Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity

Abstract

NK cells are a major component of the antitumor immune response and are involved in controlling tumor progression and metastases in animal models. Here, we show that dysfunction of these cells accompanies human breast tumor progression. We characterized human peripheral blood NK (p-NK) cells and malignant mammary tumor-infiltrating NK (Ti-NK) cells from patients with noninvasive and invasive breast cancers. NK cells isolated from the peripheral blood of healthy donors and normal breast tissue were used as controls. With disease progression, we found that expression of activating NK cell receptors (such as NKp30, NKG2D, DNAM-1, and CD16) decreased while expression of inhibitory receptors (such as NKG2A) increased and that this correlated with decreased NK cell function, most notably cytotoxicity. Importantly, Ti-NK cells had more pronounced impairment of their cytotoxic potential than p-NK cells. We also identified several stroma-derived factors, including TGF-β1, involved in tumor-induced reduction of normal NK cell function. Our data therefore show that breast tumor progression involves NK cell dysfunction and that breast tumors model their environment to evade NK cell antitumor immunity. This highlights the importance of developing future therapies able to restore NK cell cytotoxicity to limit/prevent tumor escape from antitumor immunity.

Figure 1. Phenotype of p-NK cell receptors in patients with different stages of BC at diagnosis and controls.

(A–H) Receptors were significantly altered among the different control groups and BC patients. The individual mean fluorescence intensities (MFI) (unimodal expression) or percentages of cells positive for given markers (bimodal expression) were graphed for the following receptors: (A) NKp30 (MFI); (B) NKG2D (MFI); (C) 2B4 (MFI); (D) DNAM-1 (MFI); (E) CD16 (MFI); (F) NKp46 (MFI); (G) NKG2A (percentage of cells positive for NKG2A); and (H) CD85j (MFI). For each scattered plot, the horizontal bar represents the mean value. (I) Summary of the median expression of the 22 NK cell receptors tested in the peripheral blood of controls and BC patients. MFI or percentage of positive cell median values were submitted to a hierarchical clustering program to obtain a global view of receptor expression in the different groups. HD (n = 22), B (n = 19), Tis (n = 7), LOC (n = 55), LA (n = 26), M (n = 32). Statistical analyses were done using nonparametric unpaired Mann-Whitney U test and Kruskal-Wallis (KW) ANOVA. *P < 0.05; **P ≤ 0.005; ***P ≤ 0.0005.

Figure 2. p-NK cell functions are altered in invasive BC patients.

p-NK cells isolated from the different groups of patients were exposed to K562 cells in a direct cytotoxic assay. (A) Effective killing of K562 cells. (B) Percentage of NK cells positive for CD107. (C) Percentage of NK cells positive for IFN-γ. (D) Percentage of NK cells positive for TNF-α. (E) The multipotentiality of p-NK cells was determined from the number of functions (degranulation as measured by CD107 expression, production of IFN-γ and/or TNF-α) that each p-NK cell was able to simultaneously accomplish against K562 target cells. The E/T ratio was 1:1 in cytotoxic experiments performed against K562 cells. (F) ADCC efficiency was measured against the SK-BR-3 BC cell line preincubated without or with increasing therapeutic doses (D1 to D7) of trastuzumab. Activation of NK cells was measured by the expression of CD107. The E/T ratio was 2:1. The numbers of included patients per group were the following: B (n = 10), Tis (n = 7), LOC (n = 16), LA (n = 16), M (n = 12, except for ADCC experiments where we did not obtain enough cells to perform the test). The statistical differences between groups were established using nonparametric Mann-Whitney U test. *P < 0.05; **P ≤ 0.005. Data are represented as mean ± SEM.

Figure 3. NK cells infiltrating breast tumors (Ti-NK cells) compared with healthy mammary tissue (Mt-NK cells).

(A) Immunohistochemistry staining of Ti-NK cells on a paraffin section of a breast tumor. NK cells were identified with an anti-CD56 antibody (brown staining). Other lymphocyte populations, mostly T cells, displayed condensate dark blue nucleus, while tumor cells are distinguishable by their larger size. (B) FACS analysis of the percentage of CD45⁺CD56⁺CD3^– cells found in tumor and paired healthy mammary tissue. (C) Absolute number of CD45⁺CD56⁺CD3^– cells per million cells found in tumor compared with healthy mammary tissue. (D) Percentage of CD56^Bright and CD56^Dim NK cells in the peripheral blood, tumor, and healthy mammary tissue of BC patients.

Figure 4. Phenotype of NK cells infiltrating healthy mammary tissue (Mt-NK cells), tumors (Ti-NK cells), and comparison with peripheral blood (p-NK cells) profile.

(A) Monoparametric histograms of the most important NK cell cytotoxic receptors in paired compartments, respectively, Mt-NK cells and Ti-NK cells. (B) Monoparametric histograms of NK cell receptors involved in NK cell maturation and/or cytotoxicity in paired compartments, respectively, Mt-NK cells and Ti-NK cells. (C) Hierarchical cluster representation of NK cell receptors expressed on Ti-NK, Mt-NK, and p-NK. The phenotypes of 24 Ti-NK cells, 4 paired Mt-NK, and 11 paired p-NK cells were submitted to TMEV, and data were normalized by row; then the hierarchical clustering was applied to both NK cell markers and patient samples. Markers are represented horizontally while each patient is graphed vertically. Patients’ NPI is shown below the clustering. The following color code was used: green, NPI < 3.4 (good-to-excellent prognosis); yellow, 3.4 < NPI < 5.4 (moderate prognosis); blue, NPI > 5.4 (poor prognosis). (D) Result of the contingency data for the group II metamarker. The Fisher exact test was significant (P = 0.013), and the strength of association was as follows: relative risk: 0.1477, 95% CI: 0.02170 to 1.006; odds ratio, 0.0625; 95% CI: 0.006019 to 0.6490.

Figure 5. Functionality of NK cells infiltrating tumors (Ti-NK cells) compared with paired p-NK cells.

(A) Isolated NK cells from malignant tissue (red) or peripheral blood (black) from paired sampled were used in direct cytotoxic assays on 5 paired samples from BC patients. (B) Dot plot representation of CD69, CD107, IFN-γ, and TNF-α expression in NK cells after incubation with K652 cells, according to a 1:1 E/T ratio. Isolated NK cells were incubated overnight in medium complemented with suboptimal concentrations of IL-2 and IL-15 before incubation with K562 cells for 4 hours. (C) Dot plot representation of CD69, CD107, INF-γ, and TNF-α expression in p-NK versus Ti-NK cells after exposure to SK-BR-3 cells in the presence of trastuzumab. The E/T ratio was of 2:1. These results were obtained on paired samples (n = 3).

Figure 6. Ligands of NK cell receptors are expressed by breast tumor cells.

(A) Epithelial cells isolated from malignant mammary tissue were phenotyped for the ligands of the main altered NK cell receptors. 2 examples representative of each ligand expression are illustrated here. Horizontal bars indicate the percentage of epithelial cells positive for the marker of interest (solid line, gray histogram) in comparison with the respective control isotype on the same population (dashed line, white histogram). (B) Summary of MFI obtained for each of the NK cells ligands in 5 independent experiments. Data are represented as mean ± SEM.

Figure 7. Impact of breast tumor stroma on p-NK cells.

(A) p-NK cells were cultured for 48 hours in complete medium, dissociation supernatant from tumor-free samples (H, n = 4), or dissociation supernatant from malignant samples (T, n = 12). The main NK cell receptors were then phenotyped. The resulting expressions were submitted to TMEV. The significance of the observed variation was measured with a Kruskal-Wallis (KW) test. (B) Alterations of NK cell functions were measured from 10 different tumor supernatants. CD69 expression, CD107 degranulation, IFN-γ, and TNF-α synthesis and percentage of absolute dead K562 cells were measured from p-NK cells cultured in complete medium (white bars) and p-NK cells exposed to tumor supernatant (gray bars). (C) The correlation matrix of the R coefficients was obtained with a Spearman’s test between the quantitative levels of tissue-associated soluble factors and paired NK cell receptors expression. (D) Correlation between TGF-β1 levels found in breast tumor supernatants and the respective NPI. (E) Partial restoration of NK cell functions after preincubation of tumor supernatants (n = 10) with blocking TGF-β1 antagonist antibody. Variations were evaluated with a nonparametric Mann-Whitney U test. *P < 0.05; **P ≤ 0.005; ***P ≤ 0.0005. Data are represented as mean ± SEM.

Figure 8. Reversibility of p-NK cell phenotype in patients in remission.

The phenotypes of NKp30, CD16, NKG2D, and NKG2A receptors were restored in former BC patients compared with matched (age, group, and TNM classification) BC patients at diagnosis. BC (n = 7); B (n = 12); Ex-BC (n = 7). Data are represented as mean ± SEM.