Leukocyte immunoglobulin-like receptors in human diseases: an overview of their distribution, function, and potential application for immunotherapies

Review of how LILRs modulate plasticity of myeloid cell function, and control human diseases.

Abstract

Myeloid-derived suppressor cells (MDSCs), a population of immature myeloid cells expanded and accumulated in tumor-bearing mice and in patients with cancer, have been shown to mediate immune suppression and to promote tumor progression, thereby, posing a major hurdle to the success of immune-activating cancer therapies. MDSCs, like their healthy counterparts, such as monocytes/macrophages and granulocytes, express an array of costimulatory and coinhibitory molecules as well as myeloid activators and inhibitory receptors, such as leukocyte immunoglobulin-like receptors (LILR) A and B. This review summarizes current findings on the LILR family members in various diseases, their potential roles in the pathogenesis, and possible strategies to revert or enhance the suppressive function of MDSCs for the benefit of patients by targeting LILRs.

Introduction

The LILRs, also known as CD85 and ILTs, are a family of type I transmembrane glycoproteins with extracellular Ig-like domains that bind ligands and ITAMs or ITIMs. LILRBs possess 2 or 4 Ig-like domains and a long cytoplasmic tail with ITIMs. Conversely, LILRAs have 2 or 4 Ig-like domains with short cytoplasmic tails and associate with an FcγR chain containing ITAMs. The soluble forms of LILRs without transmembrane and cytoplasmic regions are generated by alternative splicing, thereby serving as decoy regulators. The LILR family comprises primate-specific receptors in terms of sequence homology, and PIR-A and PIR-B have been proposed as the murine orthologs of LILRs.

Many LILRA/B ligands have been identified and can be categorized into 2 groups: membrane-bound proteins, such as major MHC class I or HLA class I molecules, and soluble proteins, such as ANGPTLs, myelin inhibitors, and S100A8/9. However, ligands for some LILRs, such as LILRB3 and LILRB4, remain unknown. The LILRA/B ligands are summarized in Table 1. Because of the few studies published on the interaction between LILRs and their ligands, the biologic function and clinical significance of the ligand-mediated function is still not well understood. However, it is becoming clear that LILRs, with their capacity to regulate immune responses and mediate protumor functions, represent a new class of receptors that can be targeted for the treatment of a variety of immunologic disorders and cancer.

TABLE 1.

Ligand and cellular distribution of LILRs

LILR	Functional ligands	Distribution
LILRA1	HLA-B27 [85], HLA-C free heavy chain [86]	Mac [87], Mon, and B cells [88]
LILRA2	Unknown	Mon, Mac, DC, Gr [89–91], subsets of T cells, and NK cells [1]
LILRA3	HLA-C free heavy chain [86]	Mon, subsets of T cells, and B cells [14]
LILRA4	BST2 [92]	pDC [93]
LILRA5	Unknown	Mon, PMN [2]
LILRA6	Unknown	Mon [94]
LILRB1	HLA class I [88, 31, 95–96], UL18 [43], dengue virus product [47], S100A8/9 [98]	Mon, Mac, DC, Gr, B cells, T cells, NK cells [88, 31, 99, 100], mast cells progenitor [101], osteoclasts [102], and placental stromal cells [103]
LILRB2	HLA class I [88, 95, 97, 104] CD1d [105], ANGPTL [75, 106], Aβ oligomers [9], myelin inhibitors [8]	Mon, Mac, DC, Gr [95, 104], mast cell progenitor [101], osteoclasts [102], endothelial cells [54], placental vascular smooth muscle [103], HSC [75], and neuron [9]
LILRB3	Unknown	Mon, DC, Gr [90, 91, 64], mast cells progenitor [101] and osteoclasts [102],
LILRB4	Unknown	Mon, Mac, DC [88, 107], plasmablasts [108], mast cells progenitor [101], osteoclasts [102]
LILRB5	HLA class I heavy chains [109]	Mon, NK cells [88], mast cell granules, and mast cells progenitor [101]

Abbreviations: Aβ, β-amyloid; BST2, bone marrow stromal cell antigen 2; Gr, granulocyte; Mac, macrophage; Mon, monocyte; pDC, plasmacytoid DC.

LILRs are widely expressed in hematopoietic-lineage cells (Table 1) and mediate activation or inhibition of the functions of various immune cells, primarily myeloid cells. Cross-linking of LILRA2 or LILRA5 on monocytes induces proinflammatory cytokines [1, 2]. Membrane and soluble LILRB4 are critical to the generation of T suppressor cells and the induction of immunologic tolerance. LILRB2/4 is indispensable for induction of the tolerogenic phenotype of APCs. MDSCs are a population of immature myeloid cells expanded and accumulated in tumor-bearing mice and in patients with cancer. We and other groups have reported that MDSCs induced the activation and development of T_regs and T cell anergy in tumor microenvironment [3]. Importantly, we demonstrated that PIR-B, the mouse ortholog of LILRB2, regulates the suppressive function and fate of MDSCs [4]. PIR-B deficiency promotes the acquisition of M1 functional phenotypes in MDSC, which can suppress lung metastasis of Lewis lung carcinoma.

In addition to immune cells, LILRBs and LILRB-related, ITIM-containing receptors are expressed by tumor cells. These inhibitory receptors may directly mediate the development of certain hematopoietic malignancies, such as primary human AML [5, 6] and B cell CLL [7]. Furthermore, LILRBs are expressed or up-regulated in solid cancer cells, such as lung, stomach, breast, and pancreas cancer cells. More recently, LILRB2 has been identified as a high-affinity receptor for β-amyloid oligomers and myelin inhibitors of axonal regeneration (Nogo, MAG, and OMgp) [8, 9], suggesting it has a role in the pathogenesis of neurodegenerative disorders.

In this review, we will summarize recent research on the function and potential roles of LILRs in the context of different disease conditions and cancer development and how LILRBs can be targeted to convert the immunosuppressive function of MDSCs into an immunostimulatory phenotype. Such knowledge will pave the way for future studies that focus on exploiting the immunoregulatory properties of LILRs for immunotherapies of infections, autoimmune diseases, and cancer.

LILRs IN AUTOIMMUNE DISEASES

In PIR-B (the LILRB2 ortholog)–deficient mice, the impaired maturation of DCs and the defective regulation of receptor-mediated activation in APCs lead to exaggerated Th cell type 2–prone immune responses and B cell hypersensitivity [10]. In a collagen-induced, arthritis mouse model, binding of PIR-B to HLA-G dimers mediates the long-term immunosuppressive effects [11]. These murine studies suggest that PIR-B has an indispensable role in immunologic homeostasis and maintenance of self-tolerance.

Indeed, aberrant expressions of LILRBs have been shown to be associated with human autoimmune diseases, such as SLE, RA, and MS (Table 2). Significantly less inhibitory activity by LILRB1s on T cells and reduced expression of LILRB1 on B cells were observed in patients with SLE when compared with healthy donors [12]. Phenotypic analysis of IL-10–treated, monocyte-derived DCs in patients with SLE suggested that enhanced LILRB1 expression might have a role in tolerogenic DC functions [13]. In patients with RA, the serum level of LILRA3 is correlated with disease activity [14]. Up-regulated LILRA2, LILRB2, and B3 expression was found in synovial tissue, and the number of LILRB-expressing inflammatory cells was significantly decreased in patients who responded to antirheumatic treatment, because of partial inhibition of LIRA2-mediated TNF-α production [15]. It has also been suggested that LILRB2 contributes to the pathogenesis in spondyloarthritis [16] because recognition of the different forms of HLA-B27 by LILRB2 could affect the function of inflammatory cells from both innate and adaptive immune systems [17]. MS is an inflammatory disorder of the brain and spinal cord in which focal lymphocytic infiltration leads to damage of the myelin and axons [18]. It has been reported that LILRA3 deficiency is associated with MS [19, 20]. In patients with MS, the abnormal coexpression of LILRB1 and its ligand HLA-G in CNS cells and in areas of microglia activation suggests that LILRB1/HLA-G signaling might participate in the immune regulation of the CNS [21]. Furthermore, LILRB2 has been shown to inhibit axonal regeneration through interaction with myelin inhibitors [8] and to promote the development of Alzheimer’s disease via binding to oligomeric forms of β-amyloid [9].

TABLE 2.

LILRs in infections and autoimmune and inflammatory diseases

LILRs	Disease associations
LILRA2	Up-regulated in synovial tissue of patients with RA [15]
	Polymorphism associated with susceptibility to autoimmune diseases (SLE, MPA) [27]
LILRA3	Serum level is correlated with RA activity [14]
	Homozygosity for the nondeleted allele confers susceptibility to RA, SLE, and Sjögren’s syndrome [28, 29]
	Homozygous deficiency is a risk factor for MS [19, 20]
	Up-regulated in lepromatous leprosy skin lesions; inhibits DC maturation and antimicrobial activity [25]
LILRB1	Down-regulation of inhibitory property in patients with SLE [12]
	Coexpressed with HLA-G on CNS cells and in areas of microglia activation in patients with MS [21]
	Polymorphism associated with susceptibility to autoimmune diseases (RA) [30]
	Number of LILRB1+ B cells correlates with severity of malaria [36]
	Up-regulation of NK and T cells in patients with HIV [38–40]
	Involved in human CMV and dengue virus infection [43, 44, 45, 47, 48]
	Involved in HLA-G–mediated allograft tolerance [59, 61]
LILRB2	Up-regulated in synovial tissue of patients with RA; suggested to contribute to spondyloarthritis [15]
	Inhibits axonal regeneration; promotes the development of Alzheimer’s disease [8, 9]
	Up-regulated in response to Salmonella infection [37]
	Up-regulated by IL-10 in patients with HIV [41]
	Higher binding affinity for HIV-1 viral mutant [94]
	Up-regulated on tolerogenic APC (DCs, endothelial cells)–mediated allograft tolerance [50]
	Involved in HLA-G–mediated allograft tolerance [59, 62]
LILRB3	Up-regulated in synovial tissue of patients with RA [15]
	Polymorphism associated with susceptibility to Takayasu’s arteritis [32]
	Polymorphism involved in graft-vs.-host responses and graft-vs.-leukemia activity after HSCT [65]
LILRB4	Polymorphism associated with decreased LILRB4 expression on myeloid cells in patients with SLE [33]
	Up-regulated in response to Salmonella infection [37]
	Up-regulated on tolerogenic APC (DCs, endothelial cells)–mediated allograft tolerance [50]
LILRB5	Involved in creatine kinase clearance [34]

Abbreviation: MPA, microscopic polyangiitis.

In addition to abnormal expression of LILRs in autoimmune diseases, polymorphisms of LILRs have been shown to be associated with autoimmune disorders. LILRs are polymorphic proteins [22–26]. Individuals with a splice-site SNP (rs2241524) in LILRA2, which results in a novel isoform expression on the surface of monocytes, were more susceptible to SLE and microscopic polyangiitis [27]. Moreover, nondeleted LILRA3 (functional LILRA3) confers susceptibility to RA, SLE, and Sjögren’s syndrome [28, 29]. The polymorphisms of LILRB1 are associated with susceptibility to RA in HLA-DRB1 SE-negative patients, possibly because of insufficient inhibitory signaling in their leukocytes [30]. Compared with LILRB1 and LILRB2, LILRB3 is highly polymorphic [21]. A genome-wide association study by Renauer et al. [32] identified an SNP in LILRB3 as a genetic susceptibility locus for Takayasu’s arteritis in Turkish and North American cohorts, implicating the diminished inhibitory signaling results in the augmented immune responses. LILRB4 is also highly polymorphic. A functional genetic polymorphism study [33] reported that decreased expression of LILRB4 on circulating monocytoid DCs was observed in European-derived and Hispanic-American patients with SLE with an SNP (rs11540761) in the extracellular region of LILRB4. That low-expression allele (rs11540761) and another SNP allele located in the cytoplasm (rs1048801) were also independently associated with an increased level of serum type I IFN activity, suggesting LILRB4 has an immune suppression role in the pathogenesis of SLE [33]. Although the function of LILRB5 remains poorly characterized, a recent genome-wide association study on statin users and nonusers suggested that LILRB5 present in the mononuclear phagocytic system of the liver might have a role in creatine kinase clearance [34].

LILRs IN INFECTIOUS DISEASES

Although LILRs have pivotal roles in the immunologic balance, in certain circumstances, with bacterial or viral infections, they may behave as pathogenic mediators because of their immune-modulatory properties. Genetic analysis of skin biopsy from patients with lepromatous leprosy has shown that multiple LILR members, especially LILRA2, are up-regulated, which can shift the balance of cytokine production, convert the innate response from the proinflammatory to anti-inflammatory phenotype, and inhibit TLR-induced antimicrobial activity [35]. Infection with Plasmodium falciparum can result in malaria associated with inflammatory cytokine release. Patients with severe malaria have significantly more LILRB1⁺ apoptotic B cells when compared with those with uncomplicated cases or healthy controls, and those B cells may be a contributor to such increased inflammatory cytokine production in the peripheral blood [36]. In addition, LILRB2 and LILRB4 were up-regulated in response to Salmonella infection, and LILRB4 ligation can modulate the phenotype of APCs and alter cytokine production [37].

LILRB1 and LILRB2 have been implicated in the regulation of NK cell and CD8 T cell function in HIV-infected patients. Up-regulated expression of LILRB1 inn NK cells and CD8 T cells and LILRB2 on myelomonocytic cells was observed in HIV-infected patients, especially during chronic infection [38–40]. This may be a consequence of an elevated serum level of IL-10 produced by HIV-infected monocytes, which promote the expression of LILRBs [41]. These HIV-infected monocytes exhibit enhanced LILRB2 expression and decreased Ag-presenting ability, leading to diminished antiviral T cell responses [41]. Furthermore, a recent study [42] reported that the binding strength of LILRB2 to HLA class I alleles positively correlated with viral load in a large cohort of untreated patients with HIV-1. The impaired Ag-presenting properties of DCs may be mediated by LILRB2 and HLA interaction [42]. Furthermore, LILRB1 can recognize the human CMV HLA class I homolog UL18 [43]. Although the role of an LILRB1/UL18 interaction in human CMV infection remains controversial [43, 44, 45], the correlation between up-regulated expression of LILRB1 and CMV infection after lung transplantation has been validated, implicating the level of LILRB1 expression as a prognostic biomarker for CMV disease in patients with lung transplants [46]. In addition to CMV infection, LILRB1 is also involved in the pathogenesis of dengue virus. Ab-opsonized dengue virus colligates LILRB1, which results in inhibition of FcγR signaling and down-regulation of type I IFN-stimulated genes, leading to enhancement of the viral infection [47, 48]. However, the ligand of LILRB1 on dengue virus remains unknown.

LILRBs IN TRANSPLANTATION TOLERANCE

APCs have a central role in transplant immunity. Both donor and host APCs can present allogeneic MHC Ags and minor histocompatibility Ags to T cells, resulting in the activation of alloreactive T cells that mediate allogeneic graft rejection [49]. The crucial role of LILRB2 and LILRB4 in CD8⁺ T_reg-induced tolerization of DCs has been demonstrated [50], and those receptors can also be up-regulated to generate tolerogenic DCs via the stimulation of the IL-10 and INF-α [51–53], Thus, the LILRBs may represent ideal therapeutic targets for induction of transplantation tolerance to prevent allograft rejection.

In an organ transplantation setting, LILRB-mediated initiation of the T cell suppression cascade induces immune tolerance and enables graft acceptance. CD8⁺ alloantigen-specific T suppressor cells can enhance the expression of LILRB2 and LILRB4 on monocytes and DCs and confer tolerogenicity on the APCs. Tolerogenic APCs show reduced expression of costimulatory molecules, such as CD80/86, and inhibit proliferation of alloreactive CD4⁺ Th cells [50]. In addition to professional APCs, CD8⁺ T suppressor cells can up-regulate the expression of LILRB2 and LILRB4 while down-regulating the costimulatory molecules on endothelial cells, rendering those nonprofessional APCs tolerogenic [54, 55]. Clinic studies have reported that CD8⁺ T suppressor cells can mediate their immune-suppressive function in patients with heart, kidney, and liver transplantations [50, 56, 57]. LILRB1 and LILRB2 can also mediate graft tolerance by binding to HLA-G. Elevated HLA-G levels are positively correlated with better graft acceptance in patients with renal transplants [58]. LILRB (B1 and B2) engagement with both membrane-bound and soluble HLA-G from the serum of transplant patients was associated with decreased T cell proliferation and increased T_reg populations [59]. Similar results were found in a LILRB2/HLA-G1 transgenic mouse model [60]. Furthermore, interaction between LILRB1 and HLA-G induces the expansion of the CD11b⁺Gr-1⁺ MDSCs, which are regulatory cells with strong suppressive functions. Engagement of LILRB1 by HLA-G up-regulates the expression of IL-4 and IL-13, which are directly involved in the induction of Arg-1, resulting in the inhibition of the alloreactive T cell response and prolonging skin allograft survival [61]. A recent study [62] reported that LILRB2/HLA-G interaction was involved in maternal–fetal tolerance. In pregnant women, LILRB2 recognition of soluble HLA-G mediated phosphorylation of STAT3 and induced IDO in MDSCs, leading to MDSC accumulation and activation [62]. A potential mechanism for MDSC-mediated immunosuppressive activities through LILRB1/2 and HLA-G interactions is proposed in Fig. 1.

Figure 1. Proposed potential mechanism of MDSC-mediated T cell suppression through LILRB1/2 and HLA-G interactions.

Engagement of LILRB1 and HLA-G increases the production of IL-4 and IL-13 [61], which, in turn, actives the IL-4Rα/STAT6 signaling pathway [110, 111], resulting in enhanced production and activation of arginase-1, which inhibits T cell proliferation. HLA-G also interacts with LILRB2; the consequences of which enhance phosphorylation of STAT3, leading to induction of IDO, which mediates T cell suppression [62, 112].

GVHD remains the major complication of allogeneic HSCT. Both host and donor APCs have key roles in triggering and amplifying allogeneic responses. A study on murine GVHD has demonstrated that PIR-B–deficient mice receiving allogeneic donor T cells showed exacerbated GVHD compared with wild-type recipient mice, which was due to augmented activation of PIR-B–deficient recipient’s DCs [63]. Another murine study [64] showed that PIR-B lentivirus-transfected DCs prevented acute GVHD. Our previous study [4] demonstrated that PIR-B regulates the suppressive function and fate of myeloid-derived suppressor cells. These murine studies suggest that PIR-B/LILRBs have an important role in controlling allogeneic responses post-HSCT. Because of the few studies on LILRs from patients with HSCT, the roles of LILRs post-HSCT are not well known. A study [65] has reported that LILRB3-reactive Abs were detected in 5.4% of patients with HSCT and that they can mediate the killing of LILRB3-expressing cells, suggesting that polymorphic LILRB3 may be involved in the graft-vs.-host responses and the graft-vs-leukemia activity against LILRB3-expressing leukemic cells.

LILRBs IN CANCER

In addition to immune cells, certain cancers also express LILRBs. Although there is growing evidence that LILRB may contribute to tumor progression, the exact mechanism and its role remain to be clarified. Discussed in this review is evidence that LILRBs may have a role in various hallmarks of cancer, including proliferation, metastasis, inflammation, and immune evasion.

LILRB1

LILRB1 (also known as ILT2, CD85j, LIR1, and MIR7) is expressed with a variety of cancers, including gastric cancer cell lines, metastatic breast cancer, triple-negative breast cancer, B cell lymphoma, Sézary cells (a specific type of cutaneous T cell lymphoma), AML, and ALL.

Several lines of evidence suggest that LILRB1 contributes to cancer immune evasion. LILRB1 expression has been shown to correlate with resistance to NK cytotoxicity. The expression level of LILRB1 by gastric cancer cell lines was inversely correlated with the sensitivity to cytotoxicity by the NK cell line NK92MI [66]. In another study [67], blockade of LILRB1 resulted in NK-mediated killing of AML and ALL blast cells. Overexpression of LILRB1 in triple-negative breast cancer was correlated with resistance to epidermal growth factor receptor Ab (cetuximab)–mediated NK cell ADCC [68]. Interestingly, LILRB1 blockade by an antagonist Ab restored cetuximab-mediated ADCC. Furthermore, recombinant HLA-G6, a LILRB1 ligand, enhanced the production of TGF-β by AML U937 cells, which was inhibited by LILRB1 Abs [69]. However, LILRB1 has also been shown to participate in recognition and killing of B cell lymphoma by γδ T cells via MHC class I interactions. LILRB1, as well as MHC class I, blockade of B lymphoblastoid cells by the use of MHC class I– or LILRB1-specific mAbs and LILRB1-Fc molecules inhibited cytolysis by Vγ8Vδ3 T cell clones [70].

LILRB1 may also have a role in the regulation of tumor cell apoptosis and proliferation. CD4⁺LILRB1⁺ malignant Sézary cells were more resistant to anti-CD3 mAb-induced cell death compared with CD4⁺LILRB1⁻ lymphocytes [71]. This suggests that LILRB1 may have a role in antiapoptosis in T cell lymphoma. Raji (B cell lymphoma) cell proliferation was inhibited by HLA-G aggregated on nanoparticles in a dose- and time-dependent manner [72]. The use of small interfering RNA and Ab approaches to block LILRB1 expression restored proliferation, demonstrating HLA-G–mediated inhibition was driven by LILRB1.

LILRB2

Several types of cancer have been shown to express LILRB2 (also known as ILT4, CD85d, LIR2, and MIR10). These include pancreatic cancer, breast cancer, lung cancer, and leukemia.

The ANGPTL2/LILRB2 axis has been implicated in the EMT of pancreatic cancer [73]. Activated KRAS, HER2 and short hairpin RNA knockdown of p16p14 in papilloma virus (E6E7)–immortalized human pancreatic ductal epithelial and the human telomerase reverse transcriptase–immortalized HPDE, nestin-expressing cell line increased ANGPTL2 secretion and LILRB2 expression. The HPDE and HPDE nestin cells showed decrease E-cadherin expression and increased migration in vitro. Knockdown of ANGPTL2 increased E-cadherin expression and decreased migration potential [73]. IL-10 was increased in LILRB2-expressing breast cancer tissue and was found to be associated with advanced stage and metastatic breast cancer and decreased TILs [74]. This suggests that LILRB2 and IL-10 may contribute to poor prognosis and immunosuppression in breast cancer. In NSCLC, high LILRB2 expression was correlated with a lower level of TILs, suggesting an inhibition of immune cell infiltration or apoptosis of TILs.

LILRB2 and its murine ortholog PIR-B have a role in leukemia development and maintenance of leukemia stemness. PIR-B, through binding of ANGPTLs, suppressed leukemia differentiation [75]. PIR-B deficiency resulted in inhibition of leukemia development in an MLL-AF9 AML model. More recently, LILRB2 was shown to promote migration and proliferation of lung cancer. LILRB2-deficient A549 cells, an NSCLC line, migrated more slowly compared with normal A549 in an in vitro wound-healing assay [76]. LILRB2 overexpression in A549 cells resulted in greater migration potential in both wound healing and transwell-migration assays. Stimulation or overexpression of ANGPTL2, an LILRB2 ligand, resulted in a higher level of proliferation in A549 cells, which is mediated via the SHP2/CAMK/Creb signaling axis.

LILRB3

The role of LILRB3 (also known as ILT5, CD85a, LIR3, and HL9) in cancer progression remain poorly understood, and its functional ligands (as opposed to those identified by ELISA) have not been identified. LILRB1, LILRB2, and LILRB4 were observed in various cancers, whereas LILRB3 expression is more restricted. LILRB3 expression is up-regulated in leukemia cells, such as U937 and HL-60 cells, upon treatment with PMA and chemotherapeutics (unpublished results).

The analysis of RNA-Seq data sets (The Cancer Genome Atlas, U.S. National Institutes of Health, Bethesda, MD, USA) reveals that LILRB3 expression predicts the worst survival prognosis in stage 1, estrogen receptor–positive and progesterone receptor–positive breast cancer (unpublished results). Interestingly, a chemoresistant phenotype of breast (MCF7) and colon (HCT116) cancer cells is associated with greater expression of LILRB3. LILRB3 RNA expression was up-regulated in tumor tissues from HT29 and HCT116 colon tumor–bearing mice treated with 5-fluorouracil. These results suggest that LILRB3 may have a role in the regulation of chemoresistance in cancer.

LILRB4

A number of cancers express LILRB4 (also known as ILT3, CD85k, LIR5, and HM18), including ovarian cancer, gastric cancer, and AML. LILRB4 was expressed in serous and endometrioid ovarian cancer in a white leghorn laying-hen model [77]. However, the functional significance remains unclear. In addition to LILRB1, various gastric cancer lines also express LILRB4 [66]. Similar to LILRB1, greater LILRB4 expression was also associated with resistance to killing by NK921MI NK cell line. It is not clear whether the function of LILRB1 and LILRB4 is redundant because no functional studies have, to our knowledge, been performed. FAB M4/M5 AMLs express LILRB4 [6]. KG-1 leukemia cells transfected with LILRB4 reduced the allogeneic T cell response both in vitro and in vivo, suggesting that LILRB4 may mediate immunosuppression by inhibiting T cell response [50]. In addition to tumor cells, PMN-MDSCs in patients with late-stage NSCLC express LILRB4 [78]. Interestingly, patients with an increased proportion of PMN-MDSCs and an increased fraction of the LILRB4^high subset had a shorter median survival than did patients with elevated PMN-MDSCs and a smaller LILRB4^high fraction. This suggests that LILRB4 may contribute to the suppressive function of PMN-MDSCs in patients with NSCLC.

LILRB-RELATED ITIM-CONTAINING RECEPTORS: LAIR1

LAIR1, also known as CD305, is a transmembrane, ITIM-containing glycoprotein. It acts as an inhibitory receptor and is expressed by most immune cells as well as in leukemia, including CLL, ALL, and AML [5, 78, 80]. Although dispensable for normal hematopoiesis, LAIR1 promotes leukemia development via recruitment and activation of SHP1 and its downstream signaling molecules, including CAMK1 and CREB [5]. Deletion of LAIR1 in mouse BCR-ABL1 cells led to cell death in vitro, remission in vivo, and significantly prolonged survival of transplant-recipient mice [80].

MDSC IN CANCER STEMNESS AND TUMOR PROGRESSION

The capacity of immune cells, such as MDSCs, and the tumor microenvironment to promote tumor progression has been studied intensively, yet the mechanisms imparting stemness, invasiveness, metastatic potential, and chemoresistance are still poorly understood. Although MDSCs are known to promote immunosuppression, they have also been shown to promote cancer stemness. MDSCs induced microRNA-101 expression in ovarian cancer cells to repress the corepressor CtBP2. Decreased expression of CtBP2 led to enhanced expression of stem cell core proteins, such as NANOG, OCT3/4, and SOX2, thereby resulting in increased cancer cell stemness and enhanced metastatic and tumorigenic potential [81]. MDSCs have also been found to induce human breast cancer stem cells [82]. Aldehyde dehydrogenase–positive cancer stem cells and MCF-7 tumor-sphere formation were enhanced in the presence of MDSCs. MDSC- derived IL-6 and NO were found to induce STAT3 and Notch signaling, respectively, which are suggested to drive stemness [82]. Furthermore, MDSCs promoted stemness and increased aldehyde dehydrogenase–positive populations in human pancreatic cancer via a STAT3-dependent mechanism [83]. Whether LILRBs have a direct role in the MDSC-mediated promotion of cancer stemness remains unknown. Because both MDSCs and a subpopulation of certain cancer types express LILRBs, it raises the question of whether cancer stemness could result from MDSC LILRB-mediated reprogramming alone, cancer LILRB signaling alone, or a combination of both. LILRB/PIR-B signaling promotes the activation of the alternative M2 phenotype in macrophages and MDSCs, which is anti-inflammatory, immunosuppressive, and protumorigenic in the tumor microenvironment [4, 84]. In contrast, LILRB2 and PIR-B are expressed on HSCs, leukemia stem cells, and a population of certain solid cancer types. Binding of LILRB2 by ANGPTL results in SHP1/2 recruitment and CAMK signaling, which ultimately lead to the maintenance of HSC self-renewal and leukemia stemness [75]. Furthermore, ANGPTL2/LILRB2 signaling promotes the migration in A549 lung cancer cells [76]. This suggests that, in lung cancer cells, LILRB may have a role in metastasis, a process that confers cancer cells with stem-like properties during the EMT process. The crosstalk between MDSCs and cancer cells in the tumor microenvironment is mediated by a complicated network that consists of secreted factors and cell-associated molecules. The role of LILRB in individual cell types, such as MDSCs and specific cancer cells, is becoming clearer, but the interactions among the different cells in the tumor microenvironment via LILRBs remain unclear and warrant further studies.

LILRs AS THERAPEUTIC TARGETS FOR HUMAN DISEASES

Recent success in the use of immune checkpoint inhibition for the treatment of certain cancers has ignited a new interest in searching for novel immunoregulatory pathways that can be targeted to modulate immune responses to the benefit of patients. The studies discussed in the previous sections suggest that LILRBs are ideal targets for modulation of the adaptive immunity through myeloid cells and phenotypes of cancer-initiating cells. Various approaches can be taken to achieve that goal. Recombinant LILRB extracellular domain-Ig fusion proteins can be used to compete with ligand binding, thereby preventing the activation of LILRBs and the consequent effect (induction of the suppressive phenotype) on myeloid cells. Alternatively, inhibitors of signaling components of LILRBs, such as SHP1, can be used to block the acquisition of the suppressive function by myeloid cells. High-affinity ligands for LILRBs can be used to activate LILRB signaling and to endow myeloid cells with suppressive activities. However, these approaches have some significant limitations. MHC class I, including HLA-G, has a moderate to low affinity for LILRB1, 2, and 5, when compared with ANGPTLs [75], and the high-affinity functional ligands (as opposed to those identified by ELISA) for LILRB3 and 4 have not been identified. LILRB-Ig fusion proteins only prevent activation of the receptor, whereas inhibitors of LILRB signaling components may have undesirable off-target effects because they also inhibit other signaling pathways that use the same signaling molecule.

mAbs have several advantages as immune modulators and have been used successfully as immune checkpoint inhibitors. In addition to high affinity and stability, mAbs can be screened to activate (agonists) or to block (antagonists) the function of the intended target. Agonistic LILRB Abs that induce the signaling cascade of LILRBs could promote polarization of myeloid cells toward an immunosuppressive phenotype. In contrast, anti-LILRB antagonists that compete with ligand binding without activating LILRB signaling could polarize myeloid cells toward an immune-activating phenotype. Therefore, it is conceivable that LILRBs is a target for the treatment of cancer and autoimmune/inflammatory diseases using anti-LILRBs antagonists and agonists, respectively. Furthermore, the blockade of LILRBs may prove to a be more effective treatment for various types of cancer by not only converting the suppressive phenotype of MDSCs and tumor-associated macrophages into an immune-conducive one but also directly targeting various hallmarks of cancer, such as proliferation, metastasis, and therapeutic resistance.

CONCLUDING REMARKS

Increasing evidence supports a pivotal role for LILRs in maintaining immune homeostasis and cancer stemness. Although ligands for some LILRs have been identified, the functional significance and clinical relevance of LILRs and their ligands remain to be determined. The crucial roles of LILRs in regulation of myeloid function and immune tolerance are mostly derived from the studies of PIR-B–deficient mice. Therefore, the various function of LILRs needs to be confirmed by the use of ligands, if available, or anti-LILR antagonists or agonists, while testing the therapeutic efficacy of anti-LILR in the treatment of human diseases. In the context of potential therapeutic applications, one critical consideration is the high homology among LILR family members, in particular, some LILRA and LILRB members. Consequently, targeting one member of the LILRB family with Abs might also trigger the function of other members, which may complicate the interpretation of biologic effects. Another hurdle to elucidating the functional role of LILRs is the lack of suitable mouse models because of the low homology between mouse PIRs and human LILRs. The development of novel mouse models might be needed to study the mechanistic details of how LILRs regulate myeloid function and tumor progression and to test the therapeutic efficacy of targeting LILRs for the treatment of malignant, autoimmune, and inflammatory diseases.

AUTHORSHIP

J.Z., S.M., S.H.C., and P.Y.P. conceived the idea and wrote the first draft, H.M.C. and K.K. helped with the first draft, J.Z. created the figures, and all the authors (J.Z., S.M., H.M.C., K.K., X.C.L., S.H.C., and P.Y.P.) read the draft, made inputs, and approved the final version.

Glossary

ADCC

Ab-dependent cell cytotoxicity

ALL

acute lymphoblastic leukemia

AML

acute myeloid leukemia

ANGPTL

angiopoietin-like protein

CMV

cytomegalovirus

CLL

chronic lymphocytic leukemia

CtBP2

C-terminal binding protein-2

DC

dendritic cell

EMT

endothelial-to-mesenchymal transition

GVHD

graft-vs.-host disease

HPDE

human pancreatic ductal epithelial

HSC

hemopoietic stem cell

HSCT

hematopoietic stem cell transplantation

ILT

Ig-like transcript

LAIR1

leukocyte-associated immunoglobulin-like receptor-1

LILR

leukocyte immunoglobulin-like receptor

LILRA

activating leukocyte immunoglobulin-like receptor

LILRB

inhibitory leukocyte immunoglobulin-like receptor

MDSC

myeloid-derived suppressor cell

MS

multiple sclerosis

NSCLC

non–small cell lung carcinoma

PIR

paired Ig-like receptor

PMN

polymorphonuclear

RA

rheumatoid arthritis

SHP1

Src homology region 2 domain-containing phosphatase 1

SLE

systemic lupus erythematosus

SNP

single-nucleotide polymorphism

TIL

tumor-infiltrating leukocyte

DISCLOSURES

The authors declare no conflicts of interest.