Leukocyte immunoglobulin-like receptor subfamily B: therapeutic targets in cancer.

LILRB5, also known as CD85C and LIR8, contains four extracellular immunoglobulin domains, a transmembrane domain, and two ITIMs. The expression of LILRB5 has been reported in subpopulations of monocytes, NK cells, and T cells, as well as in vitro cultured osteoclasts and mast cell granules [ 16 , 63 , 230–232 ]. A recent study showed that LILRB5 specifically binds to HLA-B7 and HLA-B27 heavy chains [ 230 ]. Due to a relative paucity of studies on LILRB5, the functional role of this receptor is not clear. Several genome-wide association studies have highlighted LILRB5 variants whose expression is correlated with serum creatine kinase and lactate dehydrogenase levels. This correlation suggests an as yet unknown role for LILRB5 in muscle repair [ 233–235 ]. Mycobacteria exposure has been shown to upregulate LILRB5 expression in APCs derived from BCG vaccinated donors, indicating a possible role for LILRB5 in bacterial infection [ 231 ]. Within mature cord blood-derived mast cells, LILRB5 is expressed in cytoplasmic granules that are released after crosslinking of high-affinity IgE receptors. This hints at a possible role in mast cell inflammatory response [ 63 ]. LILRB5 is unique among LILRBs in that it is the only LILRB that is not highly expressed by M5 AML cells, and its expression level does not correlate with the overall survival of AML patients based on the analysis of TCGA data for AML patients ( https://tcga-data.nci.nih.gov/tcga/ ). There are two mouse genes encoding proteins resembling LILRBs in human, PirB and gp49B1. Due to the expression patterns and, in some cases, the ligands they recognize, there is no clear 1:1 counterpart relationship between human LILRBs to mouse PirB and gp49B1. PirB, considered the mouse relative of LILRB2/3, contains six extracellular immunoglobulin domains, a transmembrane domain, and four ITIMs. It is expressed on HSCs [ 34 ], DCs [ 236–238 ], macrophages [ 239 ], neutrophils [ 240 ], eosinophils [ 241 ], B cells [ 240 ], T cells [ 242 , 243 ], osteoclasts [ 54 ], and neuronal cells [ 30 ]. PirB ligands include MHC-I [ 233 ], Angptls [ 37 , 64 , 244 , 245 ], β-amyloid [ 31 ], and myelin inhibitory molecules (MAG, Nogo, and OMgp) [ 30 ]. PirB can interact in cis with MHC-I expressed on the same cell [ 158 ]. The extended conformation of extracellular domains of PirB enables trans -cellular interaction with ligands, such as MAG and MHC-I [ 246 ]. PirB is expressed on mouse HSCs and multiple hematopoietic lineages [ 37 ]. On DCs, PirB regulates cytokine-mediated signaling [ 236–238 ], inhibits type I interferon secretion [ 237 ], induces peripheral tolerance within an allograft graft-versus-host disease model by suppression of alloreactive T cells [ 238 ], facilitates maturation of DCs with a hypothesized alteration of cell signaling involving granulocyte-macrophage colony-stimulating factor [ 247 ], and produces IL-27 to suppress CD4 + T cells [ 248 ]. PirB is also a negative regulator of intestinal macrophages to prevent the progression of inflammatory diseases such as Crohn’s disease and ulcerative colitis [ 239 ]. PirB inhibits alveolar macrophages and suppresses IL-4 induction of pulmonary fibrosis [ 249 ]. Moreover, the high expression of PirB in eosinophils contributes to both inhibitory and activating pathways [ 241 ], such as inhibition of IL-13-mediated eosinophil activation [ 250 ]. Differentiation of myeloid lineage cells and B cells leads to upregulation of PirB [ 240 ]. Ectopic expression of PirB in peripheral T cells contributes to the suppression of type 1 helper T-cell immune response [ 251 ]. Expression of PirB on T cells may be associated with chronic autoimmunity [ 242 , 243 ]. PirB is expressed in cortical and hippocampal neurons and regulates visual cortical plasticity [ 252–254 ]. Interaction of PirB on neurons with β-amyloid oligomer leads to recruitment of cofilin to facilitate actin depolymerization and results in synaptic loss and cognitive deficits [ 31 ]. Interaction of PirB with myelin inhibitor molecules suppresses axonal outgrowth and regeneration via activation of SHP-1/SHP-2 signaling [ 255 ], PI3K/Akt/mTOR signaling [ 256 ], and Trk signaling pathways [ 257 ]. In addition, the binding of PirB to MHC-I molecules contributes to suppression of synaptogenesis [ 258 , 259 ]. PirB is upregulated on DCs during cancer progression; knockdown of PirB on DCs increases Th17 response and decreases Treg differentiation to suppress tumor growth in a mouse lung cancer model [ 260 ]. PirB highly expressed on DCs competes with CD8 for MHC-I binding and inhibits tumor antigen-specific CD8 + T-cell proliferation and cytotoxic activity to support tumor immune escape in a syngeneic mouse lymphoma model [ 261 ]. The upregulation of PirB on tumor-infiltrating DCs can be inhibited by PD-L1 blockade [ 262 ]. It was also reported that Angptl2 binds to PirB and activates Notch signaling for activation and maturation of DCs and subsequent CD8 + T-cell cross-priming in mouse melanoma and kidney cancer models [ 263 ]. PirB expressed on MDSCs suppresses differentiation of MDSCs into M1 macrophages, which in turn inhibits regulatory T-cell activities and tumor development [ 53 ] . Interaction of glatiramer acetate with PirB on MDSCs suppresses T cell by promoting IL-10 and TGFβ release [ 186 ]. On the other hand, PirB is expressed on mouse AML cells and supports AML development by maintaining self-renewal and inhibiting differentiation of leukemia stem cells [ 37 ]. Defective PirB signaling diminishes phosphorylation of SHP-1 and SHP-2 in AML cells [ 37 ]. By recruiting SHP-1/SHP-2, PirB further activates CAMKs and the downstream CREB signaling pathway to support leukemia progression [ 264 ]. gp49B1 is a mouse protein resembling human LILRB4. It contains two extracellular Ig-like domains. Unlike human LILRB4, it contains two cytoplasmic ITIMs instead of three. It is expressed on macrophages, mast cells, DCs, neutrophils, NK cells, T cells, microglia, and cardiomyocytes [ 265–272 ]. Unlike mouse PirB and human LILRB4, gp49B1 cannot be activated by ApoE [ 196 ]. Integrin α v β 3 is the only known ligand of gp49B1, and the integrin α v β 3 /gp49B1 interaction inhibits mast cell activation [ 273 ]. Co-ligation of gp49B1 and FcγRI also blocks IgE-mediated mast cell activation [ 29 ]. The gp49B1-mediated inhibition of mast cell activation requires recruitment of SHP-1 by ITIMs [ 274 ], which may also interact with SHIP and SHP-2 [ 274–276 ]. Although gp49B1-deficient mice [ 277 , 278 ] exhibit no developmental abnormalities, mast cells in these mice exhibit hypersensitivity to ovalbumin-challenged anaphylaxis [ 277 ], elevated SCF-induced mast cell activation [ 279 ], and increased neutrophil-dependent vascular injury induced by LPS [ 267 , 280 ]. In addition, gp49B1 deficiency induces significant T helper cell type 2 immune responses and pulmonary inflammation [ 281 ]. These result from elevated expression of chemokine (C-C motif) receptor 7 (CCR7) on DCs and increased secretion of CCL21 by lung lymphatic vessels [ 282 ]. The decrease of gp49B1 on tolerogenic uterine DCs or decidual macrophages contributes to abnormal pregnancy outcomes by changing M1/M2 functional molecular expression, synthesis of arginine metabolic enzymes, and cytokine secretions [ 264 , 283 ]. Moreover, gp49B1 is upregulated on macrophages in atherosclerotic lesions from mouse aortic roots. Deficiency of gp49B1 significantly accelerates the development of atherosclerotic lesions and increased the instability of plaques [ 201 ]. Deficiency of gp49B1 in bone marrow-derived macrophages in the lung exacerbates acute lung injuries via promotion of NFκB signaling [ 284 ]. Downregulation of gp49B1 with other Treg-related genes, including Ikzf2, Ikzf4, Tigit, and Il10, is found in atherosclerosis-driven Treg plasticity [ 285 ]. Furthermore, deficiency of gp49B1 promotes cardiac hypertrophy via elevated NFκB signaling and TGFβ expression in cardiomyocytes [ 272 ]. On the other hand, overexpression of gp49B1 in cardiomyocytes inhibits angiotensin II-induced cardiomyocyte hypertrophy via interaction between gp49B1 and SHP-2 and inhibition of NFκB signaling [ 286 ]. gp49B1 also recruits SHP-1, which inhibits TRAF6 ubiquitination and subsequently inactivates NFκB signaling and MAPK cascades in nonalcoholic fatty liver disease [ 287 ]. Finally, gp49B1 expression levels are elevated in activated microglia in transgenic APP/PS1 Alzheimer’s disease mice [ 288 ] and aged mice [ 271 ]. In addition to infection, gp49B1 is also expressed on activated CD4 and CD8 effector T cells after allogeneic tumor challenge [ 269 ]. NK and T cells from gp49B1-deficient mice exhibit enhanced cell cytotoxicity activities, which suggests that gp49B1 is an inhibitory checkpoint on anti-tumor immune cells in TME [ 269 ]. Moreover, gp49B1 is increased on activated plasmacytoid DCs after toll-like receptor activation, which is correlated to the reduction of T-cell activity against leukemia cells [ 289 ]. In summary, gp49B1 may play important roles in various inflammatory diseases and cancer.

The identification of LILRBs and their downstream signaling as potential therapeutic targets has reshaped our views of how cancer cells interact with the TME and the immune system, how cancer cells differ from other cells, and how to treat cancer. Numerous studies indicate that LILRBs and their signaling in infiltrating immune cells protect tumor cells from immune surveillance and attack. In addition, LILRB signaling in cancer cells, particularly in some leukemia cells, directly support cancer development in cell autonomous and immune-related manners. Since inhibition of the signaling of specific LILRBs unleashes immune checkpoints and directly blocks cancer growth with only mild toxicities, these receptors represent promising therapeutic targets for cancer treatment.

Immunotherapy holds great promise for achieving long-lasting anti-cancer effects. In particular, immune checkpoint Programmed cell death protein 1 and ligand 1 (PD-1/PD-L1) blockade therapies have been successful for treating a small portion of cancers [ 1 ]. Developing approaches to identify more effective immune checkpoint targets is essential for successful application of immunotherapy to a broader range of cancers. Two features of PD-1 may hint us in these endeavors. First, activation of PD-1 as an immune inhibitory receptor involves the immunoreceptor tyrosine-based inhibitory motif (ITIM) and a related immunoreceptor tyrosine-based switch motif (ITSM) in its signaling domains [ 2 ]. ITIM consists of six amino acids (S/I/V/LxYxxI/V/L) [ 3 ], and ITSM is defined as TxYxx(V/I) [ 4 ]. The activation of ITIMs typically leads to the recruitment of tyrosine phosphatases SHP-1 and SHP-2 or the inositol phosphatase SHIP and the consequent inhibition of immune cell activation [ 5–7 ]. Therefore, ITIM-containing receptors represent a rich source of candidates for the next generation of immune checkpoint proteins. Second, PD-1 is expressed on exhausted T cells within the tumor microenvironment (TME). While ongoing efforts to scrutinize all inhibitory receptors on T cells are intensive, it is known that some other populations of immune cells, such as myeloid cells, are present in the TME in even larger numbers than T cells and can contribute to tumor immune evasion. For example, macrophages are the most abundant immune cell population in tumor tissues [ 8 ]. These immune cells possess the capacity to kill tumor cells and to prime or reactivate T cells. However, they become dysfunctional in the TME, turning into immunosuppressive cells that can support tumor development and suppress immune surveillance and attack. These immunosuppressive cells may include monocytic myeloid-derived suppressor cells (M-MDSCs), polymorphonuclear MDSCs, tumor-associated macrophages (TAMs), and immunosuppressive populations of dendritic cells (DCs), neutrophils, eosinophils, and B cells [ 9–14 ]. Immune inhibitory receptors on these cells may play key roles in their immunosuppressive functions. Reprogramming, removing, or blocking trafficking of these immunosuppressive cells is becoming an attractive anti-cancer therapeutic strategy [ 10 ]. To identify the next generation of immune checkpoint molecules, it is important to study the biology of ITIM-containing receptors that are expressed by immune cells in the TME. There are more than 100 ITIM-containing receptors [ 15 ], including receptor families [ 3 ] such as leukocyte immunoglobulin-like receptor subfamily B (LILRB), certain killer cell immunoglobulin-like receptors (KIRs), and several sialic acid-binding immunoglobulin-like lectins (Siglecs). These receptors contain extracellular immunoglobulin (Ig)-like domains for ligand binding and intracellular ITIM domains to negatively regulate activation signaling in immune cells. The LILRBs are a group of type I transmembrane glycoproteins with extracellular Ig-like domains that bind ligands and intracellular ITIMs that can recruit tyrosine phosphatases SHP-1, SHP-2, and the inositol phosphatase SHIP. The LILRB family contains five members LILRB1–LILRB5 ( Fig. 1A ), all of which were cloned in 1997 [ 16–19 ]. Historically, this family of receptors was also named as members of CD85, ILT, or LIR family ( Table 1 ). In 2001, the name LILRB was officially assigned [ 20 ]. Because of their immunosuppressive functions, LILRBs are considered to be immune checkpoint factors [ 21 ] and may play significant roles in human immunity and cancer development. Domain structure of (A) human LILRBs and (B) mouse relatives. Extracellular Ig domains are depicted as circles and intracellular ITIMs are depicted as boxes. Summary of ligands and expression of human LILRBs and mouse relatives, and clinical trials of antibodies targeting human LILRBs LILRBs are primate-specific. The human LILRBs are encoded in a region called the leukocyte receptor complex (LRC) at chromosomal region 19q13.4 [ 16 , 20 , 22 ]. Like the inhibitory receptor PD-1 [ 23 ], the relatives of LILRBs exist in birds and mammals [ 24 , 25 ], although by phylogenetic definition these relatives are not considered LILRB homologs [ 26 , 27 ]. Paired immunoglobulin-like receptor B (PirB) [ 28 ] and gp49B1 [ 29 ] are the mouse relatives of LILRBs ( Fig. 1B ). Due to rapid evolution, the expression pattern and, in some cases, the ligands of these LILRB relatives are different from those of their human counterparts. Therefore, the PirB or gp49B1 knockout mouse models are of limited value for building and understanding of the biology of human LILRBs. LILRBs are predominantly expressed by cells of the hematopoietic system. LILRBs may also be expressed by certain non-hematopoietic cells. For instance, LILRB2 is expressed on neurons, which has been implicated to regulate axon regeneration and is involved in the pathology of Alzheimer’s disease [ 30 , 31 ]. LILRBs and a related ITIM-containing receptor LAIR1 [ 32–35 ] are abnormally expressed by certain cancer cells [ 36–53 ]. Overall, the immune cell-expressing LILRBs have immune inhibitory functions and thus are indirectly tumor-supportive, and the cancer cell-expressing LILRBs may directly regulate cancer development [ 54 ]. Similar to regulation of PD-L1 levels by external cues from TME, the expression of LILRBs can be regulated by both immunosuppressive and proinflammatory signals. The expression of LILRB1-4 can be upregulated by the immunoinhibitory cytokine IL-10 [ 55–57 ], and the LILRB4 level can also be elevated by the immunosuppressive hormone vitamin D3 [ 58 , 59 ]. On the other hand, LILRB2 and LILRB4 are also upregulated by the proinflammatory cytokines interferon (IFN)-α [ 60 ] and IFN-β [ 61 ], analogous to upregulation of PD-L1 by IFN-γ. Together, such induced increase of LILRB levels may enhance the immunosuppressive and tumor-promoting capacities of TME. We hypothesize that the immunosuppressive myeloid cells is a key component of TME that inhibits tumor-specific immune responses and supports tumor development, and LILRBs are a major group of inhibitory receptors that regulate the immunosuppressive function of these tumor-supportive myeloid cells. Here, we review the signaling and functions of LILRBs in cancer development.

LILRBs inhibit anti-tumor immune activities and support cancer cell survival, self-renewal, and migration in various types of cancer, thus representing attractive therapeutic targets. Several key questions need to be addressed in order to better apply our knowledge to cancer diagnosis and treatment. Identification of ligands for LILRBs is a key step to understanding the biology and function of these receptors in tumor immune evasion and cancer development. The study of chicken relatives of LILRBs suggested that the ancient ligands for these Ig-containing receptors were MHC class I and β2-microglobulin [ 24 , 25 ]. However, human LILRBs interact with both HLA and non-HLA ligands. Given the fact that LILRB1 and LILRB2 each have multiple ligands, it will not be surprising if individual LILRBs have multiple binding partners. High-affinity ligands, co-ligands, or binding proteins of LILRB1, 2, and 4 may have yet to be identified. The ligand for LILRB3 is still unknown. HLA-B27, a ligand of LILRB5, needs further functional validation. Several experimental techniques could be useful in identification of LILRB ligands such as expression cloning, crosslinking followed by co-immunoprecipitation and mass spectroscopy, protein liquid chromatography fractionation followed by reporter assays and mass spectrometry [ 196 ], protein arrays, candidate screening, cell microarrays [ 290 ] and ligand-based receptor capture technologies [ 291 ]. The identification of multiple Ig-containing receptors that interact with LILRBs [ 197 ] has new implications of signaling and functions of LILRBs. If these ligand/receptor interactions happen trans among different cells, our understanding of how LILRBs act may significantly change. The signaling and functions of individual LILRBs may share common features and also differ depending on their expression in normal immune cells, immune cells in diseased individuals (such as MDSCs, TAMs, and other immunosuppressive cells), hematological malignant cells, and solid cancer cells. A major question in the study of LILRBs and other classical ITIM-containing receptors is whether these inhibitory receptors have independent signaling or whether their signaling needs to be associated with those of activating receptors. It was proposed that the activity of the ITIM-containing inhibitory receptors requires ITAM-containing receptors [ 3 , 126 ]. In this model, an ITIM-containing receptor cannot activate by itself but needs to interact with an activating receptor. When the ITAM-containing activating receptor is activated, its ITAM recruits the Src tyrosine kinase [ 126 ], which phosphorylates and thus activates the ITIMs of the nearby inhibitory receptors. The recruitment of SHP-1 may subsequently dephosphorylate the ITAM and/or associated proteins, thus preventing further activation of the activating receptors [ 126 ]. This model explains TCR-, BCR-, and FcR-coupled LILRB signaling in T and B cells. Nevertheless, in monocytic cells, LILRB4 clustering per se without crosslinking with an ITAM receptor can induce SHP-1 recruitment [ 18 ]. In fact, the Src kinase Lck can activate the ITIM- and ITSM-containing receptor in the absence of ITAM receptors in an in vitro reconstitution system [ 292 ]. Further investigations are warranted to determine LILRB signaling and functions in malignant cells, in which ITIM-containing receptors may have acquired certain independent cancer-promoting activities due to an altered signaling context [ 36 , 196 ]. The cell-context-dependent difference of LILRB signaling and functions may result from a number of factors: (1) different extrinsic cues. The diversity of ligands of each receptor contributes to distinct function of each receptor in different microenvironments. In addition, different ligand binding at different epitopes of LILRBs can lead to different conformational changes of the receptors and consequently different signaling. (2) The interaction between LILRBs and other receptors. Most recently, it was demonstrated that multiple Ig-domain receptors interact with various LILRBs [ 197 ]. Such cis or trans interactions may regulate the signaling and functions of LILRBs differently in different cells. In addition, extracellular factors that bind to other receptors on the same immune cells could affect the feedback signaling of LILRBs [ 5 ]. (3) Different signaling domains of different LILRBs. Due to variable sequences and context, not all ITIMs are equivalent. For example, it was suggested that a certain ITIM in LILRB1 was possibly an ITSM [ 139 ]. Although all LILRBs contain ITIMs, we found that certain ITIMs in different LILRB members were not interchangeable [ 224 ]. (4) Different levels of signaling molecules in different types of cells. The large number of substrates for SHP-1, SHP-2, and SHIP and divergent downstream signaling may contribute to the complexity. (5) Different transcriptional (such as LILRBs regulation by IL-10 [ 55–57 ] and LILRB4 by vitamin D3 [ 58 , 59 ]), perhaps translational, or post-transcriptional regulation of individual LILRBs may lead to formation of different interactomes. The studies of LILRB biology in cancer cells may shed new lights on better understanding of the functions of LILRBs in immune cells in TME, and vice versa. For example, monocytic AML cells and immunosuppressive monocytic cells (including M-MDSCs and TAMs with monocytic origin) in cancer patients may share several characteristics: (1) both are monocytic cells marked by LILRB4 expression [ 50 , 196 , 211 , 217 ], (2) STAT3/NFκB/Arginase-1 axis is functionally active in both populations [ 293 , 294 ], and (3) both have robust migration abilities [ 10 ]. It is therefore possible that LILRB4 signaling in monocytic AML cells and M-MDSCs is similar, and antagonizing LILRB4 signaling by blocking antibodies may have anti-tumor effects in different applications such as treatment of leukemia (by directly targeting LILRB4 in leukemia cells) and treatment of certain solid cancers (by targeting or reprogramming LILRB4 in TME). Overall, efforts to identify new ligands and study signaling and downstream effectors could lead to further determination of exact functions of LILRBs (antigen presentation, priming, activation, trafficking, reprogramming, and functions on cancer cells) in immune checkpoint biology. Elucidation of underlying mechanism of LILRBs paves the way for the development of therapeutics for human malignances. LILRB1 expressed by macrophages mediate the secondary anti-phagocytic ‘don’t eat me’ signals independently but cooperatively with the CD47-SIRPα pathway [ 141 ]. Anti-CD47 and anti-MHC class I or anti-LILRB1 might act in synergy to induce phagocytosis or immune system activation of macrophages. LILRB2 expressed by MDSCs or TAM suppresses anti-tumor immune activities in TME. Anti-LILRB2 monotherapy or combination with an anti-PD-1 antibody is in phase 1/2 clinical trial by Merck (MK-4830; Clinical Trial ID: NCT03564691 ; Table 1 ). Preliminary clinical data have shown that MK-4830 was well tolerated, and anti-cancer responses were observed in 10 patients treated with the anti-LILRB2 antibody MK-4830 in combination with pembrolizumab, 5 of whom progressed on prior anti-PD-1 therapies [ 295 ]. These data suggest that LILRB2 from immunosuppressive myeloid cells may contribute to drug resistance in the anti-PD-1 therapy. Other anti-LILRB2 therapeutics are also under preclinical development by Immune-Onc Therapeutics (IO-108) and in phase 1 clinical trial by Jounce Therapeutics (JTX-8064; Clinical Trial ID: NCT04669899 ; Table 1 ), respectively, to reprogram immune suppressive myeloid cells in solid cancers. Among all LILRB members, LILRB4 is clearly the best target for treating monocytic AML. It may also be a target for treating some other hematologic malignancies and solid cancers. By blocking ApoE-induced LILRB4 activation, an anti-LILRB4 antibody developed by Immune-Onc Therapeutics is in a phase 1 clinical trial as monotherapy for AML and CMML patients (IO-202; Clinical Trial ID: NCT04372433 ). An anti-LILRB4 antibody (h52B8) by Merck inhibits the immunosuppressive activities of monocytic MDSCs in vitro [ 296 ], and a phase 1 clinical trial for cancer treatment was announced ( Table 1 ). In addition, based on the information that LILRB4 is specifically expressed by monocyte lineage but not hematopoietic progenitor and stem cells, the CAR-engineered T (CAR-T) cell and ADC therapeutics that directly target LILRB4-expressing monocytic AML cells have been generated [ 217 , 219 ]. Preclinical studies have shown that both LILRB4-targeting CAR-T and ADC have anti-leukemia efficacy but do not affect the stem cell activities and differentiation of hematopoietic progenitor and stem cells. Other potential approaches to inhibition of LILRB signaling include targeting different segments of their downstream signaling pathways, although the signaling of ITIM-containing receptors is considered to be divergent. In addition to cancer, these drugs may also benefit patients affected by other diseases including infectious diseases, autoimmune diseases, and neurodegenerative diseases.