Leukocyte Immunoglobulin-like Receptor Research Articles

Expression of leukocyte immunoglobulin-like receptor subfamily B expression on immune cells in hepatocellular carcinoma

BackgroundLeukocyte immunoglobulin-like receptor subfamily B (LILRB) is a group of inhibitory receptors involved in innate immune mainly expressed on lymphoid and myelomonocytic cells. LILRB is proposed to serve as immune checkpoint like PD-1 and CTLA-4 for tumor treatment. We recently reported that the expression of LILRB2 in CD1c+ mDC from tumor tissue might suppress immune for HCC patients. However, the expression of all the LILRB family on other immune cells in peripheral blood and tumor microenvironment of HCC patients has not been systematically studied. MethodsThe expression of LILRB family (LILRB1, LILRB2, LILRB3, LILRB4 and LILRB5) on immune cells, including granulocytes, NK cells, NKT cells, monocyte subsets, TAMs, B cells, γδ T cells, CD4+ T cells, CD8+ T cells and MDSC subsets, was analyzed by flow cytometry in the peripheral blood of 20 HCC patients and 20 healthy donors as well as in the tumor and tumor free tissues of 10 HCC patients. ResultsLILRB1, LILRB2 and LILRB3 in granulocytes from peripheral blood were expressed increased in HCC patients compared with healthy donors. The expression of LILRB5 in NK cells and NKT cells from HCC blood were higher compared with healthy donors` blood. CD14+CD16+ monocyte subsets in blood of HCC patients expressed increased LILRB1 and LILRB4 than that in healthy donors. CD14+CD16− monocyte subsets in blood of HCC patients expressed increased LILRB3 than that in healthy donors. Compared to corresponding TFL, LILRB3, LILRB4 and LILRB5 were expressed enhanced in TAMs from HCC tumors. LILRB1 expressed on the B cells both in the blood and tumor had significantly increased compared with healthy donors or corresponding TFL. Different from peripheral blood, in the HCC microenvironment, CD4+ T cells expressed lower LILRB2, LILRB3 and LILRB4 than that from TFL and CD8+ T cells expressed decreased LILRB2. And γδ T cells expressed LILRB1 in HCC blood and microenvironment. Surprisingly, the percentage of LILRB1 expressed on MDSC from HCC peripheral blood and tumors was lower than that from healthy donors and corresponding TFL. ConclusionsThis is the first systemically examination of the LILRB family expression on a variety of immune cells from both peripheral blood and microenvironment in HCC patients. The specific increasing expression of LILRB on immune cells may regulate innate and adaptive immune and impact on HCC progression. Our findings justify further investigation of LILRB function in HCC.

A phase 1/2, open-label, dose-escalation, safety and tolerability study of NC410 in subjects with advanced or metastatic solid tumors.

TPS2659 Background: Leukocyte-Associated Immunoglobulin-like Receptor (LAIR)-1 and LAIR-2 are members of the Leukocyte Receptor Complex (LRC) (An & Brodsky, 2016). LAIR-1 is a co-inhibitory receptor expressed on several subsets of immune cells, and functions to delimit immune responses (Meyaard et al., 1997). LAIR-2 is a secreted protein with homology to the transmembrane protein LAIR-1 (Lebbink et al., 2008). In cancer, it is hypothesized that LAIR-1 expression on several subsets of leukocytes prevents optimal immune responses by limiting both innate and adaptive immune functionality. LAIR-1 serves to suppress anti-tumor immunity through the inhibition of stimulatory signaling pathways. Specifically, LAIR-1 is a checkpoint and adhesion receptor on T cells that limits T cell activation and increases adhesion to collagens (Meyaard, 2008). LAIR-2 is capable of blocking LAIR-1 functional interactions with ligands, resulting in improved immune function on multiple immune cell subsets. Dysregulation of LAIR-1 ligands in the tumor microenvironment results in excessive production of collagens and complement C1q as well as altered forms of collagens, that leads to immune inhibition through binding to LAIR-1+ immune cells. NC410 is a dimeric form of the LAIR-2 protein fused to a human Fc domain of the immunoglobulin (Ig) subtype IgG1. The rationale for developing NC410 as a cancer therapeutic is based on nonclinical data demonstrating LAIR-1 signaling blockade can improve the immune response. Because LAIR-2 binds to ligands shared with LAIR-1 with increased affinity, NC410 acts as a decoy receptor for LAIR-1 ligands releasing suppression from myeloid cells and T cells and promoting anti-tumor immunity. NC410 may also mediate remodeling of the tumor extracellular matrix, further contributing to anti-tumor activity. Methods: This is a multi-center, first in human, phase 1/2, open-label, single-armed study to determine the safety and tolerability, define maximum tolerated dose (MTD) and/or pharmacologically active dose, assess preliminary efficacy, and explore predictive and pharmacodynamic biomarkers of NC410 in subjects with advanced or metastatic solid tumors. Key eligibility criteria include measurable disease based on RECIST v1.1 and being able to consent for collection of biopsies at screening and on treatment. Phase 1 is a classic 3+3 dose escalation design to determine the safety, tolerability, DLT, MTD and recommended phase 2 dose (RP2D) (NCT04408599). Ongoing exploratory analyses include the assessment of predictive biomarkers associated with treatment benefit, and pharmacodynamic markers associated with study drug activity. Phase 2 is going to enroll ovarian, colorectal, NSCLC, H&N, and gastric carcinomas and other tumors depending on biomarker data available from the Phase 1 part of the study. Clinical trial information: 04408599.

Phase 1, first-in-human trial of JTX-8064, an anti-LILRB2/ILT4 monoclonal antibody, as monotherapy and in combination with anti-PD-1 in adult patients with advanced solid tumors (INNATE).

TPS2672 Background: Leukocyte Immunoglobulin-like receptor B2 [LILRB2; immunoglobulin-like transcript 4 (ILT4)] is an immunoinhibitory protein expressed on the surface of myeloid cells and is a therapeutic target of interest in immuno-oncology. Published data showed that antagonism of LILRB2 resulted in the repolarization of human macrophages from an M2 (suppressive) to M1 (pro-inflammatory) phenotype, and enhancement of anti-tumor immunity in a mouse model (Chen 2018). JTX-8064 is a novel humanized IgG4 monoclonal antagonist antibody that selectively binds LILRB2 and prevents it from binding its ligands, classical and non-classical MHC I molecules. By blocking the ability of LILRB2 to bind HLA-A/B and/or HLA-G, a marker of immunotolerance on cancer cells, JTX-8064 has been shown to enhance pro-inflammatory cytokine production in macrophages (Cohen 2019). Additionally, blocking HLA-A/B-LILRB2 binding with JTX-8064 may augment antigen presentation and has been shown to lead to enhanced T cell activation and IFNg production (McGrath 2021). Using an ex vivo tumor explant model, we observed an IFNg-associated pharmacodynamic response in tumor tissue treated with JTX-8064 and a PD-1 inhibitor (PD-1i) that was not observed with PD-1i alone. Biomarkers were identified that predicted this JTX-8064 driven response (Hashambhoy-Ramsay 2020). It is hypothesized that JTX-8064 is a novel macrophage immune checkpoint inhibitor that may overcome mechanisms of resistance to PD-1i in tumors not responsive to JTX-8064 or PD-1i alone. Methods: The primary objectives of this open-label, phase 1, first-in-human, multicenter trial are to determine the safety and tolerability, and the recommended phase 2 dose (RP2D) of JTX-8064 as a monotherapy and in combination with a PD-1i, JTX-4014 (a Jounce investigational agent) or pembrolizumab, in patients with advanced solid tumors (NCT04669899). The INNATE study will consist of 4 stages: 1) JTX-8064 monotherapy dose escalation, 2) JTX-8064 dose escalation in combination with a PD-1i, 3) JTX-8064 monotherapy in indication-specific expansion cohorts and 4) JTX-8064 in combination with a PD-1i in indication-specific expansion cohorts. Stages 1 and 2 will employ an innovative interval i3 + 3 design with Bayesian decision framework to guide dose escalation. Safety, pharmacokinetic and receptor occupancy data will be considered during dose escalation. INNATE will assess pharmacodynamic and potential predictive biomarkers of response, and the expansion cohorts will explore multiple patient populations, including PD-(L)1i sensitive and PD-(L)1i-resistant (primary or acquired) patients to address current unmet medical needs. Enrolment in INNATE began in January 2021. Clinical trial information: NCT04669899.

Association of the Leukocyte Immunoglobulin-like Receptor A3 Gene With Neutrophil Activation and Disease Susceptibility in Adult-Onset Still's Disease.

ObjectiveAdult‐onset Still’s disease (AOSD) is a severe autoinflammatory disease. Neutrophil activation with enhanced neutrophil extracellular trap (NET) formation is involved in the pathogenesis of AOSD. Functional leukocyte immunoglobulin‐like receptor A3 (LIR‐A3; gene name LILRA3) has been reported to be associated with many autoimmune diseases. We aimed to investigate the association of LILRA3 with disease susceptibility and neutrophil activation in AOSD.MethodsThe LILRA3 deletion polymorphism and its tagging single‐nucleotide polymorphism rs103294 were genotyped in 164 patients with AOSD and 305 healthy controls. The impact of LILRA3 on clinical features and messenger RNA expression was evaluated. Plasma levels of LIR‐A3 were detected using enzyme‐linked immunosorbent assay (ELISA), and the correlation between LIR‐A3 plasma levels and disease activity and levels of circulating NET‐DNA was investigated. LIR‐A3–induced NETs were determined using PicoGreen double‐stranded DNA dye and immunofluorescence analysis in human neutrophils and a neutrophil‐like differentiated NB4 cell line transfected with LIR‐B2 small interfering RNA.ResultsThe findings from genotyping demonstrated that functional LILRA3 was a risk factor for AOSD (11% in AOSD patients versus 5.6% in healthy controls; odds ratio 2.089 [95% confidence interval 1.030–4.291], P = 0.034), and associated with leukocytosis (P = 0.039) and increased levels of circulating neutrophils (P = 0.027). Functional LILRA3 messenger RNA expression was higher in the peripheral blood mononuclear cells (P < 0.0001) and neutrophils (P < 0.001) of LILRA3 +/+ patients. Plasma levels of LIR‐A3 were elevated in patients with AOSD (P < 0.0001) and correlated with disease activity indicators and levels of circulating NET–DNA complexes. Finally, enhanced NET formation was identified in neutrophils from healthy controls and patients with inactive AOSD after stimulation of the neutrophils with LIR‐A3. Moreover, NET formation was impaired in NB4 cells after knockdown of LILRB2 gene expression.ConclusionOur study provides the first evidence that functional LILRA3 is a novel genetic risk factor for the development of AOSD and that functional LIR‐A3 may play a pathogenic role by inducing formation of NETs.

Analysis of Intra-Tumoral Macrophages and T Cells in Non-Small Cell Lung Cancer (NSCLC) Indicates a Role for Immune Checkpoint and CD200-CD200R Interactions.

Simple SummaryLung cancer is the leading cause of cancer-related death worldwide, accounting for nearly one-fifth of all cancer-related deaths. Immunotherapy with immune checkpoint inhibitors has become one of the most promising approaches in the treatment of advanced lung cancer, although beneficial responses are seen only in a proportion of patients. To improve immunotherapy treatment responses in lung cancer, we need to identify which immunosuppression mechanisms are activated in the tumor microenvironment. In this study, we investigated gene expression profiles in intra-tumoral immune cells in lung cancer, focusing on tumor-associated macrophages, and interactions with CD4+ and CD8+ T cells. Our data highlight two newly described immunosuppressive pathways, which may represent novel innate immune checkpoints dampening the anti-tumor T cell immune response in lung cancer. Our results substantiate the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy.Non-small cell lung carcinoma (NSCLC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related deaths. Immunotherapy with immune checkpoint inhibitors shows beneficial responses, but only in a proportion of patients. To improve immunotherapy in NSCLC, we need to map the immune checkpoints that contribute immunosuppression in NSCLC-associated immune cells and to identify novel pathways that regulate immunosuppression. Here, we investigated the gene expression profiles of intra-tumoral immune cells isolated from NSCLC patients and compared them to the expression profiles of their counterparts in adjacent healthy tissue. Transcriptome analysis was performed on macrophages, CD4+ and CD8+ T cells. The data was subjected to Gene Ontology (GO) term enrichment and weighted correlation network analysis in order to identify mediators of immunosuppression in the tumor microenvironment in NSCLC. Immune cells from NSCLC revealed a consistent differential expression of genes involved in interactions between myeloid cells and lymphocytes. We further identified several immunosuppressive molecules and pathways that may be activated in tumor-associated macrophages in NSCLC. Importantly, we report novel data on immune cell expression of the newly described CD200/CD200R1 pathway, and the leukocyte immunoglobulin-like receptors (LILRs), which may represent novel innate immune checkpoints, dampening the anti-tumor T cell immune response in NSCLC. Our study substantiates the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy.

Nuclear Leukocyte Immunoglobulin-like Receptor A3 Is Monomeric and Is Involved in Multiple Layers of Regulated Gene Expression and Translation.

The leukocyte immunoglobulin-like receptor A3 (LILRA3) is a soluble protein primarily expressed by peripheral blood monocytes and is abundant in sera of healthy donors. Extracellular LILRA3 is anti-inflammatory and displays neuro-regenerative functions in vitro. However, its intracellular expression, distribution, and function(s) remain unknown. Using a combination of high-resolution confocal and super-resolution microscopy, we identified intracellular expression of native LILRA3 in the nucleus of peripheral blood monocytes and in vitro-derived macrophages. This unexpected nuclear localization of LILRA3 was confirmed in LILRA3-GFP-transfected HEK293T cells. Western blot of proteins fractionated from primary macrophages and the transfected HEK293T cells confirmed nuclear localization of the native and expressed LILRA3 proteins. Interestingly, most of the LILRA3 in the nucleus was in a monomeric form like the biologically active secreted protein, while that in the other cellular compartments was in mixed monomeric, dimeric, and oligomeric forms. The predominant presence of monomeric LILRA3 in the nucleus was independently corroborated in transfected live HEK293T cells using the number and molecular brightness (N&B) analysis method. Immunoprecipitation and mass spectrometric peptide sequencing studies revealed that nuclear LILRA3 co-immunoprecipitated with several nuclear proteins involved in host protein synthesis machinery via direct interactions to a key multifunctional RNA-binding protein, the Ewing sarcoma breakpoint region 1 protein (EWS) (data are available via ProteomeXchange with identifier PXD024602). The biological significance of the nuclear expression of LILRA3 and its interaction with these key proteins remain to be elucidated.

Development of polymorphic markers in the immune gene complex loci of cattle

The addition of cattle health and immunity traits to genomic selection indices holds promise to increase individual animal longevity and productivity, and decrease economic losses from disease. However, highly variable genomic loci that contain multiple immune-related genes were poorly assembled in the first iterations of the cattle reference genome assembly and underrepresented during the development of most commercial genotyping platforms. As a consequence, there is a paucity of genetic markers within these loci that may track haplotypes related to disease susceptibility. By using hierarchical assembly of bacterial artificial chromosome inserts spanning 3 of these immune-related gene regions, we were able to assemble multiple full-length haplotypes of the major histocompatibility complex, the leukocyte receptor complex, and the natural killer cell complex. Using these new assemblies and the recently released ARS-UCD1.2 reference, we aligned whole-genome shotgun reads from 125 sequenced Holstein bulls to discover candidate variants for genetic marker development. We selected 124 SNPs, using heuristic and statistical models to develop a custom genotyping panel. In a proof-of-principle study, we used this custom panel to genotype 1,797 Holstein cows exposed to bovine tuberculosis (bTB) that were the subject of a previous GWAS study using the Illumina BovineHD array. Although we did not identify any significant association of bTB phenotypes with these new genetic markers, 2 markers exhibited substantial effects on bTB phenotypic prediction. The models and parameters trained in this study serve as a guide for future marker discovery surveys particularly in previously unassembled regions of the cattle genome.

Plasmodium falciparum RIFIN is a novel ligand for inhibitory immune receptor LILRB2

Plasmodium falciparum causes the most severe form of malaria. Acquired immunity against P. falciparum provides insufficient protection even after repeated infections. Therefore, P. falciparum parasites might exploit inhibitory receptors for immune evasion. P. falciparum RIFINs are products of a multigene family consisting of 150–200 genes. Previously, we demonstrated that some RIFINs downregulate the immune response through the leukocyte immunoglobulin-like receptor (LILR) family inhibitory receptor, LILRB1, and leukocyte-associated immunoglobulin-like receptor 1, LAIR1. In this study, we further analyzed the expression of inhibitory receptor ligands on P. falciparum-infected erythrocytes and found that P. falciparum-infected erythrocytes expressed ligands for another LILR family inhibitory receptor, LILRB2, that recognizes HLA class I molecules as a host ligand. Furthermore, we identified that a specific RIFIN was a ligand for LILRB2 by using a newly developed RIFIN expression library. In addition, the domain 3 of LILRB2 was involved in RIFIN binding, whereas the domains 1 and 2 of LILRB2 were involved in the binding to HLA class I molecules. These results suggest that inhibitory receptor LILRB2 is also targeted by RIFIN for immune evasion of P. falciparum similar to LILRB1 and LAIR1.

Upregulated expression of leukocyte immunoglobulin-like receptor A3 in patients with severe aplastic anemia.

Severe aplastic anemia (SAA) is a rare and potentially life-threatening disease characterized by pancytopenia and bone marrow (BM) hypoplasia. In a previous study by our group, increased expression of leukocyte immunoglobulin-like receptors A (LILRA), LILRA3 in myeloid dendritic cells (mDCs) and LILRA5 in CD34+ cells in SAA was detected using proteomics techniques, highlighting their potential role in disease pathogenesis. In the present study, the expression of LILRA1-6 mRNA was assessed in the BM mononuclear cells of patients with SAA using reverse transcription-quantitative (RT-q)PCR. The expression of homogenic LILRA3 and LILRA5 isoform on mDCs, as well as CD34+, CD3+CD8+, CD19+ and CD14+ cells, was detected using flow cytometry. mDCs were then induced, cultured and sorted. The expression of LILRA3 was confirmed using RT-qPCR and western blot analyses. The serum levels of soluble LILRA3 were measured using ELISA. Furthermore, the relationship between LILRA3 expression and disease severity was assessed. The results indicated increased LILRA3 mRNA expression in patients with SAA. The percentage of LILRA3+ in BM mDCs and CD34+ cells was increased. Compared with controls, the relative LILRA3 mRNA expression and the relative protein intensity were highly increased in SAA mDCs. The serum LILRA3 levels in patients with SAA were also increased. The proportion of LILRA3+CD11C+ human leukocyte antigen (HLA)-DR+/CD11C+HLA-DR+ cells was positively correlated with the ratio of LILRA3+CD34+/CD34+ cells and the expression of LILRA3 mRNA. Taken together, the expression of LILRA3 on mDCs of patients with SAA was increased, which may affect the function of mDCs. LILRA3 may have a significant role in the immune pathogenesis of SAA.

Leukocyte Immunoglobulin-Like Receptor A3 (LILRA3): A Novel Marker for Lymphoma Development among Patients with Young Onset Sjogren's Syndrome.

Background: Primary Sjogren’s syndrome (SS) is an autoimmune disease with a strong predilection for lymphoma development, with earlier disease onset being postulated as an independent risk factor for this complication. Variations of the Leukocyte immunoglobulin-like receptor A3 (LILRA3) gene have been previously shown to increase susceptibility for both SS and non-Hodgkin B-cell lymphoma (B-NHL) in the general population. We aimed to investigate whether variations of the LILRA3 gene could predispose for lymphoma development in the context of SS. Methods: Study population, all of Greek origin, included 101 SS cases with a current or previous diagnosis of lymphoma (SS-lymphoma, SS-L) and 301 primary SS patients not complicated by lymphoma (SS-non-lymphoma, SS-nL). All SS patients fulfilled the 2016 SS American College of Rheumatology/European league against Rheumatism (ACR/EULAR) classification criteria. A total of 381 healthy controls (HC) of similar age/sex/race distribution were also included. On the basis of the age of SS onset and the presence or absence of adverse predictors for lymphoma development, SS patients were further stratified into younger (≤40 years) and older (>40 years) age of disease onset, as well as into high/medium and low risk groups. Polymerase chain reaction (PCR) was implemented for the detection of the following LILRA3 gene variants: homozygous non-deleted or functional wild type (+/+) heterozygous (+/−) and homozygous deleted (−/−). LILRA3 serum protein levels were quantitated by enzyme-linked immunosorbent assay (ELISA) in 85 individuals (29 SS-L, 35 SS-nL patients and 21 HC). Results: While no statistically significant differences were detected in the overall frequency of LILRA3 gene variants between SS-L, SS-nL and HC groups, LILRA3 serum protein levels were increased in the SS-L group compared to HC (1.27 ± 1.34 vs. 0.38 ± 0.34 ng/mL, p-value: 0.004). After stratification according to the age of SS onset and history of lymphoma, as well as the presence or absence of adverse predictors for lymphoma development, the prevalence of the functional LILRA3 gene variant was found to be significantly increased in the young onset SS-L group compared to the HC of similar age and sex distribution (100% vs. 82.9%, p = 0.03), as well as in the high/medium risk SS compared to the low risk SS (91.3 vs. 78.3%, p = 0.0012). Of note, young onset SS-L and SS-nL groups displayed higher LILRA3 serum levels compared to their older counterparts (p-values: 0.007 and 0.0005, respectively). Conclusion: The functional LILRA3 gene variant increases susceptibility to SS-related lymphoma development in patients with a disease onset of <40 years old, implying that genetically determined deranged immune responses in younger SS individuals could underly their pronounced risk for lymphoma development.

ANGPTL2 Induces Synovial Inflammation via LILRB2.

Angiopoietin-like proteins (ANGPTLs) are circulating proteins that are expressed in various cells and tissues and are thought to be involved in the repair and remodeling of damaged tissues; however, ANGPTL2 hyperfunction has been shown to cause chronic inflammation, leading to the progression of various diseases. ANGPTL2 is known to exert cellular effects via receptors such as integrin α5β1 and leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); however, their roles in ANGPTL2-induced inflammation remain unclear. In this study, we investigated the mechanisms underlying ANGPTL2-induced inflammation involving LILRB2 and various signaling pathways in human fibroblast-like synoviocytes (HFLS). The effects of ANGPTL2 and an anti-LILRB2 antibody on the gene expression of various inflammation-related factors were examined using real-time RT-PCR, while their effects on MAPK, NF-κB, and Akt phosphorylation were analyzed by western blotting. We found that the addition of ANGPTL2 enhanced the gene expression of inflammatory factors, whereas pretreatment with the anti-LILRB2 antibody for 12h decreased the expression of these factors. Similarly, ANGPTL2 addition activated the phosphorylation of ERK, p38, JNK, NF-κB, and Akt in HFLS; however, this effect was significantly inhibited by pretreatment with the anti-LILRB2 antibody. Together, the findings of this study demonstrate that ANGPTL2 induces the expression of inflammatory factors via LILRB2 in synovial cells. Therefore, LILRB2 could be a potential therapeutic agent for treating matrix degradation in osteoarthritis.

LILRA3 is related to monocyte-derived dendritic cell maturation and activation.

Objective(s):Previously we reported functional leukocyte immunoglobulin-like receptor A3 (LILRA3) leads to susceptibility and sub-phenotypes of several autoimmune diseases. LILRA3 levels in blood serum and CD14+ monocytes enhanced in systemic lupus erythematosus and resulted in disease severity. However, the mechanism of LILRA3 in the pathogenesis of autoimmunity remains elusive. This study aims to explore the potential impact of LILRA3 on the differentiation, maturation, and function of monocyte-derived DCs (MoDCs).Materials and Methods:The human monocytic cell line (THP-1) was cultured to derive MoDCs in vitro. We performed plasmid transfection to examine the impact of LILRA3 on monocyte differentiation. Surface markers on MoDCs were measured using FACS. To assess the function of mature MoDCs, IL-12p70, IFN-γ and IL-4 levels were detected after the mixed leucocyte response by enzyme-linked immunosorbent assay. Western blot assay was employed in this study to determine the signaling pathways in MoDCs activation.Results:LILRA3 promotes MoDCs maturation, our results showed significant up-regulation of CD40, CD80, CD86, CD209, and HLA-DR and increased production of pro-inflammatory cytokine IL-12. LILRA3-treated MoDCs exhibited a robust proliferation of allogeneic CD4+ T cells and induced naïve CD4+ T cell polarization into the Th1 phenotype. Furthermore, the preceding activation of MoDCs maturation and LILRA3 function might be attributed to p38 MAPK and STAT1 signaling pathway’s aberrant activation. Conclusion:This is the first study to report that LILRA3 played a critical role in promoting MoDCs maturation and directing MoDCs to modulate Th1 cell differentiation, which may have a role in the pathogenesis of autoimmune diseases.

Identification of distinct LRC- and Fc receptor complex-like chromosomal regions in fish supports that teleost leukocyte immune-type receptors are distant relatives of mammalian Fc receptor-like molecules.

Leukocyte immune-type receptors (LITRs) are a large family of immunoregulatory receptor-types originally identified in the channel catfish (Ictalurus punctatus (Ip)LITRs). Phylogenetic analyses of LITRs show that they share distant evolutionary relationships with important mammalian immunoregulatory receptors belonging to the Fc receptors family and the leukocyte receptor complex (LRC), but their syntenic relationships with these immunoglobulin superfamily members have not been investigated. To further examine the possible evolutionary connections between teleost LITRs and various mammalian immunoregulatory receptor-types, we surveyed the genomic databases of representative vertebrate taxa and our results show that teleost LITRs generally exist in large genomic clusters, which are linked to vangl2, arhgef11, and slam family genes, features that are also shared by amphibian and mammalian Fc receptor-like molecules (FCRLs). Moreover, detailed phylogenetic comparisons between the individual Ig-like domains of LITRs and mammalian FCRLs shows that these receptors share related Ig-like domains indicative of their common ancestry. However, contrary to our previous reports, no supportive evidence for phylogenetic relationships between the Ig-like domains of LITRs with the Ig-like domains of LRC-encoded mammalian immunoregulatory receptors was found. We also identified an LRC-like region in the zebrafish genome, but no expanded litr-related genes were located in this region. Similarly, no lilr-related genes were found in spotted gar, a representative basal ray-finned fish. Finally, two distantly related fcrls and an LRC-like gene were identified in the elephant shark genome, suggesting that the loss of an immunoregulatory receptor-containing LRC region may be unique to ray-finned fish.

TARM1 contributes to development of arthritis by activating dendritic cells through recognition of collagens

TARM1 is a member of the leukocyte immunoglobulin-like receptor family and stimulates macrophages and neutrophils in vitro by associating with FcRγ. However, the function of this molecule in the regulation of the immune system is unclear. Here, we show that Tarm1 expression is elevated in the joints of rheumatoid arthritis mouse models, and the development of collagen-induced arthritis (CIA) is suppressed in Tarm1–/– mice. T cell priming against type 2 collagen is suppressed in Tarm1–/– mice and antigen-presenting ability of GM-CSF-induced dendritic cells (GM-DCs) from Tarm1–/– mouse bone marrow cells is impaired. We show that type 2 collagen is a functional ligand for TARM1 on GM-DCs and promotes DC maturation. Furthermore, soluble TARM1-Fc and TARM1-Flag inhibit DC maturation and administration of TARM1-Fc blocks the progression of CIA in mice. These results indicate that TARM1 is an important stimulating factor of dendritic cell maturation and could be a good target for the treatment of autoimmune diseases.

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

Inhibitory leukocyte immunoglobulin-like receptors (LILRBs 1–5) transduce signals via intracellular immunoreceptor tyrosine-based inhibitory motifs that recruit phosphatases to negatively regulate immune activation. The activation of LILRB signaling in immune cells may contribute to immune evasion. In addition, the expression and signaling of LILRBs in cancer cells especially in certain hematologic malignant cells directly support cancer development. Certain LILRBs thus have dual roles in cancer biology—as immune checkpoint molecules and tumor-supporting factors. Here, we review the expression, ligands, signaling, and functions of LILRBs, as well as therapeutic development targeting them. LILRBs may represent attractive targets for cancer treatment, and antagonizing LILRB signaling may prove to be effective anti-cancer strategies.

PIR-B Regulates CD4+ IL17a+ T-Cell Survival and Restricts T-Cell–Dependent Intestinal Inflammatory Responses

Background & AimsCD4+ T cells are regulated by activating and inhibitory cues, and dysregulation of these proper regulatory inputs predisposes these cells to aberrant inflammation and exacerbation of disease. We investigated the role of the inhibitory receptor paired immunoglobulin-like receptor B (PIR-B) in the regulation of the CD4+ T-cell inflammatory response and exacerbation of the colitic phenotype.MethodsWe used Il10-/- spontaneous and CD4+CD45RBhi T-cell transfer models of colitis with PIR-B-deficient (Pirb-/-) mice. Flow cytometry, Western blot, and RNA sequencing analysis was performed on wild-type and Pirb-/- CD4+ T cells. In silico analyses were performed on RNA sequencing data set of ileal biopsy samples from pediatric CD and non–inflammatory bowel disease patients and sorted human memory CD4+ T cells.ResultsWe identified PIR-B expression on memory CD4+ interleukin (IL)17a+ cells. We show that PIR-B regulates CD4+ T-helper 17 cell (Th17)-dependent chronic intestinal inflammatory responses and the development of colitis. Mechanistically, we show that the PIR-B– Src-homology region 2 domain-containing phosphatase-1/2 axis tempers mammalian target of rapamycin complex 1 signaling and mammalian target of rapamycin complex 1–dependent caspase-3/7 apoptosis, resulting in CD4+ IL17a+ cell survival. In silico analyses showed enrichment of transcriptional signatures for Th17 cells (RORC, RORA, and IL17A) and tissue resident memory (HOBIT, IL7R, and BLIMP1) networks in PIR-B+ murine CD4+ T cells and human CD4+ T cells that express the human homologue leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3). High levels of LILRB3 expression were associated strongly with mucosal injury and a proinflammatory Th17 signature, and this signature was restricted to a treatment-naïve, severe pediatric CD population.ConclusionsOur findings show an intrinsic role for PIR-B/LILRB3 in the regulation of CD4+ IL17a+ T-cell pathogenic memory responses.

Potential Treatment for Multiple Myeloma: Immune Checkpoint Inhibitors

Multiple myeloma (MM) is a malignant disease in which monoclonal plasma cells proliferate abnormally. The poor prognosis of MM is always closely related to the immunosuppression of T cells. Restore the immune function of suppressed T cells may reverse the immunodeficiency in the MM microenvironment and improve prognosis. In recent years, immunosuppressive receptors such as programmed cell death receptor-1 (PD-1), T cells immunoglobulin and ITIM domain (TIGIT), Lymphocyte-Activation Gene-3 (LAG-3), T cell immunoglobulin and mucin domain–containing molecule 3 (Tim-3), Leukocyte immunoglobulin-like receptor B1 (LILRB1) and Cytotoxic T- lymphocyte antigen 4 (CTLA-4) have been discovered playing a key role in the tumor immunodeficiency microenvironment. For patients with solid tumors and some leukemia, immunotherapy targeting such receptors can significantly improve the T cells immunodeficiency. However, similar positive results were not found in MM patients, which is related to the complex immunosuppressive mechanism. At present, the understanding of immunosuppressive receptors in MM is insufficient. In this review, this paper summarized part of the studies on PD-1, TCLA-4, Tim-3, TIGIT and other popular immunosuppressive receptors in MM, in order to attract more attention, and in-depth research on the immunotherapy also to promote the immunotherapy of MM from basic research to clinical transformation as soon as possible.

The association of leukocyte immunoglobulin-like receptor subfamily B-4 expression in acute myeloid leukemia and central nervous system involvement

Central nervous system (CNS) involvement in patients with acute myeloid leukemia (AML) varies, ranging from 0.6%–46%. Leukocyte immunoglobulin-like receptor B4 (LILRB4) has been shown to be critical in orchestration of infiltration of AML cells into the CNS in animal models, however it is unknown if an association exists between LILRB4 and CNS involvement (CNS+) in human patients with AML. LILRB4 was measured by flow cytometry in a heterogeneous population of fifty-six AML patients. Patients were then followed clinically for the development of CNS + . LILRB4 was positive in 91 % of patients with CNS + compared to 38 % without CNS involvement (p < 0.002). In logistic analysis: age, BMI, serum albumin and positive LILRB4 were predictive for CNS+ [OR, 95 % CI, p-value]: 0.95, 0.92−0.99, p < 0.01; 0.85, 0.73−0.998, p < 0.05; 0.23, 0.066−0.78, p < 0.02; 16.46, 1.93–140.2, p < 0.02, respectively. This finding of the association of LILRB4 with CNS + in combination with earlier findings suggests that LILRB4 has a mechanistic role in infiltration of the CNS and may provide insight into the pathogenesis of AML seeding the CNS. Moreover, this proof of concept and the findings in the present study may lead to the development of innovative and novel therapies to improve the lives of patients with AML.

Leukocyte immunoglobulin-like receptor A3 is increased in IBD patients and functions as an anti-inflammatory modulator.

Growing evidence shows that a homozygous 6·7-kb deletion of the novel anti-inflammatory molecule leukocyte immunoglobulin-like receptor A3 (LILRA3) is associated with many autoimmune disorders. However, its effects on pathogenesis of inflammatory bowel disease (IBD) have yet not been clarified. LILRA3 is mainly expressed in monocytes, whereas its effects on biological behaviors of monocytes have not been systematically reported. In our study, to investigate the association between LILRA3 polymorphism and IBD susceptibility, LILRA3 polymorphism was assessed in 378 IBD patients and 509 healthy controls. Quantitative real time PCR (qRT-PCR), Western blot and immunohistochemistry (IHC) were employed to detect the LILRA3 expression in IBD patient blood and intestinal samples. The human U937 monocyte cell line was employed to establish LILRA3 over-expressing cells and the effects of LILRA3 on the biological behaviors of U937 cells were systematically explored. Although no association of the polymorphism with IBD development was found, LILRA3 expression was markedly increased in IBD patients compared with healthy controls. Over-expression of LILRA3 in monocytes led to significant decreases in secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-6. Additionally, LILRA3 abated monocyte migration by reducing the expression of several chemokines and enhanced monocyte phagocytosis by increasing CD36 expression. Furthermore, LILRA3 promoted monocyte proliferation through a combination of Akt and extracellular receptor kinase/mitogen-activated protein kinase (Erk/MEK) signaling pathways. We report for the first time, to our knowledge, that LILRA3 is related to IBD and functions as an anti-inflammatory modulator in U937 cells.

LILRB4-targeting Antibody-Drug Conjugates for the Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the most common and aggressive blood cancer in adults. In particular, significant unmet medical needs exist for effective treatment strategies for acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) AML subtypes. Antibody-drug conjugates (ADC) are a promising drug class for AML therapy, as demonstrated by the FDA-approved anti-CD33 ADC, gemtuzumab ozogamicin (Mylotarg). However, CD33 is expressed in normal hematopoietic stem cells, highlighting the critical need to identify AML-specific targets to minimize the risk of potential adverse effects. We have demonstrated that the leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) is expressed at significantly higher levels on monocytic M4 and M5 AML cells than on normal counterparts. Here, we test whether LILRB4 is a promising ADC target to kill monocytic AML cells while sparing healthy counterparts. To this end, we generated ADCs from a humanized anti-LILRB4 mAb and the antimitotic payload, monomethyl auristatin F. The conjugates constructed were characterized and evaluated for LILRB4-specific cell killing potency, toxicity to progenitor cells, pharmacokinetics, and therapeutic efficacy. Our ADC linker technology platform efficiently generated homogeneous anti-LILRB4 ADCs with defined drug-to-antibody ratios. The homogeneous anti-LILRB4 ADCs demonstrated the capacity for LILRB4-mediated internalization, suitable physicochemical properties, and high cell killing potency against LILRB4-positive AML cells. Importantly, our data indicate that these ADCs spare normal progenitor cells. One of our homogeneous conjugates exerted a remarkable therapeutic effect and no significant toxicity in a xenograft mouse model of disseminated human AML. Our findings highlight the clinical potential of anti-LILRB4 ADCs in monocytic AML therapy.