Tumor-associated macrophages: untapped molecular targets to improve T cell-based immunotherapy

Abstract

T cell responses are generally curtailed by suppressive mechanisms within the tumor microenvironment (TME) that prevent T cell infiltration and function. Consequently, T cell-based therapies for solid tumors have yielded limited and often non-durable clinical responses. Tumors develop a hostile TME, where tumor-associated macrophages (TAMs) that initially support T cells are converted into immune suppressive TAMs that facilitate tumor evasion from T cell control. In fact, immune suppressive TAMs represent a dominant fraction of immune cells within the TME and their presence is associated with poor prognosis and resistance to immunotherapy. Often in close contact with effector T cells, TAMs directly suppress CD8⁺ T cells through mechanisms involving metabolic mediators, co-signaling receptors, their ligands and/or cytokines. Here, we revisit molecular interactions behind TAM-mediated suppression of T cell responses and address the potential targeting of such molecules and pathways to re-boost anti-tumor T cell immunity. This perspective, focusing on molecular interactions between TAM and T cells, may aid the improvement of T cell-based therapies for solid tumors.

Background

The interplay between the immune system and cancer is considered an intricate process, where immune cells may paradoxically promote complete elimination of some tumors, while promoting immune escape and tumor growth of others [1]. When tumors arise, T cell responses are induced to clear tumor cells, yet these are subsequently kept in check by suppressive mechanisms that limit T cell infiltration and function. In line with this, immunotherapy responses in patients with solid tumors have generally been poor or non-durable despite impressive clinical responses in a few human malignancies [2, 3].

Immunotherapies approved to human use are mainly based on enhancing the function of endogenous CD8⁺ T cell responses through blockade of immune checkpoints (ICB) or engineering T cells to specifically target defined tumor antigens. While such therapies have potential to cure cancer, their effectiveness is generally limited in solid tumors. In most cases, T cells that reach the tumor fail to produce effector cytokines or cytotoxic molecules, yet quickly upregulate expression of inhibitory molecules and undergo metabolic changes [4]. This dysfunctional state of T cells can be largely attributed to the tumor microenvironment (TME), which contains immune cells that have become immunosuppressive following interaction with tumor cells and/or other suppressive cells [5, 6]. In fact, the presence of tumor-associated macrophages (TAMs) is predominantly associated with poor prognosis in multiple tumor types, where they have been shown to promote tumorigenesis as well as to suppress anti-tumor therapy [7].

Macrophages are innate immune cells with a wide range of functions, including host defense, wound healing and immune regulation. In the context of cancer, they are often referred to as TAMs and represent the most abundant immune cell type present within the TME [8]. Notably, TAMs are phenotypically and functionally highly diverse and may exert pro- as well as anti-tumor activity depending on cues they receive from the TME [9]. On the one hand, anti-tumor TAMs display functions such as antigen presentation, phagocytosis and tumor cell killing, generally boosting the function of intra-tumoral T cells, while on the other hand, pro-tumor or immunosuppressive TAMs express metabolic mediators, co-inhibitory molecules and/or suppressive cytokines to directly or indirectly render intra-tumoral T cells dysfunctional [9]. Ultimately, the success of an anti-tumor T cell response is largely determined by the ability of T cells to engage with anti-tumor TAMs or to resist pro-tumor TAMs within the TME [10, 11]. This is well illustrated by blockade of colony stimulating factor 1 receptor (CSF1R), a cytokine receptor highly expressed by TAMs, which, in combination with either checkpoint blockade or adoptive T cell transfer, shows improvement of T cell infiltration and function in lung, pancreatic and melanoma mouse models [12–14].

Current therapeutic approaches to target TAMs include elimination, inhibition of recruitment and reprogramming from pro-tumor TAMs to anti-tumor TAMs, yet up to now these approaches show limited efficacy in clinical settings [7, 15, 16]. Moreover, the phenotypic plasticity of TAMs challenges the selection of specific targets. To unlock the development of successful approaches, we need to better understand mechanisms of action that underlie TAM-mediated T cell suppression. In this review, we first highlight and discuss critical determinants of an effective anti-tumor T cell response, and review how anti- and pro-tumor TAMs relate to these determinants. Second, we revisit molecules and cellular pathways behind TAM-mediated T cell suppression and address specific interventions to strengthen anti-tumor T cell immunity rather than simply depleting TAMs. Emphasis is given to underexplored pathways related to the aryl hydrocarbon receptor, galectins, siglecs, as well as B7 homologues. While we focus on CD8⁺ T cells, many mechanisms described herein also apply to CD4⁺ T cells. Lastly, we discuss the road of such interventions to advance T cell-based therapies for solid tumors.

TAMs as determinants of T cell immunity

While TAMs can be divided into two broad functional states, pro- and anti-tumor TAMs, it should be acknowledged that these two states likely represent two extremes of a spectrum. Macrophages are highly heterogeneous and plastic immune cells, thus creating a continuum of phenotypes. In fact, recent advances in single-cell transcriptomics and spatial omics have revealed distinct TAM subsets, each having specific molecular and functional programs [17–19]. For instance, among others, the following TAM subsets have been identified: interferon-primed; inflammatory cytokine-enriched; lipid-associated; and pro-angiogenic TAMs [18]. In addition, different TAM populations have been identified according to the expression of marker genes, such as secreted phosphoprotein 1 (SPP1) and triggering receptor expressed on myeloid cells 2 (TREM2)-expressing TAMs [20–22]. This phenotypic and functional heterogeneity is shaped by ontogeny, tissue of residence as well as local cues [9]. Although leveraging TAM heterogeneity may lead to the advancement of precision therapies, studies describing molecular interactions for such TAM subsets are limited. In this review, we focus on anti-tumor and pro-tumor TAMs, which although reductive, remain relevant to address and discuss TAMs in relation to T cell immunity.

Anti-tumor TAMs promote T cell immunity

The efficacy of an anti-tumor T cell responses is generally determined by infiltration into the tumor, recognition of tumor-specific antigens and tumor killing capacity. Notably, anti-tumor TAMs have key roles in each of these steps.

T cell infiltration into tumors

Tumor-infiltrating lymphocytes (TILs), such as CD8⁺ T cells, are critical cells for an anti-tumor response and their spatial localization at the tumor site has become a prominent prognostic marker for several tumor types [23]. Based on the spatial localization of TILs, tumors can be defined as inflamed, excluded or ignored meaning that TILs are present throughout the TME, only at the margins of the tumor or nearly absent, respectively [24, 25]. Impaired T cell infiltration into the tumor may underlie ignored and excluded tumors, whereas intra-tumoral suppression of T cells most likely explains the cases where inflamed tumors are unresponsive to T cell-based therapies.

TAMs may originate from tissue-resident macrophages that are seeded at the tissue during embryogenesis and persist locally into adulthood or originate from monocytes circulating in the blood, which are recruited to the tumor by chemo-attractants, such as CSF1, chemokine (C-C motif) ligand 2 (CCL2), CCL5 and chemokine (C-X-C motif) ligand 12 (CXCL12) [8, 18]. Upon sensing tumor material or environmental cues, anti-tumor TAMs secrete inflammatory chemo-attractants, such as CXCL9 or CXCL10, to recruit T cells to the tumor via CXCR3 [26–28] (Fig. 1A). In line with this, studies have shown that intra-tumoral CD8⁺ T cells contribute to interferon-γ (IFNγ)-induced CXCL9 expression by macrophages and to accumulation of anti-tumor TAMs in the tumor via CCR5 signaling, which indicates a positive feedback loop between these two cell types [29–31].

Fig. 1.

TAMs as determinants of T cell immunity. A Anti-tumor TAMs are critical in building an effective anti-tumor T cell response. Anti-tumor TAMs secrete CXCL9 and CXCL10 that recruit T cells, which in turn contribute to the accumulation of anti-tumor TAMs and further recruitment of T cells. Anti-tumor TAMs cross-present tumor-associated antigens to CD8⁺ T cells resulting in the activation of antigen-specific T cells. T cell effector functions are sustained as anti-tumor TAMs, besides presenting antigen, provide co-stimulation through CD80/86-CD28 ligation as well as cytokine signaling through IL-12. Ultimately, cytotoxic T cells secrete cytokines and cytolytic granules to efficiently kill tumor cells. B Pro-tumor TAMs dampen anti-tumor T cell responses. After an initial T cell response, pro-tumor TAMs suppress T cell immunity, which is induced, in part, by T cell-derived IFNγ. Pro-tumor TAMs express co-inhibitory molecules, produce immunosuppressive cytokines and shift their metabolism, thereby rendering intra-tumoral T cells dysfunctional

T cell recognition of tumor cells

Once T cells are within the tumor, the nature of an anti-tumor T cell response is determined by the interaction between the T cell receptor (TCR) and the cognate antigen presented by the major histocompatibility complex (MHC) molecules. Thereafter, TCR signaling leads to activation of antigen-specific T cells. Notably, anti-tumor TAMs can cross-present antigens to CD8⁺ T cells via MHC-I [32] (Fig. 1A). Mechanistically, cross-presentation in TAMs was observed to be promoted by TIM4 and TLR4 in the context of ovarian and breast cancer, respectively, and may be negatively regulated by the RNA-binding protein YTHDF2 [18, 33, 34].

T cell-mediated killing of tumor cells

Once cytotoxic CD8⁺ T cells recognize tumor cells, they start producing cytolytic molecules, such as perforin and granzymes, as well as cytokines, such as IFNγ and tumor necrosis factor-α (TNFα), and apoptosis-inducing molecules, such as Fas ligand (FasL) or TRAIL (TNF-related apoptosis inducing ligand) [35]. Yet, in order to have persistent anti-tumor T cell responses, tumor antigen recognition must be accompanied by co-stimulation and cytokine signaling to support T cell survival, clonal expansion and differentiation [36–38]. Along these lines, anti-tumor TAMs express co-stimulatory molecules and produce cytokines that are essential for locally amplifying CD8⁺ T cell responses (Fig. 1A). For instance, anti-tumor CD8⁺ T cell responses were found to be sustained by CD28 co-stimulation in situ provided by myeloid antigen-presenting cells [39]. In addition, myeloid-derived IL-12 has been shown to induce proper anti-tumor CD8⁺ T cell activity [40].

Pro-tumor TAMs suppress T cell immunity

Macrophages are not only important to initiate and boost T cell responses but also to subsequently dampen T cell activity, a critical role to prevent tissue damage and promote tissue homeostasis. However, the mechanisms employed by macrophages to dampen T cell responses in other contexts are largely exploited by tumors to evade from T cell control [41]. For instance, while uptake of dead cells by macrophages is a central process to restore homeostasis after immune responses, it results in upregulated expression of indoleamine-2,3-dioxygenase (IDO) and programmed death-ligand 1 (PD-L1) by TAMs and consequent T cell suppression [42]. Thus, homeostasis is never reached, and cancer is said to be a wound that does not heal, which in turn might explain the abundance of TAMs within the TME [43].

Importantly, T cells may be involved in the start of their own dampening. In a seminal study, Spranger and colleagues showed that IFNγ secretion by T cells leads to upregulated expression of IDO, PD-L1 and recruitment of regulatory T cells to the tumor [44]. Furthermore, recent studies indicated that prolonged exposure to IFNγ may facilitate immune evasion through recruitment of suppressive myeloid cells and induction of terminally differentiated CD8⁺ T cells [45, 46]. This helps to explain why T cells eventually fail to eliminate tumors and why inflamed tumors, which contain a rich immune infiltrate, are nonetheless resistant to T cell responses and T cell-based therapy in some cases.

Keeping T cell number and function in check

Pro-tumor TAMs facilitate immune evasion through several mechanisms, amongst which is direct suppression of CD8⁺ T cells [9, 47, 48] (Fig. 1B). Pro-tumor TAMs secrete immune suppressive cytokines, such as interleukin-10 (IL-10) and transforming growth factor β (TGFβ), which can restrict T cell infiltration into the tumor [14, 49]. For example, TGFβ increases the binding of Smad2 to the CXCR3 promoter and downregulates CXCR3 expression by T cells, thereby limiting tumor infiltration towards CXCL10 [50]. Moreover, pro-tumor TAMs possess impaired antigen presentation ability, which may be regulated through SPP1 signaling [51]. If antigen presentation ability is maintained, pro-tumor TAMs, but not tumor cells, can drive terminal exhaustion of T cells and as a result decrease immunotherapy efficacy [52]. Furthermore, IDO and other metabolic pathways represent a metabolic shift that often occurs within the TME to meet the high metabolic demands of tumor and stromal cells, thus contributing to an unfavorable metabolic landscape for T cells [53]. For instance, pro-tumor TAMs express CD39, which degrades ATP to AMP. In turn, CD73 degrades AMP to adenosine, which binds the adenosine A2a receptor (A2AR). Activation of A2AR on CD8⁺ T cells leads to decreased production of effector cytokines and upregulated expression of PD-1 [54, 55]. This metabolic shift goes hand in hand with the upregulated expression of co-inhibitory molecules, such as PD-L1, by tumor cells and pro-tumor TAMs. These ligands, in the same way as co-stimulatory molecules, are present at the immunological synapse and their signaling integrates with the TCR signal to inhibit T cell activation and function [56, 57]. Thereby, an anti-tumor T cell response may depend qualitatively and quantitatively on the proportion of co-stimulatory and co‐inhibitory signals.

Emerging TAM pathways underlying CD8⁺ T cell dysfunction

Clinical trials aiming at the interference with above molecules/pathways have been conducted. Table 1 provides an overview of these trials and summarizes, when available, outcomes towards the crosstalk between TAMs and T cells. However, these pathways are not always restricted to TAM-mediated T cell suppression. In this section, we have revisited molecules and pathways that take part in TAM: CD8⁺ T cell interactions, which were selected according to novelty and non-redundancy with regard to common suppressive pathways. These represent underappreciated mechanisms of TAM-mediated T cell suppression which might turn into effective ways of targeting TAMs in combination with T cell-based therapies.

Table 1.

Overview of clinical trials intervening with TAM-mediated T cell suppression

Target	Treatment	Tumor type	Phase/no. patients	Clinical outcome	T cell-TAM observations	Trial ID/Ref
A2AR	Taminadenant (+ Spartalizumab)	Non-small cell lung cancer	Phase 1/1B (n = 25)	Completed. Monotherapy: 1% CR, 1% PR, 7% SD, 12%PD. Combination: 1% CR, 1% PR, 14% SD, 8% PD.	Increased expression of immune activation genes. No change of CD8+ T cells and CD33+ myeloid levels in the tumor.	NCT02403193 [58]
	Ciforadenant (+ Atezolizumab)	Renal cell cancer	Phase 1 (n = 68)	Completed. Monotherapy: 3% PR. Combination: 11% PR.	Increased frequency of CD8+ T cells in tumor. Broadening of TCR repertoire in blood. Increased expression of genes encoding macrophage inflammatory mediators.	NCT02655822 [59]
CD73	Oleclumab (+ Durvalumab)	Colorectal Cancer Pancreatic cancer Lung Cancer	Phase 1 (n = 192)	Completed. Dose escalation phase: 0% CR/PR, 14.3% SD monotherapy, 8.3% SD combination.	Decreased frequency of CD73+ T cells in blood and tumor	NCT02503774 [60]
IDO1	Epacadostat + Pembrolizumab	Melanoma	Phase 3 (n = 706)	Completed. Treatment group: median 4.7 months PFS; 4% CR, 30% PR, 17% SD, 43% PD; Control group: median 4.9 months PFS; 4% CR, 27% PR, 19% SD, 43% PD;	N/A	NCT02752074 [61]
TGFβ	Galunisertib + Durvalumab	Pancreatic cancer	Phase 1B (n = 32)	Completed. 0% CR, 3% PR, 21% SD, 47% PD	N/A	NCT02734160 [62]
	Bintrafusp alfa	Non-small cell lung cancer	Phase 3 (n = 304)	Completed. Bintrafusp alfa: median 7 months PFS; 0.7% CR, 46.1% PR, 15.8% SD, 25.7% PD; Pembrolizumab: median 11.1 months PFS; 1.3% CR, 50% PR, 19.1% SD, 20.4% PD;	N/A	NCT03631706 [63]
CSF1R	AMG 820 + Pembrolizumab	Colorectal cancer Pancreatic cancer Non-small cell lung cancer	Phase 1B (n = 15), 2 (n = 101)	Completed. Phase 1B (0% CR, 27% SD, 53% PD), Phase 2 (0% CR, 3% PR, 35% SD, 40% PD)	No change in CD68+ or CD163+ TAMs	NCT02713529 [64]
	Emactuzumab + Selicrelumab	Advanced solid tumors (i.e. TNBC, CRC, MEL)	Phase 1 (n = 37)	Completed. 0% CR, 40.5% SD	Increased frequency of CD8+T cells in blood. Decreased abundance of CD163+ TAMs	NCT02760797 [65]
	Emactuzumab + Paclitaxel	Advanced solid tumors (i.e. ovarian and breast cancer)	Phase 1 (n = 54)	Completed. 0% CR, 7% PR, 43% SD	Decreased abundance of CD163+ TAMs No change in CD8+/CD4+ T cell ratio in tumor	NCT01494688 [66]
	Pexidartinib + Paclitaxel	Advanced solid tumors	Phase 1B (n = 54)	Completed. 3% CR, 13% PR, 34% SD, 45% PD	N/A	NCT01525602 [67]
	ARRY382 + Pembrolizumab	Advanced solid tumors (i.e. TNBC, CRC, PDA)	Phase 1B (n = 19), 2 (n = 57)	Terminated. Phase 1B (0% CR, 10.5% PR, 26.3% SD, 47.4% PD); Phase 2 (0% CR, 3.7% PR, 29.8% SD, 35% PD)	N/A	NCT02880371 [68]
TREM2	PY314 + Pembrolizumab	Renal cell cancer	Phase 1 (n = 288)	Terminated. 0% CR, 5.9% PR, 23.5% SD, 70.6% PD	Inefficient TREM2+ TAM depletion	NCT04691375 [69]

Table summarizes representative trials that have been completed or terminated and published in scientific literature. Abbreviations: TNBC (triple-negative breast cancer); CRC (colorectal cancer); MEL (melanoma); PDA (Pancreatic adenocarcinoma); CR (complete response); PR (partial response); SD (stable disease); PD (progressive disease); PFS (progression-free survival); N/A (not available). Biological activity: Bintrafusp alfa (bifunctional fusion protein to target TGFβ and PD-L1); Paclitaxel (chemotherapy); Pembrolizumab (anti-PD-1 antibody); Selicrelumab (CD40 agonist antibody); Durvalumab (anti-PD-L1 antibody); Budigalimab (anti-PD-1 antibody); Spartalizumab (anti-PD-1 antibody); Atezolizumab (anti-PD-L1 antibody)

Aryl hydrocarbon receptor: a master regulator of metabolic T cell suppression

Molecular mechanism of action

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates the expression of a wide variety of genes and pathways, some leading to T cell suppression [70, 71]. AhR is expressed by tumor and immune cells and can be activated by several types of agonists, although in the context of cancer most originate from the metabolism of tryptophan (Trp) [72].

In the TME, pro-tumor TAMs are significant producers of AhR agonists (Fig. 2). IDO and interleukin-4-induced-1 (IL4I1) are two enzymes expressed by TAMs, including the newly defined interferon-primed TAMs (see the beginning of Sect. 2), that metabolize Trp into kynurenine (Kyn) and indole-3-pyruvic acid (I3P), respectively, both leading to AhR activation on CD8⁺ T cells [18, 19, 73–75]. More specifically, Liu and colleagues observed that AhR activation by Kyn leads to the upregulation of PD-1 expression on CD8⁺ T cells in a TCR-independent manner [76]. The study shows that IFNγ produced by CD8⁺ T cells induces the expression of IDO1 by tumor cells and the subsequent release of Kyn at high levels, which is then transferred to adjacent CD8⁺ T cells via the transporters SLC7A8 and PAT4. In addition, the enzyme IL4I1 generates I3P, which gives rise to indole-3-aldehyde (I3A) and kynurenic acid (KynA) that activate AhR and limit T cell proliferation [74]. Such limited T cell proliferation was mediated by the upregulation of TIPARP and CYP1B1 expression by T cells. In the same study, IL4I1 was also associated with expression of PD-1 and other dysfunctional markers by CD8⁺ T cells. Notably, in a large meta-analysis of human macrophages, Mulder and colleagues identified IL4I1⁺PD-L1⁺IDO1⁺ macrophages conserved among multiple tumor types [19]. These macrophages were associated with IFNγ⁺ CD8⁺ T cells as well as tryptophan degradation gene signatures, with authors suggesting that IFNγ reprograms TAMs to a pro-tumor phenotype through IL4I1-induced activation of AhR. Lastly, AhR can also be activated through 5-hydroxytryptophan (5-HTP), an intermediate molecule from the serotonin pathway. In a recent study, high levels of IL-2 led to upregulated expression of tryptophan hydroxylase 1 (TPH1) by CD8⁺ T cells, which in turn produced 5-HTP to activate AhR. CD8⁺ T cells were then rendered dysfunctional through upregulated expression of co-inhibitory receptors and reduced production of IFNγ and TNFα [77].

Fig. 2.

AhR signaling and CD8⁺ T cell dysfunction. Pro-tumor TAMs express IDO or IL4I1, which convert tryptophan into AhR agonists. AhR signaling in pro-tumor TAMs, upon exposure to such agonists, leads to the upregulation of CD39 expression, which together with CD73 expressed by T cells, results in the production of adenosine, thereby further suppressing T cells. AhR signaling in tumor cells leads to increased expression of PD-L1 and IDO, indicating that AhR favors a pro-tumor TME. Among the effects from AhR signaling in CD8⁺ T cells is the expression of co-inhibitory molecule PD-1, reduced production of IFNγ and TNFα and limited cell proliferation

AhR can also indirectly lead to CD8⁺ T cell suppression via TAMs (Fig. 2). For instance, AhR signaling in TAMs leads to upregulated expression of the ectonucleotidase CD39, which in cooperation with CD73 expressed by T cells, was shown to result in the accumulation of immunosuppressive adenosine [78]. In turn, adenosine may bind A2AR expressed by T cells and promote CD8⁺ T cell dysfunction. Specifically, AhR signaling in TAMs promoted expression of CCR2 and krüppel-like factor 4 (KLF4). While CCR2 drives TAM recruitment, KLF4-mediated suppression of NF-κB activity drives the expression of genes associated with an immunosuppressive TAM phenotype [79, 80]. In addition, AhR has been identified as a regulator of the checkpoint molecule VISTA, standing for V-domain Ig-containing suppressor of T cell activation, an important macrophage-expressed ligand that inhibits T cell function (and described in more detail below) [81].

Targeting in preclinical models

Blocking AhR signaling has been done through IDO and AhR inhibitors or kynureninase [82, 83]. Targeting AhR directly has shown potential for T cell-based therapies since adoptive transfer of tumor-specific T cells showed a similar therapeutic effect in a melanoma mouse model when combined with the AhR inhibitor 3,4-dimethoxyflavone (DMF) or with anti-PD-1 [76]. Similarly, when AhR expression is silenced in TAMs, this leads to increased CD8⁺ T cell infiltration and improved efficacy of T cell-based therapy in tuberculosis and pancreatic ductal adenocarcinoma mouse models, respectively [84, 85]. Furthermore, the AhR inhibitor BAY2416964 has shown anti-tumor efficacy in a melanoma mouse model, where more CD8⁺ T cells infiltrated the tumor and the frequency of suppressive myeloid cells, including pro-tumor TAMs, decreased [86]. Notably, there was no anti-tumor efficacy when using NSG immunodeficient mice, suggesting that the antitumor effects of BAY2416964 were immune-mediated. Besides, BAY2416964 downregulated the expression of CYP1A1 and TIPARP, while increasing the expression of TNF and CXCL9 by primary human monocytes exposed to KynA, indicating that BAY2416964 rescues their proinflammatory phenotype [86]. In another example, the AhR inhibitor IK-175 increased the number of CD8⁺ T cells in tumor-draining lymph nodes in a colon carcinoma mouse model, where these CD8⁺ T cells produced higher levels of IL-2, TNFα, and IFNγ [87]. Lastly, pegylated kynureninase, which catalyses the degradation of Kyn into anthranilic acid, has been shown to increase CD8⁺ T cell infiltration and to reduce tumor growth in combination with checkpoint inhibition in a melanoma mouse model [88]. More recently, kynureninase modified CAR-T cells showed enhanced killing capacity in a Kyn-enriched leukemia mouse model [89].

Targeting AhR in patient studies

In patients with multiple tumor types, a selective IDO1 inhibitor turned out to be ineffective in a phase 3 trial in combination with anti-PD-1 [61]. This might be explained by the presence of alternative enzymes and metabolites that can activate AhR, such as IL4I1. In fact, IL4I1 and IDO1 are expressed at the same time within the TME and IL4I1 associates more often with AhR activity than IDO1 [74, 90]. Other AhR inhibitors with promising preclinical data (see above) are currently in phase 1 trials (IK-175 from Ikena Oncology, NCT04200963, and BAY2416964 from Bayer, NCT04999202).

Galectin-3: a negative regulator of tumor cell recognition

Molecular mechanism of action

Galectin 3 (Gal3) is a β-galactoside-binding lectin expressed by macrophages, T cells and tumor cells [91]. Specifically, Gal3 has been associated with lipid-associated and tissue-resident alveolar macrophages [18]. Gal3 alters the spatial organization of immune synapses via galectin-glycoprotein lattices, which in turn leads to inefficient tumor cell recognition and killing by T cells (Fig. 3). In fact, activation of CD8⁺ T cells leads to changes in the glycosylation machinery that increases the number and accessibility of Gal3-binding motifs, possibly as a mean to keep CD8⁺ T cell activity in check [92]. Alternatively, the enzyme mannoside acetylglucosaminyltransferase 5 (MGAT5) generates N-glycan branching of glycoproteins, such as the TCR, CD8 and other cell surface receptors, thus enabling formation of Gal3-containing lattices [93, 94]. Demetriou and colleagues have shown that Mgat5-dependent binding of Gal3 inhibits TCR recruitment to the immunological synapse, thereby increasing the threshold for TCR activation [93]. Also in TILs, Gal3 prevents the co-localization of the TCR with CD8 co-receptor [95, 96]. In addition, Gal3 inhibits the docking of the microtubule-organizing center (MTOC) and the recruitment of adhesion protein lymphocyte function-associated antigen 1 (LFA-1) at the immunological synapse, thereby preventing CD8⁺ TILs from secreting lytic granules [97]. With this in mind, dysfunctional T cells as evidenced by low production of effector cytokines and lytic granules, may in fact be able to produce these molecules but in contrast fail to secrete them due to Gal3. Gal3 was also observed to bind the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3) in activated CD8⁺ T cells, which led to reduced IFNγ secretion in vitro [98]. The mechanism by which LAG-3 inhibits CD8⁺ T cell function remains unknown, yet this study points towards a potential crosslinking of LAG-3 by Gal3. Lastly, Gal3 can bind glycans of the extracellular matrix and glycosylated IFNγ, which can lead to decreased intra-tumoral IFNγ diffusion, CXCL9 expression and CD8⁺ T cell infiltration [99].

Fig. 3.

Galectin-3, Siglec-9 and Siglec-15 and CD8⁺ T cell dysfunction. Pro-tumor TAMs secrete Gal3 and express Siglec-9 and Siglec-15 on their surface. Gal3 inhibits the formation of functional immune synapses by binding to glycosylated TCR, CD8, LFA-1 and other proteins, slowing down their mobility within the plasma membrane. Siglec-9 and Siglec-15 bind sialoglycans to exert their suppressive action. Siglec-9 does not have a known receptor on T cells but binds sialic acids on tumor cells, which leads to upregulated expression of various suppressive genes. Siglec-15 may bind the heterodimer CD11b/CD18 on T cells thereby inhibiting IFNγ secretion

Interestingly, IL-10 induces the expression of Mgat5 and consequently decreases CD8⁺ T cell antigen sensitivity via Gal3 [100]. As pro-tumor TAMs produce IL-10 as well as Gal3, they are a critical cellular component regarding this suppressive IL-10-Gal3 axis [9, 101]. In macrophages, intracellular Gal3 induces inflammatory activity and phagocytosis, yet extracellular Gal3 has been shown to polarize macrophages into an anti-inflammatory phenotype [102]. By being pushed towards an anti-inflammatory phenotype via Gal3, pro-tumor TAMs may secrete extracellular Gal3 themselves to maintain a positive feedback loop [103, 104].

Targeting in preclinical models

The targeting of Gal3 ranges from the use of small molecule chemical inhibitors to natural polysaccharides and neutralizing monoclonal antibodies (mAbs) [105]. The chemical Gal3 inhibitor GB1107 reduced the growth of human lung adenocarcinoma in a xenograft mouse model and increased anti-tumor macrophage polarization as well as CD8⁺ T cell infiltration in these tumors [106]. GR-MD-02 or belapectin is a pectin-derived polysaccharide, a Gal3 inhibitor that in combination with the agonist anti-OX40 antibody was able to induce tumor regression, increased CD8⁺ T cell recruitment and reduced MDSC-mediated T cell suppression in different tumor mouse models [107]. Also, mAb 14D11 targeting Gal3 in MUC16-expressing cancer cells prolonged overall survival of ovarian tumor-bearing mice [108]. Finally, other Gal3-blocking mAbs, such as TB006 (from TrueBinding), are currently being developed and tested in other diseases, which paves the way for this therapeutic approach to enter the cancer field as well.

Targeting in patient studies

Following the described preclinical results, GB1211 (from Galecto) has been developed with high binding affinity for Gal3 and is currently tested in phase 1/2 trials in combination with anti-PD-L1/PD-1 to treat patients with NSCLC, melanoma and HNSCC (NCT05240131/NCT05913388) [109]. In addition, belapectin (from Galectin Therapeutics) in combination with anti-PD-1 has been tested in a phase 1 trial for patients with metastatic melanoma and HNSCC (NCT02575404), where increased effector memory T cell activation and reduced numbers of circulating MDSCs were observed in responders compared to non-responders [110].

Siglec-9 and Siglec-15: an unwanted partner for sialic acids

Molecular mechanism of action

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are cell surface proteins that bind sialoglycans, meaning all glycans containing one or more sialic acids, which are increased in multiple tumor types [111]. Siglec-9 is expressed by both T cells and macrophages, whereas Siglec-15 is expressed by TAMs, tumor cells and osteoclasts while generally absent in other immune cells and tissues [112, 113]. Recently, single-cell RNA sequencing and spatial transcriptomics from glioblastoma derived from patients treated with neoadjuvant anti-PD-1 therapy, has revealed a subpopulation of SIGLEC9⁺ TAMs that accumulated in patients not responding to treatment [114]. Similarly, in high-grade ovarian cancer, Siglec-9 was mainly expressed by TAMs and associated with a poor prognosis as well as dysfunctional CD8⁺ T cells [115]. Mei and colleagues have shown that absence of Siglec-9 in TAMs leads to activated T cells through TAM-mediated antigen presentation, chemokine secretion and co-stimulation [114]. In addition, it has been suggested that T cells can acquire Siglec-9 from myeloid cells via trogocytosis, leading to dephosphorylation of TCR-related signaling cascades and resulting in impaired T cell effector functions [116]. Despite a clear indication that Siglec-9⁺ TAMs induce dysfunctional T cells, surface molecules expressed on T cells that bind Siglec-9⁺ TAMs have yet to be elucidated (Fig. 3). Interestingly, binding of tumor-derived sialic acids by Siglec-9⁺ TAMs leads to increased expression of CD206, CD163, IDO and PD-L1, thus inducing TAMs with a pro-tumor phenotype [117–119]. In fact, Siglec-9 expressed by TAMs has been suggested to be the main receptor for cancer-associated sialic acids [120].

Siglec-15 expression is minimal in macrophages from normal tissues, whereas it is highly expressed by TAMs [121]. In fact, Siglec-15 might be a more specific marker for pro-tumor TAMs compared to CSF1R. Siglec-15 expressed on the surface of human monocytic leukemia cells (THP-1) was observed to bind the sialyl-Tn antigen expressed by tumor cells, which resulted in enhanced production of TGFβ by THP-1 cells [122]. Furthermore, Siglec-15 expression has been considered mutually exclusive with that of PD-L1, which may be a consequence of IFNγ upregulating expression of PD-L1 but downregulating expression of Siglec-15 [121, 123]. Wang and colleagues have attributed T cell suppressive functions to Siglec-15 (Fig. 3). In fact, they observed that recombinant Siglec-15 fusion protein suppressed CD8⁺ T cell proliferation and IFNγ secretion in vitro [121]. In the same study, it was found that Siglec-15⁺ TAMs suppressed antigen-specific proliferation of T cells via IL-10 and allowed for tumor growth in vivo. Interestingly, it has recently been suggested that the heterodimer of integrins CD11b/CD18 may represent a natural receptor of Siglec-15 [124].

Targeting in preclinical models

Targeting Siglec-mediated T cell suppression has mainly been addressed by antibodies and active removal of sialic acids from glycans on tumor and immune cells [125]. In a preclinical setting, desialylation of breast tumors repolarizes pro-tumor TAMs into anti-tumor TAMs, activates CD8⁺ T cells and increases the efficacy of checkpoint blockade [120]. This was achieved by using an antibody-silalidase conjugate in order to avoid systemic removal of sialic acids and potential toxicity. In another study, deletion of Siglece (murine homolog of Siglec-9) improved the anti-tumor efficacy of anti-PD-1/PD-L1 therapy in glioblastoma mouse models [114]. Likewise, blockade of Siglec-9 synergized with anti-PD-1 to enhance the cytotoxic activity of CD8⁺ T cells in high-grade ovarian cancer tissues [115]. Along the same lines, genetic ablation or antibody blockade of Siglec-15 amplified anti-tumor immunity and inhibited tumor growth in tumor mouse models [121].

Targeting in patient studies

In a clinical setting, the drug E-602 (from Palleon Pharmaceuticals), which enzymatically degrades immunosuppressive sialoglycans on hypersialylated tumors and immune cells, is currently being evaluated alone or in combination with anti-PD-1 in patients with advanced solid tumors in a phase 1/2 trial (NCT05259696). From this trial, it has already been observed that E-602 in combination with anti-PD-1 decreased the frequency of CD163⁺ TAMs within the tumor in 8 out of 10 patients [126]. In another phase 2 clinical trial with advanced non-small cell lung cancer patients (NCT04699123), a humanized anti-Siglec-15 mAb, named NC318 (from NextCure), is currently being investigated alone or in combination with anti-PD-1.

B7-H4 and VISTA: co-inhibition beyond PD-L1

Molecular mechanism of action

B7-Homologs 4 (B7-H4) and B7-H5 (also named VISTA) are emerging co-inhibitory molecules that negatively regulate T cell function [127–130] (Fig. 4). Their binding partners on T cells are not fully clear yet, although VISTA has been suggested to act as a ligand as well as a receptor [131–133]. While VISTA is highly expressed by TAMs and to a lesser extent by T cells, B7-H4 is expressed by TAMs and tumor cells [130]. A pioneering study observed that even though primary ovarian tumor cells expressed intracellular B7-H4, it was TAMs expressing cell surface B7-H4 that suppressed T cell proliferation, IL-2 production and cytotoxicity in vitro [134]. In addition, B7-H4 fusion protein is able to inhibit T cell activation in vitro by interfering with TCR/CD28 signaling pathways [135]. In concordance with these findings, a recent study demonstrated that the frequency of B7-H4⁺ TAMs correlated with the extent of CD8⁺ T cell dysfunction in human hepatocellular carcinoma [136]. At the molecular level, the suppressive capacity of B7-H4⁺ TAMs was independent of arginase, inducible nitric oxide synthase or PD-L1, and could possibly be through upregulated expression of the transcription factor Eomes [134, 136].

Fig. 4.

B7-H4 and VISTA and CD8⁺ T cell dysfunction. B7-H4 is expressed by pro-tumor TAMs and binds an unknown receptor on T cells. VISTA is expressed on both pro-tumor TAMs and T cells and binds to PSGL-1 in acidic environments and LRIG1 in both acidic and non-acidic environments. VISTA and LRIG1, when expressed by the same T cells, can also interact in cis. B7-H4 and VISTA-driven interactions inhibit TCR signaling and downregulate many T cell effector functions; they also lead to upregulated expression of PD-1

VISTA can suppress T cells by engaging its receptor on T cells or by eliciting intrinsic suppression when expressed on T cells [137, 138] (Fig. 4). P-selectin glycoprotein ligand-1 (PSGL-1) is expressed by T cells and has been proposed to selectively bind VISTA at acidic pH, which is a main feature of the TME [139, 140]. This interaction leads to suppression of proliferation, decreased production of IFNγ, TNFα and IL-2 as well as upregulated expression of PD-1 by CD8⁺ T cells [139, 141]. More recently, leucine-rich repeats and immunoglobulin-like domain 1 (LRIG1) was identified as another receptor expressed by T cells that interacts with VISTA at both neutral and acidic pH [142]. Again, this engagement led to reduced proliferation, survival and effector function of T cells, likely by disrupting proximal and distal TCR signaling pathways. Moreover, Ta and colleagues demonstrated that VISTA, besides acting in trans through TAM: T cell interactions, may also act in cis through T cells expressing both VISTA and LRIG1 [142]. Other molecules that have been reported to bind VISTA include V-set and immunoglobulin domain-containing protein 3 (VSIG-3) and Galectin-9 (Gal9), which upon binding lead to reduced production of cytokines in vitro as well as T cell apoptosis, respectively [113, 143]. Yet, in these latter two interactions VSIG-3 and Gal9 act as ligands expressed by tumor cells and VISTA acts as a receptor expressed by T cells. Regarding VISTA⁺ TAMs, these have been observed in multiple tumor types, amongst which pancreatic tumors, where they were responsible for inhibition of IFNγ and TNFα production in vitro by intra-tumoral CD8⁺ T cells to a greater extent than PD-L1-mediated inhibition [144]. In contrast to other co-inhibitory receptors that restrain T cell responses in every tissue, VISTA appears to have similar suppressive effects but in acidic environments such as tumors. Notably, a recent study identified AhR as a regulator of VISTA expression in macrophages, underpinning the crosstalk between these two molecules towards T cell dysfunction [81]. In fact, AhR may as well regulate VISTA expression on T cells but further studies are needed to elucidate it.

Targeting in preclinical models

Targeting of B7-H4 and VISTA is generally performed through antibody-drug conjugates, bispecific antibodies, mAbs and small molecule inhibitors. For instance, the antibody-drug conjugate SGN-B7H4V was able to shift the TME towards antigen presentation pathways and demonstrated strong anti-tumor activity in xenograft models of breast and ovarian cancer, which was further enhanced by anti-PD-1 [145]. Furthermore, a bispecific antibody targeting B7-H4 and CD3 has shown anti-tumor activity in humanized mouse tumor models, which was accompanied by increased CD8⁺ T cell infiltration [146]. With a different approach, Li and colleagues demonstrated that mAb-mediated blockade of B7-H4 and PD-1 showed synergic effects on inhibition of tumor growth [136]. Similarly, mAb-mediated co-blockade of VISTA and PD-1 has shown inhibited tumor growth in vivo, enhanced T cell infiltration and reduced expression of co-inhibitory receptors by intra-tumoral T cells [139]. Notably, the pH-selective VISTA mAb SNS-101, in combination with anti-PD-1, showed in vivo anti-tumor efficacy, while increasing CD8⁺ T cell numbers and shifting TAMs from a pro-tumor to an anti-tumor phenotype [147]. Also, two other anti-VISTA mAbs, HMBD-002 and KVA12123, were able to revert myeloid-mediated T cell suppression in vitro and led to strong anti-tumor activity, as single agent or in combination with anti-PD-1 treatment in several mouse models [148, 149]. Beyond antibodies, the small molecule inhibitor CA-170 (from Curis/Aurigene) is able to bind to VISTA as well as PD-L1 and improved anti-tumor T cell responses in a number of immunocompetent mouse tumor models [150].

Targeting in patient studies

The B7-H4/CD3 bispecific antibody GEN1047 (from Genmab) as well as the antibody-drug conjugates AZD8205 (from AstraZeneca) and SGN-B7H4V (from Pfizer) are being tested in patients with multiple types of solid tumors in phase 1/2 trials (NCT05180474/NCT05123482/NCT05194072). Regarding VISTA, the mAbs HMBD-002 (from Hummingbird Bioscience) and KVA12123 (from Kineta) are currently being tested in patients with advanced solid tumors, alone or in combination with anti-PD-1 in phase 1 and 2 trials (NCT05082610/NCT05708950). Finally, the pH-selective VISTA mAb SNS-101 has recently entered into phase 1/2 clinical trials as monotherapy or in combination with anti-PD-1 for patients with advanced solid tumors (NCT05864144).

Rescuing T cells from TAM-mediated suppression

The efficacy of T cell-based therapy varies between cancer types and even between patients with the same type of tumor, which indicates that the exact mechanisms of T cell suppression, and in particular pro-tumor TAM-mediated T cell suppression, may be overlooked. In fact, targeting TAMs is already in clinical practice yet often needs to be combined with other therapies to show modest efficacy at best (Table 1) [7, 16, 151]. One explanation for the current limited effect is that depletion of macrophages, currently the most used strategy to target TAMs, not only eliminates pro-tumor TAMs, but potentially also eliminates anti-tumor TAMs as well as tissue-resident macrophages in healthy tissues, thereby adversely affecting the functioning of the patient’s immune system. Secondly, therapies using CSF1R or TREM2 inhibitors may lead to an inefficient depletion of TAM subsets since the expression of these receptors and consequently the sensitivity towards these therapies is variable among different TAM subsets [69, 152]. Finally, the use of anti-CSF1R may lead to the unintended activation of regulatory T cells, recruitment of suppressive myeloid cells and activation of the tumor-promoting phosphoinositide 3-kinase (PI3K) pathway, all negatively impacting an anti-tumor T cell response [153–155]. To improve the efficacy of strategies that target TAMs, it is imperative to understand and molecularly exploit the mechanism of action used by pro-tumor TAMs to suppress CD8⁺ T cells. At the same time, new therapeutic approaches should spare anti-tumor TAMs, which not only promote anti-tumor CD8⁺ T cell responses but also play a role in tertiary lymphoid structures that support and amplify anti-tumor T cell responses [156]. Along these lines, there is now considerable evidence that T cell-based therapy itself leads to the polarization of TAMs towards an anti-tumor phenotype and eventually depends on such anti-tumor TAMs to be durable and effective [26, 31, 157–159]. Taken these premises into account, we are here emphasizing new avenues and interventions to selectively provide T cells with resistance to macrophage suppression.

To overcome AhR-mediated T cell suppression, one can target AhR itself, the ligands or their sources. To date, this is mainly done through IDO1 inhibitors, which are generally ineffective in larger clinical studies, although AhR inhibitors are emerging as promising alternatives. However, targeting AhR is challenging due to its ligand- and context-dependent effects. For example, AhR signaling in CD8⁺ T cells has also been shown to promote T cell memory differentiation as well as to enhance anti-tumor immunity and improve checkpoint inhibitor efficacy in melanoma [160–162]. Bearing this in mind, targeting AhR directly could be a double-edged sword. Instead, IL4l1 is selectively used by TAMs and represents a novel target to revert TAM-mediated T cell suppression. IL4I1 has been proposed to be the main enzyme responsible for Trp catabolism, which is a prominent metabolic mechanism of T cell suppression [74]. Furthermore, IL4I1 expression and AhR activation were induced in advanced melanoma after treatment with anti-PD-1, suggesting that IL4I1-AhR pathway may take part in resistance against ICB therapy [74]. In fact, IL4I1 and IDO1 inhibitors may be used in combination to block two distinct pathways of Trp catabolism, yet whether this leads to synergistic effect still needs to be elucidated. Other questions remain, such as: how do AhR⁻ CD8⁺ T cells perform in pre-clinical tumor models?; and, how important is AhR signaling in pro-tumor TAMs to indirectly suppress CD8⁺ T cells?. In the end, T cell-based therapy may benefit from context-specific AhR inhibition or from blocking TAM-mediated production of AhR agonists, such as IL4I1 inhibitors.

Targeting of Gal3, Siglec-9 and Siglec-15 can be achieved by monoclonal antibodies or chemical inhibitors. Gal3 has been suggested to be part of resistance mechanisms following PD-1/PD-L1 blockade, therefore holding promise to be targeted in combination therapies [109]. Current targeting approaches focus on Gal3 itself, while an alternative is to specifically target the N-glycan branching of T cell proteins to prevent Gal3 binding. Such an approach has given promising results in boosting the efficacy of adoptive transferred T cells against solid tumors [163]. A similar rationale can be applied to overcome Siglec-mediated suppressive mechanisms through desialylation. Indeed, Siglec-9 can be highly expressed by human TILs, which can lead to inhibited T cell responses when binding to sialoglycans expressed by tumor cells or other immune cells [164, 165]. Therefore, targeted desialylation may offer an unique opportunity to protect T cell function from Siglec-9 and Siglec-15 inhibition. Hence, T cell based-therapy can be combined with agents that locally disrupt N-glycan branching and/or desialylation. However, more studies into these pathways in the context of TAM: T cell interactions are needed. In example, to improve T cell-based therapy, it may be beneficial to know silencing MGAT5 expression in T cells could boost their activity or lead to unwanted effects, and which receptors expressed by T cells actually bind Siglec-9 and Siglec-15. Interestingly, Siglec-15 expression and its mechanism of action are non-redundant to the PD-1/PD-L1 axis, which warrants further research into this mechanism.

B7-H4 and VISTA are predominantly expressed by pro-tumor TAMs and are primarily targeted by antibodies, which have shown great promise to be combined with T cell-based therapies. Antibody-drug conjugates directed towards B7-H4 are mainly intended to kill tumor cells, yet it might be an alternative to focus on TAM-centered approaches, such as bispecific or monoclonal antibodies. In this regard, the pH-selective VISTA mAb SNS-101 may be at forefront as it mitigates cytokine release syndrome and avoids rapid drug clearance, common issues generally encountered by other mAbs targeting VISTA [147]. Interestingly, VISTA and PD-L1 were found to be expressed in different lesions of the same tumor and pro-tumor TAMs express either VISTA or PD-L1 after anti-CTLA-4/PD-1 therapy, but rarely both [166–168]. In addition, in a colorectal cancer mouse model, anti-VISTA led to the induction of T cell pathways highly distinct from and complementary to those induced by anti–PD-1 therapy [169]. These studies indicate that VISTA and PD-1 are non-redundant pathways and can be targeted in combination with ICB or gene-engineered T cells [170]. Nevertheless, a caveat for exploring these targets therapeutically may be that the receptors on T cells that bind either B7-H4 or VISTA expressed by TAMs are still unclear. In addition, it is critical to elucidate cis-mediated VISTA suppression in order to facilitate the design of effective therapies.

Importantly, given the novelty of the selected targets, it is still unclear to which extent the tumor type and the immune context affect the above described molecular interactions between pro-tumor TAMs and CD8⁺ T cells.

Finally, we advocate for further mechanistic studies to bring forward new and better understanding concerning the role of TAMs in dysfunctional anti-tumor T cell responses. Recent omics technologies are contributing to the development of next generation immunotherapies by enabling the study of TMEs to a deeper level [171, 172]. These technologies are already being used for characterization of pan-cancer TMEs, for definition of intra-tumoral T cell states and for uncovering macrophage complexity within specific contexts [17–19, 173–176]. Although these studies are definitely elucidating the complexity of tumors, they mainly provide a descriptive snapshot instead of mechanistic understanding of cell interaction within the TME. For that purpose, functional genomics and mechanistic studies may generate a more complete understanding of tumor heterogeneity towards therapy [177–179]. For instance, Abdrabou and colleagues have used gene editing approaches to identify regulators of VISTA expression in macrophages, coming then to realize that by targeting those genes and pathways they could shift macrophage phenotype towards a pro-inflammatory state [81]. Similarly, Sheban and colleagues used gene editing, single-cell sequencing and a deep generative model to find out that ZEB2 suppresses interferon response and antigen presentation in TAMs, thus driving them to a pro-tumor phenotype [180]. By targeting ZEB2, they were able to reprogram TAMs to anti-tumor phenotype, enhance anti-tumor T cell responses and led to tumor control. In another example, Carnevale and colleagues identified RASA2 as a signaling checkpoint in suppressed human CD8⁺ T cells and its ablation enhanced the efficacy of T cell-based therapy [181]. Such studies are expected to aid the development of combination and synergistic therapies, aimed at counteracting TAM-mediated T cell suppression.

Conclusion

TAMs are essential to build and sustain an effective anti-tumor T cell response, yet they are often subverted to a pro-tumor phenotype to keep T cell function in check and limit the efficacy of T cell-based therapies. Thus far, the success of TAM-targeting therapies is limited, which implies the need for alternative targets that are linked to exact mechanisms of action underlying pro-tumor TAM-mediated suppression of T cells. Here, we revise how T cell immunity depends on co-existence with TAMs, and which molecular interactions between these two cell types steer either a stimulatory or suppressive T cell response against tumors. Examples of emerging targets expressed by pro-tumor TAMs to boost anti-tumor T cell immunity include the transcription factor AhR, the lectins Gal3, Siglec-9 and Siglec-15 as well as the co-inhibitory molecules B7-H4 and VISTA. AhR regulates the expression of many T cell suppressive pathways, providing multiple targeting options. Gal3 can interfere with T cell proteins essential for immune synapse formation and tumor cell recognition. Siglec-9, Siglec-15, B7-H4 and VISTA are immune checkpoint molecules with T cell inhibitory functions. Notably, some of these targets, such as IL4I1, Siglec-15 and VISTA, seem to be highly restricted to TAM-mediated, but not tumor cell-mediated, T cell suppression. In addition, Siglec-15 and VISTA and their pathways are non-redundant to PD-1/PD-L1 pathways and targeting those molecules may enhance the efficacy of anti-PD-1 blockade. To move the development of these targets further, more explorative studies on binding partners and mechanisms of action are still needed, to which end we require functional genomics and intervention studies. Ultimately, to move the development of these targets further, more explorative studies on binding partners and mechanisms of action are still needed, to which end we require functional genomics and mechanistic studies. Besides offering novel targets for antibodies and small molecules, such developments would enable the use of next generation T cells, engineered not only with a tumor-specific CAR or TCR but also engineered to be resistant against TAM-mediated suppression. This approach may overcome suppressive pathways without systemic depletion of cell subsets or administration of blocking monoclonal antibodies, and is anticipated to enhance the efficacy of T cell therapies in a larger population of cancer patients.

Abbreviations

A2AR

Adenosine A2a Receptor

AhR

Aryl hydrocarbon Receptor

B7-H4

B7 Homolog 4

CAR

Chimeric antigen receptor

CD8

Cluster of differentiation 8

CSF1R

Colony Stimulating Factor 1 Receptor

CTLA-4

Cytotoxic T Lymphocyte Antigen 4

CXCL10

C-X-C Motif Chemokine Ligand 10

CXCR3

C-X-C Motif Chemokine Receptor 3

CYP1B1

Cytochrome P450 Family 1 Subfamily B Member 1

Eomes

Eomesodermin

IDO

Indoleamine-2,3-Dioxygenase

IFNγ

Interferon Gamma

Gal3

Galectin-3

ICB

Immune checkpoint blockade

I3A

Indole-3-Aldehyde

I3P

Indole-3-Pyruvic Acid

IL-2

Interleukin-2

IL4I1

Interleukin-4-Induced 1

KLF4

Kruppel-Like Factor 4

Kyn

Kynurenine

KynA

Kynurenic Acid

LAG-3

Lymphocyte-Activation Gene 3

LFA-1

Lymphocyte Function-Associated Antigen 1

LRIG1

Leucine-Rich Repeats And Immunoglobulin-Like Domain 1

mAb

monoclonal Antibody

MGAT5

Mannoside acetylglucosaminyltransferase 5

MHC-I

Major histocompatibility complex class I

MTOC

Microtubule-Organizing Center

NF-κB

Nuclear Factor Kappa-light-chain-enhancer of Activated B cells

NSG

NOD scid gamma

PAT4

Proton-assisted Amino Acid Transporter 4

PD-1

Programmed Cell Death 1

PD-L1

Programmed Death-Ligand 1

PSGL-1

P-Selectin Glycoprotein Ligand-1

RASA2

Ras P21 Protein Activator 2

Siglec-15

Sialic acid-binding Immunoglobulin-type Lectin 15

SLC7A8

Solute Carrier Family 7 Member 8

SPP1

Secreted Phosphoprotein 1

TAM

Tumor-associated macrophage

TCR

T cell receptor

TGFβ

Transforming Growth Factor Beta

TIL

Tumor-Infiltrating Lymphocyte

TIM4

T cell Immunoglobulin And Mucin Domain-Containing 4

TIPARP

TCDD-Inducible Poly(ADP-Ribose) Polymerase

TLR4

Toll-Like Receptor 4

TME

Tumor microenvironment

TNFα

Tumor Necrosis Factor Alpha

Trp

Tryptophan

TPH1

Tryptophan Hydroxylase 1

VISTA

V-domain Ig Suppressor of T cell Activation

VSIG-3

V-Set And Immunoglobulin Domain-Containing Protein 3

YTHDF2

YTH N6-Methyladenosine RNA Binding Protein 2

ZEB2

Zinc Finger E-Box Binding Homeobox 2

Authors’ contributions

R.M.L.C. was responsible for literature collection, its interpretation and writing of the original draft of the manuscript. R.M.L.C. created the figures. All authors conceived the structure of the manuscript, processed literature, revised the manuscript and approved the final version.

Funding

This work was funded by KWF-TKI 2024-PPS-1/16295.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.