Targeting macrophage-driven NK cell immunosuppression to improve cancer immunotherapy

Abstract

Natural killer (NK) cells are critical effectors of antitumor immunity, however their cytotoxic function is frequently impaired within the tumor microenvironment (TME). Tumor-associated macrophages (TAMs), the most abundant immune stromal population in solid tumors, play a central role in shaping NK cell responses through a broad range of mechanisms, including receptor–ligand interactions, immunosuppressive cytokine signaling, metabolic reprogramming, and engagement of immune checkpoint pathways. Here, we review current insights into the bidirectional crosstalk between TAMs and NK cells and discuss therapeutic strategies aimed at restoring NK cell activity by targeting TAMs. These include macrophage depletion and reprogramming approaches, modulation of metabolic and transcriptional pathways, and interventions targeting cytokine networks and immune checkpoints. We further examine emerging strategies that reshape the TME to enhance NK-macrophage cooperation, such as induction of inflammatory cell death, modulation of innate immune signaling pathways, and the development of synthetic NK cell engagers. In addition, we highlight the impact of macrophage ontogeny, tissue residency, and spatial organization on NK cell function, emphasizing how distinct microanatomical niches within the TME regulate immune cell interactions and influence therapeutic responses. Finally, we summarize translational advances and ongoing clinical efforts aimed at integrating TAM-targeted therapies with NK cell-based approaches. Collectively, these findings provide a conceptual and mechanistic framework for the rational design of combination immunotherapies that leverage macrophage-NK cell interactions to enhance innate immune responses and improve cancer treatment outcomes.

Introduction

The tumor microenvironment (TME) plays a decisive role in shaping immune responses and critically determines the success of immunotherapeutic strategies across cancer types.¹ While cytotoxic CD8⁺ T lymphocytes have traditionally been the primary focus of cancer immunotherapy, natural killer (NK) cells have emerged as powerful innate immune effectors capable of eliminating malignant cells through death receptor-mediated apoptosis and the release of cytolytic granules.^{2 3} Despite this potential, NK cell activity is frequently compromised in solid tumors, largely due to immunosuppressive signals orchestrated by tumor-associated macrophages (TAMs), the most abundant immune stromal population within the TME.^{4 5}

TAMs display pronounced functional plasticity, dynamically adopting proinflammatory or immunosuppressive phenotypes in response to environmental cues.^{6 7} Their impact on NK cell function is therefore highly context dependent and influenced by activation state, developmental origin, and spatial localization within the tumor.

A detailed understanding of TAM-NK cell crosstalk is essential for the development of therapeutic strategies that overcome innate immune suppression and fully exploit NK cells for cancer treatment. In this review, we summarize current insights into TAM-mediated regulation of NK cell function, discuss approaches to deplete or reprogram TAMs to restore NK cell activity, examine the metabolic and spatial determinants of TAM-NK interactions, and explore translational opportunities for combining NK cell-based therapies with TAM-targeted interventions in clinical settings.

Role of TAMs in regulating NK cell function

TAMs exert a dual role in regulating NK cell function, thereby profoundly influencing their antitumor potential.⁸ Depending on their activation state and microenvironmental cues, TAMs can either enhance NK cell development, maturation, and receptor expression or suppress NK cell activity through downregulation of activating receptors and engagement of inhibitory pathways. The dynamic balance between these opposing effects is a critical determinant of effective antitumor immunity and immunotherapeutic efficacy.

NK cell activation by macrophages

Exposure of macrophages to inflammatory stimuli, such as interferon-gamma (IFNγ) or Toll-like receptor (TLR) agonists, including polyinosinic-polycytidylic acid (poly I:C) and lipopolysaccharide (LPS), promotes robust NK cell activation, although this effect is not macrophage-specific and likely reflects the broader activation of innate immune pathways within the TME. For instance, tissue-resident macrophages (TRMs) treated with poly I:C enhance NK cell cytotoxicity against tumor cells by inducing the expression of the activating receptor NKG2D on NK cells through the secretion of IFNβ and interleukin (IL)-15.⁹ In parallel, poly I:C stimulation upregulates the NKG2D ligands RAE1 and MULT1 on macrophages, resulting in a marked reduction of melanoma metastasis in murine models through direct NK cell-macrophage interaction.⁹ Consistently, alveolar proinflammatory macrophages isolated from tumor-bearing mice and treated with poly I:C promote NK cell cytotoxicity in vitro.^{9 10} Similarly, immunosuppressive macrophages derived from the ascites of patients with ovarian cancer can be reprogrammed toward an inflammatory phenotype on LPS stimulation, thereby enhancing NK cell-mediated cytotoxicity.¹¹ In human systems, LPS-activated monocyte-derived macrophages (mo-Ms) promote NK cell proliferation, IFNγ production, and cytotoxic activity through direct interaction between CD48 expressed on macrophages and its receptor 2B4 on NK cells.^{12 13} Co-culture of NK cells with mo-Ms treated with LPS and IFNγ further increases activation markers, including CD69 and CD107, along with IFNγ production. These stimulated macrophages also produce IFNβ and IL-23, which upregulate activating receptors such as NKG2D and NKp44 on NK cells and express high levels of membrane-bound IL-15/IL-15Rα complexes, further enhancing NK cell activation.¹⁴

Collectively, these findings demonstrate that TAMs, when appropriately polarized toward an inflammatory phenotype by stimulatory signals, can promote potent NK cell activation and antitumor responses.

NK cell inhibition by macrophages

Immunosuppressive TAMs restrain NK cell function through several interconnected mechanisms, including persistent engagement of activating ligands leading to NK cell desensitization, signaling through immune checkpoint pathways, and inhibition mediated by non-immune checkpoint molecules.

Persistent ligand engagement and NK cell desensitization

Although activating receptor–ligand interactions are essential for NK cell effector function, sustained engagement can paradoxically induce NK cell hyporesponsiveness.¹⁵ In melanoma mouse models, TAMs upregulate the NKG2D ligand RAE1, resulting in downregulation of NKG2D on tumor-infiltrating NK cells through direct NK cell–macrophage interaction, an effect that is absent in Rae1-deficient mice.^{5 12} Prolonged contact with RAE1⁺ macrophages diminishes NK cell activation, a phenotype that can be reversed by blocking recombinant NKG2D ligands.¹²

A similar phenomenon has been observed in human cancers, where short-term interactions with TAMs enhance NK cell activation markers such as CD69, TRAIL and granzyme B, whereas prolonged exposure suppresses NK cell function. This inhibition can be reversed by blocking the coactivating receptor 2B4, which undergoes internalization following sustained macrophage contact.¹⁶ Together, these findings support a two-phase model in which TAMs initially activate NK cells but ultimately induce receptor downregulation and functional tolerance through chronic ligand engagement.

Immune checkpoint-mediated inhibition

NK cells express several inhibitory receptors, including killer-cell immunoglobulin-like receptors, leukocyte immunoglobulin-like receptors (LILRs), and NKG2A/CD94, which recognize major histocompatibility complex (MHC) class I molecules.¹⁷ TAMs in melanoma, liver, and lung cancers often display high levels of MHC class I expression.18,20 However, the role of TAM-derived MHC class I in NK cell suppression appears to be context dependent. Monocytes or bone marrow-derived macrophages (BMDM) with elevated MHC class I expression do not necessarily impair NK cell cytotoxicity.²¹ In some settings, MHC class I upregulation in poly I:C-stimulated macrophages may enhance NK cell activation while simultaneously protecting macrophages from NK cell-mediated lysis.⁹ Thus, macrophages MHC class I expression may function primarily as a self-protective mechanism rather than a dominant inhibitory signal for NK cells.

Beyond classical NK cell inhibitory receptors, immune checkpoint molecules traditionally associated with T cell exhaustion, including programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4, LAG-3, TIM-3, and TIGIT, also modulate NK cell activity.²² Although PD-1 expression is low on NK cells from healthy donors, it is increased in NK cells isolated from patients with cancer.²³ In lymph nodes from patients with lymphoma, elevated PD-1 expression on NK cells and programmed death-ligand 1 (PD-L1)/PD-L2 expression on TAMs correlate with impaired NK cell activation, which can be reversed by PD-1 blockade.²⁴ These findings suggest that TAMs directly suppress NK cell responses within the TME by exploiting immune checkpoint pathways, although the relative contribution of individual checkpoints remains to be fully defined.

Inhibition of NK cells by non-immune checkpoint pathways

Beyond classical immune checkpoint interactions, TAMs suppress NK cell function through several alternative mechanisms. One of such pathways involves growth arrest-specific gene 6 (GAS6), which encodes a ligand for the receptor tyrosine kinases AXL, TYRO3, and MER expressed on NK cells and involved in their maturation.²⁵ In murine models of breast and colon cancer, TAM-derived GAS6 attenuates NKG2D signaling, inhibiting IFNγ production and reducing NK cell degranulation.^{26 27} Pharmacological or genetic inhibition of the GAS6-TYRO3/AXL/MER axis enhances NK cell activation and significantly reduces metastatic burden in breast cancer and melanoma models.^{27 28}

Similarly, activation of the stimulator of interferon genes (STING) pathway via lipid nanoparticle-delivered agonists stimulates antigen-presenting cells to produce type I IFN and proinflammatory cytokines, promoting NK cell recruitment, cytotoxicity, and cross-talk with macrophages and dendritic cells (DCs) (figure 1).^{29 30} Type I IFNs also favor macrophage polarization toward inflammatory phenotypes, further supporting NK cell activation and antitumor responses. However, STING-induced inflammatory signals are tightly regulated by compensatory pathways in the TME.31,33 Notably, GAS6 contributes to a negative feedback loop by promoting immunoregulatory macrophages and suppressing type I IFN signaling and inflammatory cytokine production, which can directly or indirectly limit NK cell activity.34,37 Therapeutically, this suggests that STING pathway activation may be potentiated by concurrent inhibition of the GAS6-TYRO3/AXL/MER axis, as blockade of AXL or MER enhances type I IFN production and improves NK cell-mediated tumor control in preclinical models.³⁸

Figure 1. Strategies to target macrophages for enhancing NK cell activity. Schematic overview of therapeutic approaches aimed at modulating macrophages within the TME to potentiate NK cell-mediated antitumor immunity. Strategies include interventions that limit TAM recruitment or survival, selectively deplete protumor TAM populations, or reprogram TAMs toward an immunostimulatory phenotype that supports NK cell infiltration, activation, and cytotoxic function. Created by Biorender. IFN-γ, interferon-gamma; IL, interleukin; NK, natural killer; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; STING, stimulator of interferon genes; TAMs, tumor-associated macrophages; TLR, Toll-like receptor; TME, tumor microenvironment.

Transforming growth factor β (TGFβ) is another key mediator of TAM-driven NK cell suppression. In gastric cancer, TGFβ blockade restores TAM-inhibited NK cell IFNγ production.³⁹ Similarly, immunosuppressive TAMs from murine mammary tumors and IL-4-polarized BMDMs inhibit NK cell activation via TGFβ secretion.⁴⁰ Targeting TGFβ signaling can restore NK cell cytotoxicity and limit early metastatic dissemination in breast cancer models, though its efficacy in advanced metastatic disease is limited, highlighting the need for combinatorial approaches.²¹ Mechanistically, TGFβ downregulates NKG2D expression on NK cells, thereby reducing their cytotoxic potential.

Serum amyloid A1 (SAA1) further reinforces TGFβ-mediated immunosuppression. Highly expressed in ovarian cancer, SAA1 promotes immunosuppressive macrophages through TGFβ1 activation, reducing NK cell cytotoxicity both in vitro and in vivo.⁴¹ In neuroblastoma, TGFβ1 produced via TAMs-cancer-associated fibroblasts (CAFs)-tumor cell interactions similarly impairs NK cell function, contributing to therapeutic resistance and highlighting the role of the TME in immune evasion.⁴²

Collectively, these studies illustrate the multifaceted mechanisms by which TAMs suppress NK cell activity, encompassing GAS6-mediated signaling, TGFβ-driven modulation of activating receptors, and SAA1-amplified immunosuppressive circuits. Understanding how these pathways integrate within the TME is critical to developing effective strategies for restoring NK cell-mediated antitumor immunity.

TAM targeting strategies to enhance NK cell efficacy

Although numerous strategies have been developed to target TAMs in cancer,⁵ their specific impact on NK cell-mediated immunity has only recently begun to emerge. Preclinical evidence indicates that modulating TAM abundance or function can enhance endogenous NK cell activity and improve the efficacy of NK cell-directed therapies.

Depleting TAMs

Colony-stimulating factor 1 (CSF1) signaling is essential for macrophage survival, making CSF1 receptor (CSF1R) blockade a widely explored strategy to reduce TAM numbers within tumors (figure 1). However, the effects of TAM depletion are highly context-dependent. In a breast cancer model, Csf1r deletion decreased TAM accumulation, increased NK cell activation at metastatic sites, and improved control of early metastases by adoptively transferred NK cells.²¹ In a lung cancer mouse model, CSF1R inhibition reduced NK cell numbers within the TME and paradoxically promoted metastasis.⁴³ This effect was likely due to the concomitant loss of TRMs, which support NK cell function and also depend on CSF1 signaling. Clinically, CSF1R-targeting antibodies have demonstrated efficacy in diffuse-type giant cell tumor through effective TAM depletion.⁴⁴ In contrast, small-molecule CSF1R inhibitors do not always eliminate TAMs but may instead reprogram them. For example, in lung cancer models, CSF1R inhibition did not reduce TAM numbers but promoted functional crosstalk between IFNγ-producing NK/T cells and IL-12-producing DCs, contributing to tumor control.⁴⁵ Consistently, in a Phase 1/2 trial, the brain-penetrant CSF1R inhibitor BLZ945 induced partial responses and disease stabilization in patients with glioblastoma (table 1).⁴⁶ Transcriptomic analysis of tumor biopsies revealed a shift from TAM-rich to T cell-inflamed profiles,⁴⁶ suggesting the emergence of a microenvironment permissive for NK cell activation. Alternative depletion strategies, such as myeloid-specific ERK5 knockout, suppress TAM proliferation and reduce melanoma tumor metastasis in vivo,⁴⁷ although their effects on NK cell responses remain to be determined.

Table 1. Clinical trials targeting macrophages/NK cells crosstalk.

Target	Drug	Clinical trials ID	Phase	Other therapy	Indications	Clinical outcome	Refs
CSFR1	BLZ945	NCT02829723	1/2	Spartalizumab	ST	/	46
TREM2	PY314	NCT04691375	1a/1b	Pembrolizumab	ST	PR: 6%, SD: 24%	62 63
Sumoylation	TAK-981	NCT03648372	1/2	NA	Adv ST, Lym	PR:1.3%	81
		NCT04074330	1/2	Rituximab	RR-NHL	ORR: 27.6%	82
		NCT04776018	1b/2	Mezagitamab	RR-MM	/
		NCT04381650	1b/2	Pembrolizumab	Adv M ST	/
IDO1	Epacadostat	NCT02752074	3	Pembrolizumab	Mel	/	90
CD47/SIRPα	Hu5F9-G4	NCT02216409	1	NA	Adv ST, L	PR: 3.2%	93
		NCT02953782	1/2	Cetuximab	CRC, ST	ORR: 6.3% (KRASwt), DCR: 38.1% (KRASmut)	94 95
		NCT03558139	1b	Avelumab	OC	SD: 56%	96
		NCT02953509	1b	Rituximab	NHL	CR: 36%	97
CD30	AFM13	NCT01221571	1	NA	RR-HL	/	118
		NCT04101331	2	NA	RR-PTCL	ORR: 32.4%	120
		NCT05883449	2	All NK	RR-HL	/	/
		NCT03192202	1b/2a	NA	RR-L	ORR: 50%	150
NKG2A	Monalizumab	NCT02671435	1/2	Durvalumab	Adv ST	ORR: 10%	102
LILRB1	BND-22	NCT04717375	1/2	Pembrolizumab	Adv ST	/	109
		NCT06651593	2	Cemiplimab	ST	/	109
	AGEN1571	NCT05377528	1	Balstilimab	Adv ST	/	108
	ADA-011	NCT05601219	1	NA	Adv ST	/	/
HER2	Trastuzumab	NCT02129556	1b/2	Pembrolizumab	BC	/	111
EGFR	AFM24	NCT04259450	1/2a	NA	Adv ST	SD: 28.6%	117
		NCT05109442	1/2	Atezolizumab	Adv ST	/
		NCT05099549	1	Aut. NK	Adv ST	/

Adv, advanced; All NK, allogenic NK cells; Aut.NK, autologous NK cells; BC, breast cancer; CR, complete response; CRC, colorectal cancer; DCR, disease control rate; HL, Hodgkin’s lymphoma; HM, Hematologic Malignancy; Lym, lymphomas; M, metastatic; Mel, melanoma; MM, multiple myeloma; NA, not applicable; NHL, non-Hodgkin’s lymphoma; NK, natural killer; OC, ovarian cancer; ORR, objective response rate; PR, partial response; PTCL, peripheral T-cell lymphoma; RR, relapsed or refractory; SD, stable disease; ST, solid tumors.

Reprogramming TAMs

Given their remarkable plasticity, TAMs represent attractive therapeutic targets for functional reprogramming rather than physical depletion. Inducing a proinflammatory phenotype can restore their ability to support NK cell activation and antitumor immunity. Among the most effective strategies to achieve this reprogramming, TLR agonists have emerged as potent modulators of TAM function.

In squamous cell carcinoma, intratumoral TLR stimulation combined with immune checkpoint blockade (ICB) drives a pronounced reprogramming of TAMs toward a proinflammatory phenotype. This shift enhances their antigen-presenting capacity, promotes the recruitment of CD8⁺ T cells, and results in a significant suppression of tumor growth in vivo (figure 1). Importantly, the therapeutic efficacy of this approach depends on the presence of both NK cells and CD8⁺ T cells, underscoring the cooperative interplay between innate and adaptive immunity in mediating effective antitumor responses.⁴⁸ Consistent with these findings, systemic administration of the TLR7/8 agonist resiquimod (R484) enhances antitumor immunity by modulating both innate and adaptive immune compartments.⁴⁹ R484 treatment upregulated TLR7 expression in DCs, promoted their maturation and significantly enhanced NK cell and CD8⁺ T cell infiltration while reducing regulatory T cells (Tregs) within TME, accompanied by elevated levels of IFNγ, tumor necrosis factor-alpha and IL-2. While the overall frequency of macrophages remained unchanged, R848 selectively reduced the proportion of immunosuppressive TAMs and increased proinflammatory macrophages. These observations align with earlier studies⁵⁰ showing that R848-loaded nanoparticles reprogram TAM polarization and enhance the efficacy of cancer immunotherapy. More recently, it has been shown that activation of TLR5 synergizes with PD-1 blockade to reshape the TME and suppress tumor growth in murine models.⁵¹ Treatment with a TLR5 agonist shifted TAM polarization toward a proinflammatory phenotype and upregulated the expression of costimulatory molecules on macrophages. This phenotypic switch was associated with enhanced infiltration and activation of CD8⁺ T cells. Combination therapy also significantly increased the frequency of activated NK cells producing high levels of IFNγ and granzyme B, indicating enhanced cytotoxic capacity. Although the foundational reference for TLR5 agonism in cancer immunotherapy dates back to 2017, these recent findings further substantiate its relevance in combinatorial strategies.

Beyond TLR-based strategies, targeting macrophage surface receptors involved in immunosuppression has shown considerable promise. The macrophage receptor with collagenous structure (MARCO), which is highly expressed on immunosuppressive TAMs within lipid-rich TME, facilitates the uptake of modified lipids (figure 1). Antibody-mediated MARCO blockade polarizes TAMs to a proinflammatory state, limits tumor growth in melanoma models, and synergizes with ICB.⁵² Similarly, myeloid-selective deletion of 3-phosphoinositide-dependent kinase 1 reprograms TAMs toward an inflammatory phenotype, enhances NK cell-derived IFNγ production, and suppresses breast tumor growth in murine models, although clinical translation of this strategy remains to be established.⁵³

In parallel, macrophage lipid metabolism has emerged as a key determinant of TAM function and their crosstalk with NK cells. In colorectal and prostate cancers,54,56 lipid accumulation driven by long-chain fatty acids or tumor-derived signals such as IL-1β promotes the expansion of MARCO-expressing, immunosuppressive TAMs (figure 1). This metabolic state is characterized by mTOR activation, enhanced mitochondrial respiration, reduced NK cell infiltration, and promoted expansion of Treg cells, collectively contributing to a tolerogenic TME (figure 1). Accordingly, pharmacologic inhibition of lipid droplet formation has been proposed as a selective strategy to target protumor myeloid cells and restore antitumor immunity. Notably, MARCO targeting not only limits lipid accumulation but also induces a metabolic shift toward glycolysis in TAMs, indirectly enhancing NK cell cytotoxicity through increased TRAIL expression. Consistent with this mechanism, combined MARCO blockade and ICB achieve superior tumor control in melanoma models.⁵²

Overall, therapeutic strategies aimed at reprogramming TAM polarization, modulating TAM metabolism, or inhibiting lipid-sensing receptors such as MARCO represent promising avenues to restore NK cell infiltration and effector function. These approaches are particularly compelling when integrated with ICB, offering a rational framework for overcoming tumor-induced immunosuppression.

Modulating the TME to enhance TAM-NK cell crosstalk

Beyond direct TAM targeting, broader strategies aimed at reprogramming the TME can strengthen macrophage-NK cell interactions and restore innate antitumor immunity. These approaches converge on three major regulatory axes: macrophage surface receptor signaling, transcriptional control of TAM identity, and cytokine-mediated NK cell activation.

Targeting surface receptors on TAMs

Macrophage surface receptors play highly context-dependent roles in shaping antitumor immunity, with profound consequences for NK cell function. Triggering receptor expressed on myeloid cells 2 (TREM2) exemplifies this functional duality.57,59 While TREM2 has been reported to exert protective roles in certain malignancies, such as hepatocellular carcinoma,⁶⁰ its expression in non-small cell lung cancer (NSCLC) is associated with immune suppression. In these tumors, TREM2⁺ mo-Ms suppress NK cell infiltration and effector function by limiting IL-18-dependent NK cell activation (figure 1).⁶¹ In preclinical NSCLC models, TREM2 blockade, particularly when combined with IL-15, restores NK cell recruitment, enhances IFNγ production, and improves tumor control.⁶¹ However, early clinical translation has yielded modest efficacy despite favorable safety profiles. In a Phase 1a study involving 28 patients with advanced solid tumors, TREM2-targeting antibodies administered either as monotherapy or in combination with pembrolizumab (anti-PD-1) resulted primarily in stable disease (11 patients), highlighting the need for biomarker-driven patient selection and rational combination strategies (table 1).⁶² Similarly, a phase 1b trial in 17 patients with ICB-resistant metastatic renal cell carcinoma (RCC) showed modest clinical benefit, with one partial response and four cases of stable disease, despite favorable tolerability (table 1).⁶³ Notably, the impact of TREM2 inhibition on NK cell function in these clinical settings remains insufficiently characterized. Mechanistically, the therapeutic relevance of TREM2 targeting is tightly linked to its role in myeloid-mediated suppression of NK cell immunity within the TME.⁵⁷ In myeloid-rich tumors, including NSCLC, breast, colorectal, prostate cancer, melanoma and gliomas TREM2 is highly expressed not only on TAMs, but also on myeloid-derived suppressor cells, and senescent neutrophils.2061 64,67 TREM2 blockade disrupts this immunosuppressive axis, reduces the accumulation of suppressive myeloid cells, restores NK cell activity and improves antitumor responses in preclinical models, where NK cells are required for therapeutic efficacy. In contrast, in tumor types where TREM2⁺ macrophages are less abundant or preferentially involved in tissue repair rather than immune suppression, such as certain pancreatic cancer or hepatocellular carcinoma outside therapy-induced contexts, NK cell dysfunction is maintained by alternative suppressive mechanisms, limiting the benefit of TREM2 inhibition alone.6168,70 These divergent findings likely reflect heterogeneity in TREM2 expression among TAMs, tumor-specific immune landscapes, and tissue-dependent microenvironmental factors. A deeper understanding of how TREM2 shapes macrophage functional states will be essential to refine therapeutic strategies and overcome resistance to cancer immunotherapy. Recent advances in spatial transcriptomics and single-cell profiling have revealed close physical and functional interactions between TAMs and lymphocytes within human tumors, providing a rationale for the development of myeloid-targeted immunocytokines (MiTEs). These next-generation molecules combine TREM2-antagonistic antibodies with a masked IL-2 payload that is selectively activated within the TME by macrophage-specific matrix metalloproteinase 14, thereby minimizing systemic toxicity.⁷¹ MiTEs suppress tumor growth by reprogramming TAMs and DCs toward inflammatory phenotypes while simultaneously enhancing the proliferation and cytotoxic activity of both T and NK cells. Ex vivo studies using patient-derived RCC samples demonstrated robust activation of effector CD8⁺ T and NK cells without concomitant expansion of Treg cells. When combined with ICB, MiTEs further amplified CD8⁺ T cell-mediated antitumor responses, highlighting their potential to overcome resistance in immunotherapy-refractory tumors.

Conversely, other macrophage surface receptors can actively promote NK cell-mediated antitumor immunity. MS4A4A, a macrophage-expressed tetraspanin, promotes NK cell-dependent control of melanoma metastasis by engaging Dectin-1 and activating IRF3-INAM signaling, which induces IL-15 and IL-18 production (figure 1).⁷² This pathway potentiates NK cell cytotoxicity through increased IFNγ, perforin and granzyme B expression.⁷² Despite being expressed in immunosuppressive macrophage subsets,⁷³ MS4A4A exerts a protective function in this context, underscoring the importance of tissue-specific and disease-specific immune regulation of macrophage-NK cell crosstalk.

Transcriptional reprogramming of TAMs

Targeting transcriptional regulators that define TAM identity represents an additional strategy to restore NK cell activity. Zinc finger E-box-binding homeobox 2 (ZEB2), a transcription factor highly expressed in TAMs, suppresses type I IFN signaling and antigen presentation, and its high expression correlates with poor prognosis in TAM-rich tumors. In colon and bladder cancer models, ZEB2 inhibition reversed TAM-mediated immunosuppression, enhanced systemic NK cell responses, and induced tumor regression (figure 1).⁷⁴

Similarly, inhibitor of DNA binding 3 (ID3), a helix-loop-helix transcriptional cofactor that regulates gene expression by inhibiting DNA binding of bHLH transcription factors, is critical for the differentiation of liver-resident Kupffer cells from pre-embryonic macrophages in the liver (figure 1).⁷⁵ In murine models, ID3 deficiency resulted in increased expression of the inhibitory receptor SIRPα and reduced Dectin-1 levels, along with diminished production of cytokine (IL-12, IL-15, IL-18) and chemokine (CCL3, CCL4, CCL5). This impaired the infiltration and cytotoxic function of both NK and CD8⁺ T cells.⁷⁵ Conversely, human induced pluripotent stem cell (iPSC)-derived macrophages overexpressing ID3 showed enhanced phagocytosis of pancreatic tumor cells and promoted NK and CD8⁺ T cell activation, supporting the translational relevance of this pathway.⁷⁵

Enhancing cytokine signaling

Many TAM-targeting strategies ultimately converge on restoring cytokine-driven NK cell activation. IL-12, IL-15 and IL-18 emerge as central mediators of NK cell cytotoxicity across multiple regulatory pathways, reinforcing a shared downstream axis that can be therapeutically exploited (figure 1).^{61 72}

Collectively, these findings underscore the complexity of TAM-NK interactions and highlight multiple entry points for therapeutic intervention. Whether through modulation of surface receptor signaling (TREM2 and MS4A4A), transcriptional reprogramming (ZEB2, ID3), or cytokine enhancement (IL-15, IL-18, or blocking TGFβ), the overarching goal is to rewire the TME in favor of effective NK cell-mediated tumor clearance. Rational combination strategies and precision targeting, guided by tissue context, TAM ontogeny, and immune composition, will be critical for translating these insights into durable clinical benefit.

Multiple approaches to model the TME and enhance NK-macrophage interactions

Beyond macrophage-specific targets, several strategies aim to reshape the TME to enhance NK cell activation and support their functional cooperation with TAMs. These approaches can be broadly grouped into several mechanistic categories including i) induction of pyroptosis, ii) enhancement of type I IFN-driven and cytokine-driven innate immune responses, iii) targeting metabolic regulators, “don’t eat me” signals, and immune check points, and iv) development of synthetic NK cell engagers and bispecific antibodies.

Induction of pyroptosis

Gasdermin E (GSDME) is a pore-forming protein that triggers pyroptosis, a highly inflammatory form of programmed cell death.⁷⁶ Although GSDME expression is frequently silenced in cancer cells via epigenetic repression or mutation, chemotherapy-induced DNA damage can activate caspase-3, leading to GSDME cleavage and pyroptosis in GSDME-expressing tumors.⁷⁶

In murine breast cancer and melanoma models, ectopic GSDME expression promoted macrophage phagocytosis and increased NK and CD8⁺ T cell infiltration and cytotoxic function, resulting in tumor growth suppression (figure 1).^{77 78} Conversely, GSDME knockout in breast and colon tumor models accelerated tumor progression and reduced immune infiltration, underscoring an endogenous role for pyroptosis in contrasting tumor growth.⁷⁷ Together, these findings support pyroptosis induction as a strategy to remodel the TME and promote both innate and adaptive antitumor immunity.

Enhancing type I interferon and cytokine-mediated innate immune activation

Type I IFN signaling is a central driver of innate immune activation within the TME. In many tumors, SUMOylation is upregulated and suppresses IFN signaling; pharmacological inhibition of SUMOylation using agents such as TAK981 restores DC activation and enhances both macrophage and NK cell function (figure 1).⁷⁹ In preclinical lymphoma models, combining TAK981 with therapeutic antibodies (anti-CD20 or anti-CD38) significantly augmented antibody-dependent cellular phagocytosis (ADCP) and NK cell-mediated cytotoxicity.⁸⁰ Consistently, a Phase 1 trial of TAK981 demonstrated rapid innate immune activation, with increased CD69⁺ NK cells and elevated CXCL10 levels within 24 hours, and combination with rituximab elicited objective responses in patients with relapsed/refractory CD20⁺ non-Hodgkin lymphoma (table 1).^{81 82} These results highlight the potential of strategies that enhance type I IFN-mediated innate immune activation to boost NK cell function and improve antitumor responses. Such approaches may provide a foundation for combinatorial therapies that maximize immune effector activity in the TME.

Targeting metabolic regulators, “don’t eat me” signals and immune checkpoints

TAMs restrain NK cell activity in the TME through a combination of metabolic, inhibitory receptor, and “don’t eat me” pathways, creating a multifaceted immunosuppressive network.

Metabolic regulation represents an important mechanism by which TAMs restrain NK cell activity. Indoleamine 2,3-dioxygenase 1 (IDO1) is a central mediator of immune tolerance, linking inflammatory cues to metabolic immune suppression.^{83 84} IDO1 degrades tryptophan into kynurenine metabolites that impair both innate and adaptive immune cells. TAMs are a major source of IDO1 in many tumors, and its expression can be further induced by IFNγ or tumor-derived signals. Elevated IDO activity limits NK cell proliferation, cytokine production, and cytotoxicity reducing metabolic fitness and activating the aryl hydrocarbon receptor (AhR) pathway, which reinforces immunosuppressive transcriptional programs and immunosuppressive TAMs.^{85 86} Pharmacological inhibition of IDO1 restores tryptophan levels, reduces kynurenine accumulation, and promotes NK cell activation, IFNγ production, and tumor cell cytotoxicity. IDO blockade promotes macrophage reprogramming toward a proinflammatory phenotype, relieving immunosuppressive constraints within the TME. This shift is associated with enhanced IL-12 production and improved antigen-presenting capacity, ultimately facilitating NK cell recruitment and activation.^{87 88} These observations provided the rationale for the clinical development of several IDO inhibitors, including epacadostat, indoximod, and navoximod, often in combination with ICB.⁸⁹ However, the phase III ECHO-301 trial combining epacadostat with pembrolizumab in melanoma failed to improve clinical outcomes versus ICB alone (table 1),⁹⁰ highlighting the complexity of tryptophan metabolism and the presence of compensatory mechanisms, including tryptophan-2,3-dioxygenase (TDO) activity and persistent AhR signaling. These findings emphasize the need for biomarker-guided patient selection and rational combinations with therapies that enhance NK cell activation or reprogram TAMs to fully exploit metabolic interventions. A recent review highlights advances in targeting IDO and TDO pathways, while noting ongoing challenges related to tumor heterogeneity and variable patient responses that must be addressed to optimize the clinical efficacy of IDO/TDO inhibitors.⁹¹

TAMs also contribute to immune suppression through inhibitory receptor pathways. The CD47-SIRPα “don’t eat me” axis suppresses both macrophage-mediated phagocytosis and NK cell-mediated cytotoxicity. While SIRPα is mainly expressed on myeloid cells, it can be induced on NK cells by IL-2 stimulation (figure 1).⁹² Tumor overexpression of CD47 engages SIRPα on macrophages and NK cells, particularly in MHC class I-deficient tumors, suppressing phagocytosis and NK cell-mediated cytotoxicity. Blockade of this interaction restores NK cell function and macrophage-mediated ADCP.⁹² Clinically, the anti-CD47 antibody Hu5F9-G4 demonstrated acceptable safety and partial clinical responses in ovarian and fallopian tube cancers in a phase 1 trial,⁹³ with additional efficacy reported in combination with avelumab (anti-PD-L1) and cetuximab (anti-EGFR) or rituximab (anti-CD20) across solid tumors and relapsed or refractory lymphomas (table 1).94,97 These findings suggest that targeting CD47-SIRPα can simultaneously enhance macrophage ADCP and NK cell-mediated cytotoxicity.

The HLA-E–NKG2A axis represents a central inhibitory checkpoint that links tumor and myeloid HLA-E expression to the inhibitory receptor NKG2A on NK cells.⁹⁷ Engagement of this pathway suppresses NK cell-mediated cytotoxicity and cytokine production.⁹⁸ Notably, many tumors upregulate HLA-E as an immune evasion mechanism, thereby sustaining inhibitory signaling even when classical MHC class I expression is reduced.^{99 100} Within the TME, TAM further reinforce this axis. TAM-derived cytokines, such as IFNγ, can enhance HLA-E expression, while chronic inflammatory conditions promote the expansion of NKG2A⁺ NK cells characterized by reduced cytotoxicity. Together, these processes establish an inhibitory circuit in which macrophage-derived signals contribute to the suppression of NK cell effector functions (figure 1).^{100 101} Targeting the HLA-E–NKG2A axis has therefore emerged as a promising therapeutic strategy. The monoclonal antibody Monalizumab blocks NKG2A, restoring cytotoxic lymphocyte activity and enhancing both NK cell and CD8⁺ T cell effector functions. Early-phase trials have shown encouraging activity, particularly in combination regimens (table 1),^{102 103} and a comprehensive overview of ongoing trials is available in a recent review.¹⁰⁴ Importantly, NKG2A blockade may synergize with macrophage-targeting approaches that reprogram the TME toward a proinflammatory state, thereby promoting NK cell recruitment and amplifying innate immune-mediated tumor control.

LILRB1 constitutes an inhibitory axis modulating both macrophages and NK cells. LILRB1, expressed on TAMs, DCs, and NK cell subsets, transduces inhibitory signals via ITIMs recruiting SHP-1/2 phosphatases. Engagement by ligands such as HLA-G promotes immunoregulatory TAM phenotypes, suppresses phagocytosis, and limits NK cell recruitment and activation (figure 1).^{105 106} Blockade of LILRB1 on macrophages enhances phagocytosis of tumor cells, reprograms TAMs toward a proinflammatory state, and relieves NK cell inhibition, with preclinical evidence showing synergy with antibody-dependent cellular cytotoxicity (ADCC) and other macrophage-directed therapies.¹⁰⁷ Therefore, targeting HLA-G/LILRB pathways with antibodies, alone or in combination with other ICIs, might constitute a promising strategy for breaking down tolerance in tumors and promoting rejuvenation of exhausted tumor-infiltrating immune cells.^{108 109}

In HER2⁺ breast cancer, trastuzumab (anti-HER2) exemplifies the convergence of these mechanisms: it promotes macrophage ADCP while inducing PD-L1 and IDO expression via AIM2-mediated sensing of tumor-derived DNA, suppressing NK and T cell cytotoxicity (figure 1). Co-targeting HER2 and immune checkpoints can reverse this feedback, highlighting TAM plasticity and its impact on NK cell function.¹¹⁰ Clinically, a Phase 1b/2 trial combining trastuzumab with pembrolizumab demonstrated objective responses in PD-L1⁺ HER2⁺ patients and increased T-cell infiltration in responders (table 1),¹¹¹ although the specific contribution of NK cells remains to be fully defined.

Collectively, these metabolic and checkpoint pathways underscore the central role of TAMs in shaping NK cell responses. Therapeutic strategies targeting IDO, CD47–SIRPα, HLA-E–NKG2A, and LILRB1 hold promise to simultaneously relieve NK cell inhibition, reprogram macrophages toward inflammatory states, and enhance coordinated innate antitumor immunity.

Synthetic NK cell engagers and bispecific antibodies

NK cells mediate ADCC via FcγRIII (CD16); however, polymorphisms in CD16 can limit the efficacy of therapeutic antibodies.¹¹² To overcome this limitation, bispecific (BiKe) and trispecific (TriKe) NK cell engagers have been developed to directly link NK cells to tumor antigens, independently of CD16 affinity.^{113 114} Several of these agents are currently in phase 2 trials for hematologic and solid tumors, demonstrating more sustained ADCC compared with conventional monoclonal antibodies.¹¹⁵

AFM24 is a bispecific innate cell engager that targets EGFR on tumor cells and CD16 on both NK cells and macrophages, promoting both ADCC and ADCP (figure 1).¹¹⁶ Phase 1/2 trials are evaluating AFM24 as monotherapy or in combination with ICB in patients with advanced solid tumors, including RCC, NSCLC and colorectal cancers (table 1). As a single agent, AFM24 has shown a favorable safety profile, immune activation, and disease stabilization in a subset of patients with multiple solid tumors, supporting its further clinical development in combination with ICB.¹¹⁷

Other NK cell-redirecting antibodies have also demonstrated the ability to potentiate innate immune responses. AFM13 is a tetravalent bispecific antibody that binds CD30, a tumor-associated antigen in lymphoid malignancies, and CD16A on NK cells (figure 1).¹¹⁸ By bridging NK cells to CD30⁺ tumor cells, AFM13 induces robust ADCC, triggering NK cell degranulation, release of perforin and granzymes, and production of inflammatory cytokines such as IFNγ, leading to efficient tumor lysis.¹¹⁹ Although AFM13 primarily activates NK cells, its effects may extend to macrophages, as CD16 is also expressed on subsets of these cells. Cytokines released by activated NK cells, particularly IFNγ, can promote macrophage polarization toward inflammatory phenotypes, reinforcing reciprocal activation between NK cells and TAMs. Clinically, AFM13 has demonstrated encouraging activity in relapsed or refractory Hodgkin lymphoma and other CD30⁺ lymphomas, with evidence of NK cell activation and a favorable safety profile.¹¹⁸ Adoptive transfer strategies combining AFM13 with ex vivo expanded NK cells have further highlighted its potential to overcome tumor-induced immune suppression; however, AFM13 has shown only limited single-agent efficacy in hematologic malignancies, underscoring the challenges of translating NK cell engagers into effective monotherapies.¹²⁰ These observations emphasize the importance of rational combination strategies and provide a framework for next-generation innate cell engagers like AFM24, designed to recruit both NK cells and macrophages within the TME.¹¹⁷

Collectively, synthetic immune engagers represent a promising approach to reinforce innate immune surveillance by coordinating NK cell and macrophage activation. Integrating these agents with complementary therapies, such as ICB or cytokine-based strategies, may further enhance macrophage-NK cell crosstalk and improve innate immune-mediated tumor control.

Ontogeny, tissue residency and spatial organization of TAMs and NK cells in the TME

Foundational studies on innate immune cell development and tissue localization have established that both ontogeny and spatial distribution critically shape the functions of TAMs and NK cells within the TME.^{121 122} Beyond local environmental cues, developmental origin and tissue residency imprint distinct functional programs, generating heterogeneous subsets with context-dependent roles in tumor progression and immune surveillance. These programs are further organized within spatially restricted microanatomical niches that act as regulatory hubs coordinating innate and adaptive immune responses.^{123 124}

Macrophages exert divergent effects on tumor initiation, growth, and metastasis, with specific TAM subsets correlating with favorable clinical outcomes.⁶⁵ While mo-Ms frequently dominate immunosuppressive populations within tumors, embryonically derived TRMs can sustain antitumor immunity. However, this dichotomy is tumor-type dependent: in pancreatic cancer, mo-Ms and TRMs display opposing functions, whereas in lung cancer both populations can promote tumor progression.^{125 126} Spatial localization further refines macrophage function by organizing TAM subsets into specialized microdomains with distinct immunological outputs.^{127 128}

A key example is the perivascular niche, where macrophages accumulate around tumor blood vessels and interact with endothelial and stromal cells as well as infiltrating immune populations. These perivascular TAMs promote angiogenesis and tumor cell intravasation, while also regulating the recruitment and retention of cytotoxic lymphocytes, including NK cells.¹²⁹ Integrated single-cell and spatial transcriptomic analyses have shown that venular smooth muscle cells produce CCL19 and CCL21, which are presented within the vascular lumen via endothelial transcytosis. This mechanism enables the targeted recruitment of CCR7⁺ NK and T cells and supports the formation of specialized immune hubs in close proximity to macrophages, facilitating coordinated cellular crosstalk.¹³⁰ Within these hubs, macrophages orchestrate NK cell behavior through chemokines such as CXCL9, CXCL10, and CCL5, and cytokines including IL-12, IL-15, and TGF-β. The balance of these signals determines whether NK cells acquire cytotoxic effector functions or become suppressed, underscoring how spatial context can impose functional states independently of lineage.¹³¹

Additional interaction hubs are found at the tumor invasive margin, where malignant, stromal, and immune compartments interface.^{124 132} In this region, macrophage-NK cell clusters contribute to early immune surveillance: macrophages promote NK cell recruitment but may simultaneously restrain their cytotoxicity through inhibitory ligands or immunosuppressive metabolites. Spatial analyses in gallbladder cancer further highlight this context dependency, showing that macrophage-driven CXCL9-CXCR3 signaling at the tumor-liver interface supports the accumulation of effector T and NK cells within high-density immune regions. However, as these hubs shift toward the tumor core, tumor-derived factors such as LGALS4 induce T-cell exhaustion, illustrating a spatial transition from immune activation to dysfunction.¹³³

Within the tumor core, particularly in hypoxic or perinecrotic niches, macrophages adopt metabolically adapted phenotypes characterized by arginase activity, adenosine production, and lipid metabolism.¹²⁸ These programs create a suppressive environment that limits NK cell function by restricting nutrients, generating inhibitory metabolites, and altering cytokine gradients. As a result, NK cells in these regions often exhibit impaired effector function. Conversely, NK-macrophage interactions can also occur within tertiary lymphoid structure-like aggregates or other organized immune cell clusters resembling secondary lymphoid tissues. In these sites, macrophages, DCs, NK cells, and T cells form coordinated networks that facilitate antigen presentation, cytokine exchange, and amplification of antitumor immunity.¹³⁴ NK cells within such hubs may further enhance immune recruitment through the secretion of chemokines such as CCL5 and XCL1.¹³⁵

NK cell biology in tumors is similarly influenced by spatial organization. Although NK cell infiltration often correlates with CD8⁺ T-cell abundance,¹²⁶ high NK cell density does not necessarily predict favorable outcomes. On entering solid tumors, NK cells frequently undergo phenotypic reprogramming toward tissue-resident-like phenotypes, characterized by increased inhibitory receptor expression and reduced cytotoxicity.¹³⁶ Nonetheless, tissue-resident NK cells can retain potent antitumor activity depending on their localization. For example, in melanoma, CCL5-producing resident NK cells within stromal niches enhance responsiveness to IL-2-based immunotherapy and help overcome treatment resistance.¹³⁷

Recent advances in single-cell RNA sequencing, imaging mass cytometry (IMC), spatial transcriptomics, and computational modeling have enabled integrated analyses of macrophage and NK cell ontogeny, tissue residency, and spatial organization within the TME.121138,145 These approaches confirm that TAMs derive from both TRMs and recruited mo-Ms, each displaying distinct transcriptional and proteomic programs that underlie their functional plasticity.142,144 Importantly, spatial mapping technologies now allow systematic identification of NK-TAM interaction hubs, revealing how cell-cell interaction and localized cytokine networks coordinate immune responses across distinct microanatomical niches.¹³² Spatially resolved studies are highlighting tumor-type-specific patterns of NK-TAM crosstalk. In lung cancer, interactions vary by histological subtype: in squamous cell carcinoma, NK cells engage TAMs via CD96-NECTIN1 interactions, whereas in adenocarcinoma NK cells frequently express inhibitory receptors such as TIGIT and TIM-3 and show limited spatial proximity to their ligands, consistent with impaired activation.¹⁴¹ In glioblastoma, spatial analyses distinguish resident microglia, recruited mo-Ms, and CD163⁺ anti-inflammatory TAMs, with their relative abundance dictated by tumor genotype.¹⁴⁶ Notably, IDH-wild-type gliomas display reduced NK cell infiltration, enrichment of immature CD16⁻ NK cell subsets, and increased recruitment of peripheral mo-Ms, illustrating how macrophage ontogeny and spatial distribution converge to suppress NK cell-mediated antitumor immunity.¹⁴⁷

Emerging evidence suggests that the spatial organization of immune hubs may be more prognostically relevant than overall immune cell abundance. In lung cancer, specialized myeloid-lymphoid niches support antitumor responses through intensive NK-macrophage crosstalk. For example, NLRP4-driven secretion of CCL5 and CXCL2 promotes colocalization of NK cells and macrophages enabling reciprocal activation through NAMPT-mediated signaling.¹⁴⁸ Similarly, in SCLC, the MT2 niche-enriched in CD11c⁺ inflammatory macrophages-forms at tumor boundaries and supports the recruitment of cytotoxic lymphocytes, contributing to effective immune containment.¹⁴⁹ These studies highlight that the formation of a cohesive immunological barrier represents a key prognostic indicator associated with improved overall survival.

Collectively, these findings demonstrate that TAM and NK cell functions are shaped not only by tumor-derived signals but also by developmental origin and spatial positioning within the TME. Defining the anatomical niches and immune hubs that govern NK-TAM interactions will be essential for understanding how innate immune networks regulate tumor progression and for designing therapies that selectively target suppressive regions while reinforcing antitumor cooperation.

Discussion

TAMs emerge as central regulators of NK cell dysfunction within the TME. However, their heterogeneity, spanning developmental origin, tissue residency, spatial localization and transcriptional state remains poorly understood. Advancing therapeutic strategies will therefore require a deeper understanding of how TAM ontogeny and spatial organization shape NK cell suppression or activation in distinct tumor contexts. Integrated approaches combining spatial transcriptomics, IMC, and advanced computational modeling will be essential to map NK-TAM interactions in situ and to identify macrophage subsets that either restrain or promote NK cell function. In parallel, epigenetic and metabolic profiling of TAMs may uncover targetable pathways able to reprogram macrophages toward proinflammatory states that favor NK cell activation and cytotoxicity. From a therapeutic perspective, combinatorial strategies that integrate TAM-directed interventions with NK cell-based therapies and tumor-targeting antibodies hold substantial promise. However, rigorous longitudinal immune profiling in clinical trials will be necessary to distinguish direct effects on NK cells from indirect influences mediated by other components of the TME, including tumor cells or CAFs. Finally, systematic classification of tissue-resident and tumor-infiltrating NK cell subsets across cancer types, integrated with clinical outcome data, may establish NK cells as robust prognostic and predictive biomarkers. Such efforts will be critical for guiding the rational design of precision immunotherapies that simultaneously target macrophages and NK cells to restore effective antitumor immunity.