Non-HLA-specific activating receptors include the natural cytotoxicity receptors (NCRs) NKp46, NKp30 and CD16 (FcγRIII), as well as Natural Killer Group 2D (NKG2D) and DNAX accessory molecule-1 (DNAM-1). In addition, certain killer cell immunoglobulin-like receptors (KIRs) and Natural Killer Group 2, member C (NKG2C) serve as HLA-specific activating receptors that promote NK cell effector functions upon engagement. In BC, these activating receptors are frequently down-regulated, specifically in tumour-infiltrating NK cells (Ti-NKs). Expression of NKG2D and NKp30 is reduced, which has been closely associated with impaired cytotoxic activity and poor immune surveillance, largely driven by immunosuppressive cues within the TME, highlighting the TME-mediated regulation of NK cell function and limiting antitumour responses in breast cancer 25,26. Furthermore, tumour-derived podocalyxin-like protein-1 (PCLP1), a CD34-family glycoprotein, has been shown to down-regulate the expression of activating receptors, leading to reduced NK cell activity and further hindering NK cell cytotoxicity in BC 27.

Counterbalancing these activating signals are several inhibitory receptors exploited by tumour cells to evade immune destruction. A key example is Natural Killer Group 2, member A (NKG2A), a C-type lectin-like receptor that forms a heterodimer with CD94 and interacts with its ligand HLA-E, a non-classical MHC class I molecule with limited polymorphism, thereby suppressing NK cell activity 28. Upon ligand engagement, immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in NKG2A recruit phosphatases such as SHP-1, which suppress downstream activation pathways and inhibit NK cell cytotoxic function 29.

In the BC TME, both NKG2A and HLA-E are commonly up-regulated. Over-expression of HLA-E is driven by tumour cells and tumour-associated subsets, including CD141⁺ conventional dendritic cells (DCs) and tumour-associated macrophages (TAMs) 30. This widespread expression enables tumour cells to engage NKG2A-expressing NK cells and deliver inhibitory signals that suppress NK cell activation, degranulation and cytokine production. Notably, Ti-NKs exhibit significantly higher levels of NKG2A compared with their circulating counterparts, suggesting that the TME promotes the up-regulation of inhibitory receptors to facilitate immune evasion 31.

MHC class I-related chain A and B (MICA/B) and UL16-binding proteins (ULBPs) are important stress-induced ligands that engage the activating receptor NKG2D on NK cells. Their expression on tumour cells typically marks them for immune-mediated elimination. However, in BC, the expression of MICA/B is often down-regulated 32. The primary down-regulation mechanism of these ligands involves microRNAs (miRNAs), particularly those from the miR-17-92 cluster. Notably, miR-20a and miR-93, which regulate MICA/B, target the 3′-untranslated region (3′-UTR) of MICA/B mRNA, leading to transcript degradation and reduced protein expression 33. This post-transcriptional suppression diminishes NK cell activation, impairs NK cell-mediated cytotoxicity and enhances tumour growth, migration and invasion, thereby facilitating BC progression and metastasis 34.

In parallel, BC cells often up-regulate inhibitory ligands that bind to NK inhibitory receptors. HLA-E, a non-classical MHC class I molecule, acts as an inhibitory ligand that binds to the inhibitory receptor NKG2A, transmitting inhibitory signals through ITIMs that suppress NK cell activation and block NK cytotoxicity 35.

Moreover, alterations in the expression of another inhibitory ligand, HLA-G, in BC have been linked to reduced survival rates and an increased capacity of tumours to evade immune surveillance. HLA-G binds both KIR2DL4 and LILRB1 (at lower levels), initiating complex signalling that suppresses NK effector functions. KIR2DL4 contains both activating and inhibitory motifs, modulating NK cell activity depending on the context of HLA-G engagement 36. The molecular mechanisms underlying HLA-G over-expression in BC remain incompletely elucidated. To investigate this, Zhang et al. (2019) 38 conducted a study using MCF-7 BC cells in which they induced epigenetic modification, specifically DNA demethylation in the HLA-G promoter region, by targeting DNMT and TET enzymes with DMOG, a non-specific 2-oxoglutarate (2-OG)-dependent dioxygenase inhibitor. This process suppressed NK cell cytotoxicity and contributed to BC progression 36.

The inhibitory receptor PD-1 is best known for modulating T-cell responses; however, it is also up-regulated on tumour-infiltrating NK cells (Ti-NKs) in breast cancer (BC) under the influence of immunosuppressive cytokines such as IL-10 and TGF-β within the tumour microenvironment (TME), and is associated with an exhausted NK phenotype 39. Interaction between PD-1 and its ligand PD-L1, which is frequently over-expressed by BC cells, tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), suppresses NK-cell cytotoxicity and cytokine secretion, allowing tumour cells to evade immune destruction 40. PD-1 expression on human NK cells has similarly been linked to immune escape in melanoma, lung, pancreatic, bladder and ovarian cancers 39.

In early-stage BC, PD-1 is often co-expressed with other checkpoints, including CTLA-4, particularly on CD16⁺CD56dim NK cells, suggesting a coordinated mechanism of immune evasion in aggressive subtypes such as HER2-positive or highly proliferative tumours 41. Pro-inflammatory mediators, notably IFN-γ, further augment PD-L1 expression in the TME, thereby amplifying PD-1-mediated suppression. Humanised mouse models (hu-CB-BRGS) of triple-negative BC (TNBC) demonstrate that PD-1 blockade can restore NK-cell function and promote tumour regression 42.

CTLA-4 is an immune checkpoint receptor mainly expressed on activated T cells and regulatory T cells (Tregs), where it inhibits activation by competing with CD28 for CD80/CD86 binding 43. Although CTLA-4 expression on NK cells is controversial, some studies indicate that IL-2 stimulation can induce its expression concomitant with IFN-γ production 43. CTLA-4 expression on CD8⁺ tumour-infiltrating lymphocytes in BC has been correlated with favourable outcomes in luminal A and B subtypes 44. Intriguingly, higher CTLA-4 levels are detected more frequently in younger patients and in advanced-stage disease, implying a complex role in anti-tumour immunity 41.

TIM-3 is an inhibitory receptor expressed on multiple immune subsets, including NK cells 45. It is considered a marker of NK-cell maturation; the cytotoxic CD56dimCD16⁺ subset displays higher TIM-3 levels than the immature CD56bright population, typically acquired together with killer-cell immunoglobulin-like receptors (KIRs) 43. Under chronic stimulation in the TME, persistent TIM-3 up-regulation on Ti-NKs is associated with diminished cytotoxicity, reduced IFN-γ production and, in some contexts, apoptosis, particularly in BC 46.

TIM-3 mediates inhibition through several ligands—most prominently Galectin-9 (Gal-9), but also phosphatidylserine (PtdSer), high-mobility-group box-1 (HMGB1) and carcinoembryonic-antigen-related cell-adhesion molecule-1 (CEACAM-1)—all of which trigger immunosuppressive cascades that disrupt NK-cell activation 47,48. Pro-inflammatory cytokines such as IL-2, IL-15 and IL-18, abundant in inflamed or tumourigenic tissues, further up-regulate TIM-3, particularly on immature NK cells 49,50. In early-stage BC, elevated TIM-3 on tumour-infiltrating lymphocytes correlates with increased PD-1 and PD-L1 expression, underscoring a cooperative mechanism of immune evasion 51.

TIGIT (also known as WUCAM, Vstm3 or VSIG9) is an inhibitory receptor present on NK cells, CD8⁺ T cells and Tregs (Zhang et al., 2023). In BC, TIGIT engages its high-affinity ligand CD155 (poliovirus receptor, PVR), which is over-expressed on tumour and antigen-presenting cells in the TME 53. This interaction initiates inhibitory signalling that suppresses NK-cell activation, cytokine release and tumour lysis 46. TIGIT contains both an ITIM and an ITT-like motif; following CD155 binding, phosphorylation recruits SH2-domain phosphatases such as SHIP-1, thereby interfering with PI3K and MAPK pathways 52. The adaptor β-arrestin-2 further links phosphorylated TIGIT to SHIP-1, inhibits TRAF6 auto-ubiquitination and attenuates NF-κB signalling, resulting in reduced IFN-γ production 54. Consequently, NK-cell cytotoxicity, IFN-γ/TNF-α secretion and degranulation (CD107a) are diminished.

TIGIT also promotes suppression by out-competing the activating receptor DNAM-1 (CD226) for CD155 binding 28. In patients with advanced BC, tumour cells frequently over-express CD155, whereas NK cells exhibit increased TIGIT and decreased CD226, leading to an exhausted phenotype 55. In TNBC, high TIGIT expression on CD8⁺ and CD4⁺ TILs correlates with poor immune responses, highlighting TIGIT as a promising immunotherapy target 56.

LAG-3 (CD223) is a transmembrane protein of the immunoglobulin superfamily with potent inhibitory effects on T-cell immunity. Besides activated CD4⁺/CD8⁺ T cells, LAG-3 is expressed on NK cells, Tregs, B cells, invariant NKT cells, dendritic cells and TILs 53. By binding MHC class II molecules with higher affinity than CD4, LAG-3 negatively regulates immune responses 57. Although its function in NK cells is less defined, emerging evidence suggests that chronic activation, as in cancer, may up-regulate LAG-3 and dampen NK-cell effector functions.

Alimandi et al. (1997) demonstrated that stimulation of HER2/HER3 heterodimers by neuregulin-1β (NRG1β) leads to increased expression of MICA/B via downstream activation of both PI3K/AKT and RAS/MAPK pathways in the BC cell lines T-47D and MDA-MB-453. Although increased MICA/B expression should enhance NK-cell-mediated cytotoxicity via NKG2D engagement, tumour cells circumvent recognition by releasing soluble forms of MICA/B through proteolytic shedding. HER3 is also critical to this axis, as its knock-down significantly reduces NRG1β-induced MICA/B up-regulation 32.

In addition to regulating MICA/B, the PI3K/AKT signalling axis contributes to NK-cell dysfunction associated with metastatic progression via the inactivation of glycogen synthase kinase-3β (GSK-3β). AKT phosphorylates GSK-3β at Ser9, leading to increased accumulation of reactive oxygen species (ROS) in BC cells. This subsequently reduces the phosphorylation of eIF2B at Ser535, a translation-initiation factor required for the expression of NKG2D and its ligands, including MULT-1 and RAE-1. Reduced expression of these molecules limits NK-cell recognition and cytotoxicity in BC. Conversely, studies have shown that active GSK-3β maintains higher Ser535-phosphorylated eIF2B levels and promotes the expression of NKG2D ligands, thereby enhancing NK-cell-driven tumour surveillance 59,60.

In breast cancer, NK cells display both anti-tumor activity and, in some cases, pro-tumor functions, highlighting their complex role in disease progression. CD56brightCD16+ NK cells have been shown to promote tumor progression by secreting matrix metalloproteinase-9 (MMP-9) and pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and angiogenin, thereby driving angiogenesis in breast cancer (BC) and immune suppression through the expansion of regulatory immune cells, impairment of T-cell infiltration, and induction of T-cell exhaustion 61,62. Metalloproteinases such as ADAM17 can be activated by cytokine stimulation (e.g., IL-2 and IL-15), cleaving CD16 from NK-cell surfaces. This shedding impairs antibody-dependent cellular cytotoxicity (ADCC), weakening NK-mediated tumor surveillance 63,64.

As key stromal cells within the BC TME, cancer-associated fibroblasts (CAFs) often impede NK-cell infiltration and activity 65, primarily through secretion of immunosuppressive mediators such as TGF-β, which down-regulates NK-cell receptor expression and effector functions 66. They also reduce production of cytotoxic molecules (perforin, granzyme B) and inhibit IFN-γ synthesis via the SMAD3–T-BET signalling axis. CAFs induce miR-183, which suppresses DAP12 67, and locally produce Dickkopf-related protein-1 (DKK1), a Wnt-pathway antagonist that attenuates NK activity 68,69. In addition, CAFs remodel the extracellular matrix via CXCL12/SDF-1 and MMPs, collectively creating barriers to NK-cell trafficking and cytotoxicity 70.

MDSCs within the BC TME exert potent immunosuppression through the release of IL-6, IL-10 and TGF-β, which impairs the function of immune cells, including T cells 71,72 and NK cells 73,74, and through the generation of reactive oxygen/nitrogen species (ROS/RNS) 62. This biochemical suppression is reinforced by cellular crosstalk that down-regulates key NK-cell activation receptors, such as NKG2D and CD247, an essential signalling subunit of NKp30, NKp46 and FcγRIII/CD16. Co-culture experiments have shown that MDSC–NK-cell interactions result in diminished NK-cell degranulation capacity and reduced IFN-γ secretion 75. A central pathway involved in immature MDSC-mediated suppression is the STAT3 signalling cascade, which drives NF-κB-dependent production of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme that suppresses NK-cell proliferation and cytotoxicity. Studies in murine models have shown that STAT3 inhibition restores NK-cell activation markers—including NKG2D, CD69, FasL, granzyme B, perforin and IFN-γ—and reduces tumor progression in BC 76,77.

TAMs constitute a major component of innate immune infiltrates in the solid-tumor TME and, in BC, can polarize into two principal phenotypes: pro-inflammatory, tumor-suppressive M1-like macrophages, and immunosuppressive, tumor-promoting M2-like macrophages 78. In TNBC, M2-like TAMs are linked to enhanced tumor-cell proliferation, metastatic potential and poor prognosis (59.0 % vs 40.4 %, p = 0.02) 62,78. They secrete IL-10, TGF-β, chemokines (CCL2, CCL17, CCL22), pro-angiogenic molecules such as VEGF-A, and matrix-remodelling enzymes, thereby facilitating tumor invasion, angiogenesis and metastasis 79. Similarly, epidermal growth factor receptor (EGFR) signalling supports macrophage recruitment and polarization, reinforcing immunosuppression in the TME 80,81.

TGF-β acts at multiple levels: it down-regulates activating receptors such as NKG2D and NKp30 on NK cells 82; induces metabolic exhaustion characterised by mitochondrial dysfunction and reduced glycolysis, linked to impaired mTOR signalling, especially in metastatic settings 83,84,85; and thereby weakens tumour–NK-cell interactions, limiting NK-cell activation in advanced BC. TGF-β also inhibits IFN-γ production 86 and promotes the recruitment of immunosuppressive cells, providing an effective escape mechanism for BC 6. In metastatic BC, elevated systemic and local TGF-β correlates with impaired NK-cell bioenergetics, rendering them ineffective in tumour lysis 84,87.

IL-10 is a cytokine with complex, sometimes contradictory, roles in cancer immunity. Although generally immunosuppressive and associated with poor clinical outcomes in BC 88, it also shows anti-tumour potential. IL-10 inhibits angiogenesis by down-regulating pro-angiogenic factors such as VEGF, IL-6, TNF-α and MMP-9 89. Experimental studies indicate that IL-10 can enhance NK-cell activation and cytolytic activity in a dose-dependent manner, thereby promoting target-cell destruction. Conversely, systemic IL-10 administration in wild-type mice suppresses antigen-specific immune responses, blunting the broader adaptive response 90. This includes suppression of key cytokines such as IL-2 and IFN-γ, both of which are essential for NK-cell functional activation 91.

IL-15 generally promotes NK-cell-mediated cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC) and cytokine production. It up-regulates activating receptors (e.g., NKG2D, NKp46) and cytolytic molecules (perforin, granzyme B, TRAIL) within the BC tumour microenvironment 92,93. Through the IL-2Rβ (CD122) and γc receptor subunits, IL-15 activates the JAK/STAT pathway. By limiting expression of the pro-apoptotic protein Bim and sustaining Mcl-1 expression—processes that involve Erk1/2 activity and the transcription factor Foxo3a—IL-15 preserves NK-cell viability in BC 94,95.

IL-6 impairs NK-cell function via STAT3 activation and promotes oncogenesis through epithelial–mesenchymal transition (EMT) in ER-positive BC. IL-6 production is often triggered by IL-1β, which is over-expressed in luminal-type BC subtypes that otherwise do not secrete IL-6 96. STAT3 activation down-regulates natural cytotoxicity receptors (NCRs) on NK cells, reducing tumour-killing capacity 65,97. IL-6 not only fosters chronic inflammation but also expands MDSCs, which inhibit T-cell activity and secrete additional immunosuppressive mediators, forming a feedback loop that further suppresses NK-cell function and accelerates tumour progression 98.

PGE2, a bioactive lipid synthesised by cyclo-oxygenase-2 (COX-2) in both immune and tumour cells, leads to NK-cell suppression within the BC microenvironment 99. It reduces IL-2-driven lymphokine-activated killer (LAK) cell activity via the EP2 receptor, one of four G-protein-coupled PGE2 receptors 100. Activated NK cells normally secrete IFN-γ to promote anti-tumour immunity; however, selective EP2 activation with agonists such as butaprost diminishes IFN-γ production by 66 %, whereas EP4 agonism produces an even greater (86 %) inhibition 101,102. Cancer-associated fibroblasts (CAFs) secrete higher levels of PGE2 than normal fibroblasts—particularly in response to NK-cell presence—creating an inhibitory loop 103,104.

Emerging studies underscore the promise of immune checkpoint blockade (ICB) in rejuvenating exhausted T cells. PD-1/PD-L1 inhibition with anti-PD-1 and anti-PD-L1 monoclonal antibodies (mAbs) also enhances NK-cell–mediated antitumor responses in breast cancer (BC) 48,111. The IgG1 anti-PD-L1 mAb avelumab has been shown to enhance NK-cell functionality, particularly in triple-negative breast cancer (TNBC), by promoting the production of cytokines such as IFN-γ and TNF-α. It also mediates antibody-dependent cellular cytotoxicity (ADCC), and its efficacy is linked to PD-L1 expression levels, which correlate with increased therapeutic sensitivity 112,113,114,115.

TIM-3 is an emerging immune checkpoint receptor whose function is comparable to that of PD-1 and CTLA-4 and represents a novel immunotherapeutic target in BC 116,117. Blocking TIM-3 with antibodies such as MBG453 or TSR-022 has shown preclinical promise by restoring NK-cell proliferation, persistence, and cytotoxicity 118,119. In addition, TIM-3 inhibition synergizes with other checkpoints, such as PD-1 blockade, suggesting that dual checkpoint inhibition may elicit a more robust NK-cell-based immune response against BC 120.

Another important checkpoint is TIGIT. Inhibitors of TIGIT are currently undergoing phase I/II clinical evaluation (e.g., NCT05867771). The bispecific antibody PM1022, which targets TIGIT and PD-L1, exerts multipronged antitumor effects by (i) depleting suppressive Tregs, (ii) mobilizing myeloid effector functions, and (iii) enhancing NK- and T-cell-mediated cytotoxicity 121.

NKG2A has emerged as another actionable checkpoint. Monalizumab, a first-in-class anti-NKG2A antibody, can reverse HLA-E–mediated inhibition and thereby enhance NK-cell function. Early clinical evaluation of monalizumab (NCT04307329) in HER2-positive BC suggests potential benefit, particularly in combination with trastuzumab, by augmenting NK-cell-mediated ADCC and overcoming resistance mechanisms. Consequently, the NKG2A–HLA-E axis represents a promising, druggable checkpoint pathway for NK-based immunotherapy in BC 122,123.