As cancer progresses, NK cells express lower levels of activating receptors and are functionally suppressed by tumor microenvironment.

The progression of breast cancer is associated with mechanisms that inhibit the antitumor activity of immune system’s natural killer (NK) cells, researchers claim.They found that both peripheral and tumor invasive NK cells in breast cancer patients demonstrate altered phenotype and function and that invasive tumors build strong inhibitory microenvironments to escape NK cell antitumor immunity.

The research group was led by a team at Inserm UMR U891 and the Institut Paoli-Calmette in Marseille. Reporting in The Journal of Clinical Investigation, Emilie Mamessier, Ph.D., Daniel Olive, Ph.D., and colleagues, state that their findings highlight the need to develop new breast cancer therapies designed to restore NK cell cytotoxicity to limit or prevent tumor escape from antitumor immunity. Their paper is titled “Human breast cancer cells enhance self tolerance by promoting evasion from NK cell antitumor immunity.”

NK cells carry both activating and inhibitory receptors that are triggered during target-cell recognition to respectively induce a positive or negative cell signaling pathway, the researchers explain. Previous research has suggested that breast cancer progression is linked to antitumor immunity efficiency, particularly in relation to NK cells. However, the researchers continue, the precise relationships between NK cells and breast cancer progression in humans has not been studied to date.

The main activating receptors or coreceptors of NK cells include NKG2D and the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, DNAM-1, CD2, NKp80, 2B4, and NTBA. CD16 is a low-affinity receptor that, when cross-linked induces antibody-dependent cellular cytotoxicity (ADCC). Inhibitory receptors include the killer immunoglobulin receptors (KIRs), NKG2A, CD85j, and LAIRs, which are specific for different HLA-class I molecules. This means NK cells should be effective against tumor cells such as breast cancer cells, which have lost or express only low amounts of HLA-class I molecules.

To investigate the relationship between breast cancer and NK cell activity or suppression, Drs. Mamessier, Olive et al. enrolled 121 breast cancer patients at different stages of disease. The patients’ tumors were classified as nonvasive (in situ) BCs (Tis) or invasive BCs. Invasive BCs included localized (LOC), locally advanced (LA), and metastatic (M). Patients with benign mammary tumors and healthy donors were included as controls.

An initial evaluation of the expression of 22 different NK receptors on cells from fresh blood samples showed that a number of activating receptors including NKp30, NKG2D, DNAM-1, and 2B4 were downregulated in patients with invasive tumors compared with normal patients and those with Tis tumors. Conversely, the inhibitory receptors NKG2A and CD85j were upregulated in the M group compared with control groups and the Tis group. Analysis of expression data showed that the degree of receptor alteration clearly progressed with tumor stage, notably in the invasive tumors, which demonstrated clear underexpression of activating receptors and overexpression of inhibitory receptors in more advanced stages.

To assess whether receptor expression changes were associated with altered NK cell function, the team assessed the ability of peripheral blood NK (p-NK) cells to kill a target cell, effect degranulation, produce IFN-γ and TNF-α, and mediate ADCC. The results showed that effective killing and degranulation efficiency were altered in the invasive groups compared with the noninvasive groups, and IFN-γ and TNF-α levels were also reduced in the M group.

Interestingly, NK cells from the M group had lost their multifunctionality and either retained degranulating capability or IFN-γ or TNF-α production. NK cells from B and Tis patients were more efficient in mediating ADCC than NK cells from LOC and LA patients, corresponding with their different levels of CD16 expression. Unfortunately not enough p-NK cells from the M group could be obtained to evaluate their ability to mediate ADCC.

The researchers next moved on to evaluate tumor-infiltrating NK (Ti-NK) cells. To identify changes that were specifically tumor-induced rather than just tissue-specific, they compared Ti-NK cells isolated from different LOC and LA breast tumors, with paired p-NK cells and/or with tumor-free NK cells isolated from symmetric normal breast samples (Mt-NK cells).

Analyses suggested the Ti-NK cells were true activated NK cells, but they demonstrated increased expression of inhibitory receptors and decreased expression of activating receptors and cytotoxicity-related molecules when compared with p-NK cells and Mt-NK cells. This strongly suggested the activated Ti-NK cells had poor cytotoxic potential, the authors state.

Further analysis of the expression profiles of the different NK cells showed that while there was a similarity between the profiles of Mt-NK cells and p-NK cells, the Ti-NK cell profiles were heterogeneous. The exception to this was the profile of cells from a group of patients with poor prognosis. Ti-NK cells from these patients showed underexpression of nearly all NK cell receptors.

Ti-NK marker expression was further categorized into three groups according to type of marker. Group I comprised cytotoxic-related effectors, group II comprised receptors involved in NK cell activation, and group III was categorized as molecules related to NK cell maturation markers. When grouped in this way it became evident that downregulation of group I molecules was observed in all Ti-NK cells compared with Mt-NK and p-NK cells, suggesting an alteration induced by the tumor. 

The next test was to assess whether compromised Ti-NK cells were still capable of carrying out a helper function. CD107 functional assays comparing paired Ti-NK and p-NK cells showed that Ti-NK cells were activated more slowly, had a decreased degranulation potential, displayed strong alterations of IFN-γ and TNF-α production, and were unable to mediate ADCC.

Having confirmed the compromised state of NK cells in cancer, the team turned to look at the expression of NK cell ligands on breast tumor cells. They found that breast tumors expressed heterogeneous levels of ligands for NK cell receptors, particularly with respect to HLA ligands and NKp30-ligand. Breast cancer cells also frequently expressed high levels of DHAM-1-L and NKG2D-L.

“Most importantly,” the team continues, “we found deregulated transcriptional expression of NK cell ligands in a large set of breast cancers compared with healthy mammary tissues previously profiled using DNA microarrays, suggesting that an editing process was indeed involved.”

The researchers went on to show that the tumor environment was at least in part responsible for the NK cell changes. Exposure of normal NK cells to tumor breast tumor supernatants induced changes in NK cell receptors that mirrored those observed on Ti-NK cells.

Moreover, when p-NK cells were exposed to the supernatants and then used in cytotoxic assays, there was a marked suppression of all NK cell functions, particularly IFN-γ secretion, compared with nonexposed p-NK cells. “These results suggest that soluble factors released by tumors (epithelial or stromal components) affect both NK cell phenotype and functions,” they state.

Analysing tumor supernatant levels of soluble factors known to be increased in other cancers and to alter lymphocyte functions demonstrated a positive correlation with NKG2A expression but a negative correlation with molecules related to NK cell cytotoxicity, the researchers state. The highest negative correlations were obtained with TGF-β1 and PGE2, while TGF-β1 also displayed the highest correlation with the Nottingham Prognostic Index (NPI), showing that more aggressive breast tumors contain more TGF-β1-producing cells. In fact, blocking TGF-β1 in tumor supernatants partially restored NK cell functionality by about 20%.

As a final test to show that NK cell alterations are tumor induced, the researchers looked at the phenotypes of p-NK cells from former invasive breast cancer patients who had undergone surgery more than five years ago and had not relapsed since. The NK cells from these ex-patients expressed similar levels of Kp30, CD16, NKG2D, and NKG2A receptors as individuals with benign breast tumors. These receptors were respectively up- and downregulated compared with matched NK cells isolated from the breast cancer groups, “indicating that a normal NK cell phenotype was restored after tumor removal.”

The overall results indicate that “invasive breast tumors induce self tolerance in NK cells, resulting in attenuated malignant cell immunogenicity and the creation of a multifaceted immunosuppressive microenvironment that blunts NK cells’ cytotoxicity and prevents their final maturation process,” the authors conclude.

The data suggests that at least four mechanisms are associated with the ability of invasive breast cancers to escape from NK cell antitumor immunity: the modulation of NK surface receptors associated with invasive tumor characteristics and a poor prognosis; changes in the protein and mRNA profiles of NK cell ligands on breast cancer cells; the reversitibility of Ti-NK cell alteration, which suggests that the tumor induces its own tolerance to NK cell antitumor activity; and the impact of the tumor microenvironment on the final maturation of NK cells in tissues.

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