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Leukocyte immunoglobulin (Ig)-like receptors (LILRs) constitute a family of 11 structurally related receptors predominantly expressed on immune cells. Despite their functional diversity, LILRs show a high degree of sequence homology within their extracellular domains, with amino acid sequence identities ranging from approximately 50% to nearly 100%. This molecular similarity poses a significant challenge for antibody-based detection, often resulting in cross-reactivity and misinterpretation of expression profiles. Nevertheless, a comprehensive assessment of antibody specificity across the entire LILR family has not been previously conducted. In this study, we performed a rigorous validation of commercially available antibodies to evaluate their specificity and cross-reactivity against all LILR family members. Using flow cytometry with LILR-transfected K562 cells, we reliably identified specific antibodies for LILRA2, LILRA4, LILRA5, LILRB1, LILRB2, and LILRB4. Notably, more than half of the commercial antibodies showed cross-reactivity. Given the lack of a reliable antibody for LILRA3, the only known soluble member of the family, we generated novel anti-LILRA3 monoclonal antibodies in mice. Three hybridoma clones exhibiting high specificity for LILRA3 were successfully isolated and validated. Using these antibodies, we established a sensitive sandwich ELISA, which successfully detected LILRA3 in individuals carrying functional alleles, while no protein was detected in those with the 6.7-kb deletion or premature termination codons. Moreover, serum concentrations of LILRA3 were substantially higher than previously reported. These findings not only provide essential tools for accurate detection of LILRA3 but also underscore the importance of rigorous antibody validation in LILR-related research.

Human leukocyte immunoglobulin (Ig)-like receptors (LILR) are a family of 11 innate immunomodulatory receptors, primarily expressed on lymphoid and myeloid cells. LILRs are either activating (LILRA) or inhibitory (LILRB) depending on their associated signalling domains (D). With the exception of the soluble LILRA3, LILRAs mediate immune activation, while LILRB1-5 primarily inhibit immune responses and mediate tolerance. Abnormal expression and function of LILRs is associated with a range of pathologies, including immune insufficiency (infection and malignancy) and overt immune responses (autoimmunity and alloresponses), suggesting LILRs may be excellent candidates for targeted immunotherapies. This review will discuss the biology and clinical relevance of this extensive family of immune receptors and will summarise the recent developments in targeting LILRs in disease settings, such as cancer, with an update on the clinical trials investigating the therapeutic targeting of these receptors.

There are ten leukocyte immunoglobulin (Ig)-like receptor ( LILR ) genes, i.e., five genes encoding activating receptors ( LILRA1, LILRA2, LILRA4, LILRA5 , and LILRA6 ) characterized by their truncated cytoplasmic tails, and five genes encoding inhibitory receptors ( LILRB1, LILRB2, LILRB3, LILRB4 , and LILRB5 ) characterized by their extended cytoplasmic tails containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Among these, LILRB3 , LILRA6 , and LILRA3 are known for harboring high frequencies of copy number variations (CNVs). However, the presence of CNVs in the leukocyte receptor complex (LRC) region complicates single nucleotide polymorphism (SNP) association analysis within commercially available SNP microarray datasets. This study introduces LILR Genotype Imputation with Attribute Bagging (LIBAG), a novel method for determining CNVs in LILRB3, LILRA6 , and LILRA3 from commercially available SNP genotyping array datasets. LILRA6 CNV imputation accuracy peaked at 98.0% for the Infinium Japanese Screening Array, followed by 97.4% for Axiom Japonica V2, 97.3% for Axiom Japonica Array NEO, and 94.3% for Axiom Japonica V1, with the lowest recorded accuracy of 93.6% for the Axiom Genome-wide ASI1 array. For the 1000 Genomes Project (1kGP) dataset, LILRA6 CNV imputation achieved peak accuracies of 94.5% for 1kGP-EAS (East Asian), 86.6% for 1kGP-AMR (Admixed American), 83.8% for 1kGP-EUR European), and 75.0% for 1kGP-AFR (African), particularly after the 20 kb flanking region. Similarly, imputation accuracy for LILRA3 CNV progressively increased, peaking at the 80 kb flanking region. Accuracy reached 1kGP-AMR, reaching 99.2% and 98.9% for 1kGP-AFR, 98.7% for 1kGP-EUR, and 97.5% for 1kGP-EAS. Investigating the LILR copy number (CN) in diseases associated with HLA class I molecules will provide further insights into disease pathogenesis.

Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule that has an immunosuppressive effect mediated by binding to immune inhibitory leukocyte immunoglobulin-like receptors (LILR) B1 and LILRB2. A conventional HLA-G isoform, HLA-G1, forms a heterotrimeric complex composed of a heavy chain (α1-α3 domains), β2-microglobulin (β2m) and a cognate peptide. One of the other isoforms, HLA-G2, lacks a α2 domain or β2m to form a nondisulfide-linked homodimer, and its ectodomain specifically binds to LILRB2 expressed in human monocytes, macrophages, and dendritic cells. The administration of the ectodomain of HLA-G2, designated the soluble HLA-G2 homodimer, showed significant immunosuppressive effects in mouse models of rheumatoid arthritis and systemic lupus erythematosus, presumably by binding to a mouse ortholog of LILRB2, paired immunoglobulin-like receptor B. However, the refolded soluble HLA-G2 homodimer used in these studies tends to aggregate and degrade; thus, its stability for clinical use has been a concern. In the present study, we improved the stability of the refolded soluble HLA-G2 homodimer via a site-directed PEGylation method. PEGylation at an original free cysteine residue, Cys42, resulted in increased lyophilization and thermal and serum stability. Furthermore, the PEGylated soluble HLA-G2 homodimer could better suppress atopic symptoms in mice than the non-PEGylated homodimer. These results suggest that PEGylated soluble HLA-G2 homodimers could be candidates for immunosuppressive biologics that specifically target LILRB2-positive myelomonocytic antigen-presenting cells.

The leukocyte immunoglobulin-like receptor (LILR) family, a group of primate-specific immunoreceptors, is widely expressed on most immune cells and regulates immune responses through interactions with various ligands. The inhibitory type, LILRB, has been extensively studied, and many ligands, such as HLA class I, have been identified. However, the activating type, LILRA, is less understood. We have previously identified microbially cleaved immunoglobulin as a non-self-ligand for LILRA2. In this study, we identified fibrinogen as an endogenous ligand for LILRA2 using mass spectrometry. Although human plasma contains fibrinogen in abundance in its soluble form, LILRA2 only recognizes solid-phase fibrinogen. In addition to the activating LILRA2, fibrinogen was also recognized by the inhibitory LILRB2 and by soluble LILRA3. In contrast, fibrin was recognized by LILRB2 and LILRA3, but not by LILRA2. Moreover, LILRA3 bound more strongly to fibrin than to fibrinogen and blocked the LILRB2-fibrinogen/fibrin interaction. These results suggest that morphological changes in fibrinogen determine whether activating or inhibitory immune responses are induced. Upon recognizing solid-phase fibrinogen, LILRA2 activated human primary monocytes and promoted the expression of various inflammation-related genes, such as chemokines, as revealed by RNA-seq analysis. A blocking antibody against LILRA2 inhibited the fibrinogen-induced inflammatory responses, indicating that LILRA2 is the primary receptor of fibrinogen. Taken together, our findings suggest that solid-phase fibrinogen is an inflammation-inducing endogenous ligand for LILRA2, and this interaction may represent a novel therapeutic target for inflammatory diseases.

Inhibitory receptors play a pivotal role in fine-tuning immune responses. The leukocyte receptor complex (LRC) encodes multiple receptor families, including the leukocyte immunoglobulin-like receptor (LILR) family, which next to activating receptors involves several inhibitory receptors. The LILRB1 and LILRB2 receptors are considered immune checkpoint inhibitors, which may interact with MHC class I molecules, and are expressed mainly on monocytes, B- and T-cells. In this study, we characterized and at the genomic and transcriptomic level in three Old World monkey species, namely rhesus and long-tailed macaques and Hamadryas baboon, using SMRT sequencing on PacBio platforms. We describe 71 and 58 alleles in the two macaque species, of which only one allele was previously published. In contrast, less polymorphism is observed in the Hamadryas baboon, with only six and seven alleles characterized. Phylogenetic analysis, including known human data, revealed extensive diversification of the and in macaques, with allelic variation clustering into nine and twelve distinct lineages, respectively. This contrasts with the more conserved repertoires observed in humans and Hamadryas baboons. Compared with our experience analyzing and transcriptome data, the and transcriptomes were dominated by alternatively spliced isoforms. Alternative 3' splice sites near exons 10 and 15 and/or skipping of exon 15, were encountered for most alleles. In , the deletion of exon 9 is the most prominent event, next to deletion of exon 10 and the use of alternative 3' splice sites near exons 10 and 15. The exons that encode the extracellular domains remain largely intact, suggesting that alternative splicing predominantly affects the stem region and the signaling capacity of the LILRB1 and LILRB2 receptors.

The mammalian Leukocyte Receptor Complex (LRC) chromosomal region may contain gene families for the killer cell immunoglobulin-like receptor (KIR) and/or leukocyte immunoglobulin-like receptor (LILR) collections as well as various framing genes. This complex region is well described in humans, mice, and some domestic animals. Although single KIR genes are known in some Carnivora, their complements of LILR genes remain largely unknown due to obstacles in the assembly of regions of high homology in short-read based genomes. As part of the analysis of felid immunogenomes, this study focuses on the search for LRC genes in reference genomes and the annotation of LILR genes in Felidae. Chromosome-level genomes based on single-molecule long-read sequencing were preferentially sought and compared to representatives of the Carnivora. Seven putatively functional LILR genes were found across the Felidae and in the Californian sea lion, four to five genes in Canidae, and four to nine genes in Mustelidae. They form two lineages, as seen in the Bovidae. The ratio of functional genes for activating LILRs to inhibitory LILRs is slightly in favor of inhibitory genes in the Felidae and the Canidae; the reverse is seen in the Californian sea lion. This ratio is even in all of the Mustelidae except the Eurasian otter, which has a predominance of activating LILRs. Various numbers of LILR pseudogenes were identified. The structure of the LRC is rather conservative in felids and the other Carnivora studied. The LILR sub-region is conserved within the Felidae and has slight differences in the Canidae, but it has taken various evolutionary paths in the Mustelidae. Overall, the process of pseudogenization of LILR genes seems to be more frequent for activating receptors. Phylogenetic analysis found no direct orthologues across the Carnivora which corroborate the rapid evolution of LILRs seen in mammals.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) on human chromosome 19q13.4 encode 11 immunoglobulin superfamily receptors, exhibiting genetic diversity within and between human populations. Among the LILR genes, the genomic region surrounding LILRB3 and LILRA6 has yet to be fully characterized due to their significant sequence homology, which makes it difficult to differentiate between them. To examine the LILRB3 and LILRA6 genomic region, a tool named JoGo-LILR CN Caller, which can call copy number from short-read whole genome sequencing (srWGS) data, was applied to an extensive international srWGS dataset comprising 2,504 samples. During this process, a previously unreported loss of both LILRB3 and LILRA6 was detected in three samples. Using long-read sequencing of these samples, we have discovered a novel large deletion (33,692 bp) in the LILRB3 and LILRA6 genomic regions in the Japanese population. This deletion spanned three genes, LILRB3 , LILRA6 , and LILRB5 , resulting in LILRB3 exons 12-13 being located immediately downstream of LILRB5 exons 1-12 with the loss of LILRA6 , suggesting the potential expression of a hybrid gene between LILRB5 and LILRB3 ( LILRB5-3 ). Transcription and subsequent translation of the LILRB5-3 hybrid gene were also verified. The hybrid junction was located within the intracellular domain, resulting in an LILRB5 extracellular domain fused to a partial LILRB3 intracellular domain with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), suggesting that LILRB5-3 acquired a novel signaling function. Further application of the JoGo-LILR tool to srWGS samples suggested the presence of the LILRB5-3 hybrid gene in the CEU population. Our findings provide insight into the genetic and functional diversity of the LILR family.

MHC class I (MHC-I) polymorphisms are associated with the outcome of some viral infections and autoimmune diseases. MHC-I proteins present antigenic peptides and are recognized by receptors on natural killer cells and cytotoxic T lymphocytes, thus enabling the immune system to detect self-antigens and eliminate targets lacking self or expressing foreign antigens. Recognition of MHC-I, however, extends beyond receptors on cytotoxic leukocytes. Members of the leukocyte Ig-like receptor (LILR) family are expressed on monocytic cells and can recognize both classical and non-classical MHC-I alleles. Despite their relatively broad specificity when compared to the T cell receptor or killer Ig-like receptors, variations in the strength of LILR binding between different MHC-I alleles have recently been shown to correlate with control of HIV infection. We suggest that LILR recognition may mediate MHC-I disease association in a manner that does not depend on a binary discrimination of self/non-self by cytotoxic cells. Instead, the effects of LILR activity following engagement by MHC-I may represent a \"degrees of self\" model, whereby strength of binding to different alleles determines the degree of influence exerted by these receptors on immune cell functions. LILRs are expressed by myelomonocytic cells and lymphocytes, extending their influence across antigen-presenting cell subsets including dendritic cells, macrophages, and B cells. They have been identified as important players in the response to infection, inflammatory diseases, and cancer, with recent literature to indicate that MHC-I recognition by these receptors and consequent allelic effects could extend an influence beyond the immune system.