CD39+PD-1+CD8+ T cells mediate metastatic dormancy in breast cancer

Abstract

Some breast tumors metastasize aggressively whereas others remain dormant for years. The mechanism governing metastatic dormancy remains largely unknown. Through high-parametric single-cell mapping in mice, we identify a discrete population of CD39⁺PD-1⁺CD8⁺ T cells in primary tumors and in dormant metastasis, which is hardly found in aggressively metastasizing tumors. Using blocking antibodies, we find that dormancy depends on TNFα and IFNγ. Immunotherapy reduces the number of dormant cancer cells in the lungs. Adoptive transfer of purified CD39⁺PD-1⁺CD8⁺ T cells prevents metastatic outgrowth. In human breast cancer, the frequency of CD39⁺PD-1⁺CD8⁺ but not total CD8⁺ T cells correlates with delayed metastatic relapse after resection (disease-free survival), thus underlining the biological relevance of CD39⁺PD-1⁺CD8⁺ T cells for controlling experimental and human breast cancer. Thus, we suggest that a primary breast tumor could prime a systemic, CD39⁺PD-1⁺CD8⁺ T cell response that favors metastatic dormancy in the lungs.

Fig. 1. 4T07 but not 4T1 breast cancer cells are dormant after dissemination to the lungs.

a Diagram of the origin of 4T1 and 4T07 cell lines. b Experimental design. 4T07-mCh cells (10⁵) or PBS were injected into the mammary fat pad of female BALB/c mice. Analysis was performed 30 d later. c Quantification of disseminated 4T07-mCh cells by quantitative pathology, ****p < 0.0001. d 4T07-mCh cells (10⁵) were injected into the mammary fat pad of female BALB/c mice. Analysis was performed 35 d later. Representative section of the primary tumor (upper panels) and lung (lower panels) at the endpoint. Disseminated 4T07-mCh cells were detected as single, Ki67-negative cells in the lungs of 5 out of 5 mice. Disseminated 4T07-mCh cells are shown in red, Ki67 in green, DAPI in blue. Scale bar indicates 50 µm. e Experimental design. 4T07-mCh cells (10⁵) were injected into the mammary fat pad of female BALB/c mice. The tumor was resected 20 d after injection and analysis was performed 21 d after resection (endpoint). f Quantification of disseminated 4T07-mCh cells by quantitative pathology. ****p < 0.0001. g Representative section of the lung at the endpoint. Disseminated 4T07-mCh cells were detected as single, Ki67-negative cells in the lungs of 5 out of 5 mice. Disseminated 4T07-mCh cells are shown in red, Ki67 in green, DAPI in blue. Scale bar indicates 20 µm. Each symbol represents an individual mouse. Five mice per group. 2-tailed Student’s t-test. The bar represents the mean ± SD. Results are representative of three independent experiments.

Fig. 2. Induction of protective immunity by the primary tumor mediates metastatic dormancy.

a Experimental design. 4T1 or 4T07 cells (3 × 10⁵) were injected i.v. into female BALB/c mice. Analysis 25 d later. b Number of lung metastatic nodules. 4T1, n = 5; 4T07, n = 4. c Experimental design. 4T1 or 4T07 cells (10⁵) were injected into the mammary fat pad of female BALB/c or BALB/c Foxn1^nu/nu mice. Analysis 30 d later. Lungs of “WT Late” mice were analyzed when the 4T07 tumor size reached the size of the BALB/c Foxn1^nu/nu group (d 35). d Weight of primary tumors. Left panel with closed symbols, 4T1. Both groups n = 5. Right panel with open symbols, 4T07. Foxn1^nu/nu, n = 7; WT, n = 5; WT late, n = 4, **p = 0.006 (Foxn1^nu/nu vs. WT), **p = 0.0027 (Foxn1^nu/nu vs WT Late). e Number of lung metastatic nodules. Left panel with closed symbols, 4T1; right panel with open symbols, 4T07, **p = 0.071 for both comparisons. f Experimental design. Female BALB/c mice received 0.5 mg anti-CD8 or isotype control i.p. at days −1 and +5 relative to injection of 10⁵ 4T07-mCh cells in the mammary fat pad. Analysis 25 d later. g Lung metastatic load measured by bioluminescence, ****p < 0.0001. Anti-CD8, n = 9; isotype n = 10. h Bioluminescence images. Anti-CD8, n = 9; isotype n = 10. i Experimental design. 4T07 cells (10⁵) or PBS were injected into the mammary fat pad of female BALB/c mice on d 0. On d 11, 3 × 10⁵ 4T07-LZ cells were injected i.v.; analysis of lung metastatic load on d 25. j Quantification of lung metastatic load by bioluminescence. 4T1, n = 5; 4T07, n = 4, ***p = 0.0021. k Bioluminescence images. l 4T07 cells (10⁵) or PBS were injected into the mammary fat pad of female BALB/c mice on d 0. On day 8, mice were injected i.p. with 500 µg anti-CD8 or isotype control. On d 15, 3 × 10⁵ 4T07-LZ cells were i.v. injected and lung metastatic load was quantified on d 25. m Quantification of the lung metastatic load by bioluminescence on d 25. All groups n = 5, ****p < 0.0001. Each symbol represents an individual mouse. ns = not significant. 2-tailed Student’s t-test with Welch’s correction (b, d left panel, e left panel, g, j); 2-sided ANOVA with Bonferroni correction (d right panel, e right panel, m). The bar represents the mean ± SD. Results are representative of 3 (f, g, h) or 2 (all other) independent experiments.

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.

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.

Fig. 5. CD39⁺PD-1⁺CD8⁺ T cells have a unique transcriptional signature.

a Hierarchical clustering of significantly differentially expressed genes between sorted CD44⁺CD39⁺PD-1⁺CD8⁺ and CD44⁺CD8⁺ T cells. b Volcano plot comparing transcripts in CD44⁺CD39⁺PD-1⁺CD8⁺ T cells with those in other CD44⁺CD8⁺ T cells sorted from 4T07 breast tumors. The red symbols represent transcripts that are significantly over-expressed in CD44⁺CD39⁺PD-1⁺CD8⁺ T cells, whereas the blue symbols represent significantly under-expressed transcripts. c Heat map showing relative expression of selected transcripts in CD44⁺CD39⁺PD-1⁺CD8⁺ (three top rows) and other CD44⁺CD8⁺ T cells (three bottom rows) identified by differential gene expression analysis. d Gene set enrichment analysis comparing the transcriptional profile to the data published by Goldrath et al. (left panel) and Savas et al. (right panel).

Fig. 6. High density of intra-tumoral CD39⁺PD-1⁺CD8⁺ but not total CD8⁺ T cells correlates with disease-free survival in human breast cancer.

a Representative images of 5-color multiplex immunofluorescence on human breast cancer. Staining shows epithelial cells (PanCK + EpCAM, yellow), CD8⁺ T cells (CD8, magenta), PD-1 (green), CD39 (red) and nuclear staining (DAPI, blue). Scale bar is 50 µm. b Disease-free survival of 54 patients with high or low number of intra-tumoral CD39⁺PD-1⁺CD8⁺ T cells. c Disease-free survival of 54 patients with high or low number of intra-tumoral CD8⁺ T cells. The threshold for separating patients with high and low CD39⁺PD-1⁺CD8⁺ T cell densities was defined using ROC curve analysis. Survival was compared for patients with high and low density of cells to be compared as indicated for the individual graphs by Kaplan–Meier curves. Confidence intervals are indicated as shaded areas surrounding survival curves. Significance was calculated by log-rank test. Patient numbers at risk are displayed for each 15 years of follow-up.