. Proliferating 4T0-mCh7 cells were detected in the lungs from 4 out of 8 mice.