Natural Killer Cells Promote Fetal Development through the Secretion of Growth-Promoting Factors.

Natural killer (NK) cells are present in large populations at the maternal-fetal interface during early pregnancy. However, the role of NK cells in fetal growth is unclear. Here, we have identified a CD49a+Eomes+ subset of NK cells that secreted growth-promoting factors (GPFs), including pleiotrophin and osteoglycin, in both humans and mice. The crosstalk between HLA-G and ILT2 served as a stimulus for GPF-secreting function of this NK cell subset. Decreases in this GPF-secreting NK cell subset impaired fetal development, resulting in fetal growth restriction. The transcription factor Nfil3, but not T-bet, affected the function and the number of this decidual NK cell subset. Adoptive transfer of induced CD49a+Eomes+ NK cells reversed impaired fetal growth and rebuilt an appropriate local microenvironment. These findings reveal properties of NK cells in promoting fetal growth. In addition, this research proposes approaches for therapeutic administration of NK cells in order to reverse restricted nourishments within the uterine microenvironment during early pregnancy.

TET2 and LILRB1 mutations are frequent in Epstein-Barr virus-positive diffuse large B-cell lymphoma especially in elderly patients.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) harboring Epstein-Barr virus (EBV) primarily occurs in patients who have underlying immunodeficiency or in elderly patients but is also reported in young, immunocompetent patients. The authors investigated the pathologic differences in EBV-positive DLBCL in these three groups of patients. METHODS: In total, 57 patients with EBV-positive DLBCL were included in the study; of these, 16 patients had associated immunodeficiency, 10 were young (younger than 50 years), and 31 were elderly (aged 50 years or older). Immunostaining for CD8, CD68, PD-L1, and EBV nuclear antigen 2, and panel-based next-generation sequencing was performed on formalin-fixed, paraffin-embedded blocks. RESULTS: Immunohistochemistry revealed EBV nuclear antigen 2 positivity in 21 of the 49 patients. The degree of CD8-positive and CD68-positive immune cell infiltration and PD-L1 expression did not differ significantly in each group. Extranodal site involvement was more common in young patients (p = .021). In mutational analysis, the genes with the highest mutation frequency were PCLO (n = 14), TET2 (n = 10), and LILRB1 (n = 10). For the TET2 gene, all 10 mutations were found in elderly patients (p = .007). Compared with a validation cohort, both TET2 and LILRB1 showed a higher mutation frequency in EBV-positive patients than in EBV-negative patients. CONCLUSIONS: EBV-positive DLBCL occurring in three different age and immune status groups showed similar pathologic characteristics. Notably, a high frequency of TET2 and LILRB1 mutations was characteristic of this disease in elderly patients. Further studies are needed to determine the role of TET2 and LILRB1 mutations in the development of EBV-positive DLBCL along with immune senescence. PLAIN LANGUAGE SUMMARY: Epstein-Barr virus-positive diffuse large B-cell lymphoma occurring in three different groups (immunodeficiency-associated, young, and elderly) showed similar pathologic characteristics. The frequency of TET2 and LILRB1 mutations was high in elderly patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

Elevated LILRB1 expression predicts poor prognosis and is associated with tumor immune infiltration in patients with glioma.

BACKGROUND: Leukocyte immunoglobulin-like receptor subfamily B1 (LILRB1) is regarded as an inhibitory molecule. However, the importance of LILRB1 expression in glioma has not yet been determined. This investigation examined the immunological signature, clinicopathological importance and prognostic value of LILRB1 expression in glioma. METHODS: We used data from the UCSC XENA database, the Cancer Genome Atlas (TCGA) database, the Chinese Glioma Genome Atlas (CGGA) database, the STRING database, the MEXPRESS database and our clinical glioma samples to perform bioinformatic analysis and used vitro experiments to examine the predictive value and potential biological roles of LILRB1 in glioma. RESULTS: Higher LILRB1 expression was considerably present in the higher WHO grade glioma group and was linked to a poorer prognosis in patients with glioma. Gene set enrichment analysis (GSEA) revealed that LILRB1 was positively correlated with the JAK/STAT signaling pathway. LILRB1 combined with tumor mutational burden (TMB) and microsatellite instability (MSI) may be a promising indicator for the effectiveness of immunotherapy in patients with glioma. Increased LILRB1 expression was positively linked with the hypomethylation, M2 macrophage infiltration, immune checkpoints (ICPs) and M2 macrophage makers. Univariate and multivariate Cox regression analyses determined that increased LILRB1 expression was a standalone causal factor for glioma. Vitro experiments determined that LILRB1 positively enhanced the proliferation, migration and invasion in glioma cells. MRI images demonstrated that higher LILRB1 expression was related with larger tumor volume in patients with glioma. CONCLUSION: Dysregulation of LILRB1 in glioma is correlated with immune infiltration and is a standalone causal factor for glioma.

Natural LILRB1 D1-D2 variants show frequency differences in populations and bind to HLA class I with various avidities.

Leukocyte immunoglobulin-like receptor B1 (LILRB1) is widely expressed on various immune cells and the engagement of LILRB1 to HLA class I and pathogen-derived proteins can modulate the immune response. In the current study, 108 LILRB1 alleles were identified by screening the LILRB1 locus from the 1000 Genomes Phase 3 database. Forty-six alleles that occurred in three or more individuals encode 28 LILRB1 allotypes, and the inferred LILRB1 allotypes were then grouped into 9 LILRB1 D1-D2 variants for further analysis. We found that variants 1, 2, and 3 represent the three most frequent LILRB1 D1-D2 variants and the nine variants show frequency differences in populations. The binding assay demonstrated that variant 1 bound to HLA class I with the highest avidity, and all tested LILRB1 D1-D2 variants bound to HLA-C with lower avidity than to HLA-A and -B. Locus-specific polymorphisms at positions 183, 189, and 268 in HLA class I and dimorphisms in HLA-A (positions 207 and 253) and in HLA-B (position 194) affect their binding to LILRB1. Notably, the electrostatic interaction plays a critical role in the binding of LILRB1 to HLA class I as revealed by electrostatic analysis and by comparison of different binding avidities caused by polymorphisms at positions 72 and 103 of LILRB1. In this paper, we present a comprehensive study of the population genetics and binding abilities of LILRB1. The data will help us better understand the LILRB1-related diversity of the immune system and lay a foundation for functional studies.

LILRB1 Intron 1 Has a Polymorphic Regulatory Region That Enhances Transcription in NK Cells and Recruits YY1.

LILRB1 is a highly polymorphic receptor expressed by subsets of innate and adaptive immune cells associated with viral and autoimmune diseases and targeted by pathogens for immune evasion. LILRB1 expression on human NK cells is variegated, and the frequency of LILRB1+ cells differs among people. However, little is known about the processes and factors mediating LILRB1 transcription in NK cells. LILRB1 gene expression in lymphoid and myeloid cells arises from two distinct promoters that are separated by the first exon and intron. In this study, we identified a polymorphic 3-kb region within LILRB1 intron 1 that is epigenetically marked as an active enhancer in human lymphoid cells and not monocytes. This region possesses multiple YY1 sites, and complexes of the promoter/enhancer combination were isolated using anti-YY1 in chromatin immunoprecipitation-loop. CRISPR-mediated deletion of the 3-kb region lowers LILRB1 expression in human NKL cells. Together, these results indicate the enhancer in intron 1 binds YY1 and suggest YY1 provides a scaffold function enabling enhancer function in regulating LILRB1 gene transcription in human NK cells.

Analysis of the expression and prognosis for leukocyte immunoglobulin-like receptor subfamily B in human liver cancer.

BACKGROUND: Leukocyte immunoglobulin-like receptor subfamily B (LILRB), including 5 subtypes, is a group of inhibitory receptors in the immune system. The LILRB family is known to be involved in the tumor progression of various cancer types, especially liver cancer. However, the expression patterns and prognostic values of LILRB family members in liver cancer tissues remain unclear. METHODS: We used the Oncomine database, GEPIA database, Kaplan-Meier Plotter, Timer, and TISIDB to assess the expression and prognostic value of the LILRB family in liver cancer patients. We also verified the expression of the LILRB family in tumor tissues and tumor-free liver tissues at the protein level by using immunohistochemistry. The STRING website was used to explore the interaction between the LILRB family and their related genes. The DAVID database was used to perform the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Flow cytometry was used to assess the infiltrated NK cells in liver cancer tissues. RESULTS: Our study revealed that the mRNA expression of LILRB1, LILRB2, LILRB3, and LILRB5 was downregulated, while compared with normal tissues, the mRNA expression of LILRB4 was upregulated in liver cancer tissues. Survival analysis revealed that LILRB2 and LILRB5 mRNA expression levels were significantly positively associated with overall survival (OS) and disease-free survival (DSS) and that the mRNA expression of all LILRB family members was significantly positively correlated with recurrence-free survival (RFS) and progression-free survival (PFS). Next, we further found that the mRNA expression of all LILRB family members was significantly associated with the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells in liver cancer. Finally, GO and KEGG analyses found that the LILRB family and its related genes were involved in antigen processing and presentation and natural killer cell-mediated cytotoxicity pathways. CONCLUSIONS: Our study suggested that LILRB family expression was associated with the prognosis of liver cancer patients and infiltrated immune cells. The LILRB family might be involved in antigen processing and presentation and natural killer cell-mediated cytotoxicity pathways.

Transcriptomics of type 2 diabetic and healthy human neutrophils.

OBJECTIVES: Chronic inflammatory diseases, including diabetes and cardiovascular disease, are heterogeneous and often co-morbid, with increasing global prevalence. Uncontrolled type 2 diabetes (T2D) can result in severe inflammatory complications. As neutrophils are essential to normal and aberrant inflammation, we conducted RNA-seq transcriptomic analyses to investigate the association between neutrophil gene expression and T2D phenotype. As specialized pro-resolving lipid mediators (SPM) act to resolve inflammation, we further surveyed the impact of neutrophil receptor binding SPM resolvin E1 (RvE1) on isolated diabetic and healthy neutrophils. METHODS: Cell isolation and RNA-seq analysis of neutrophils from N = 11 T2D and N = 7 healthy individuals with available clinical data was conducted. Additionally, cultured neutrophils (N = 3 T2D, N = 3 healthy) were perturbed with increasing RvE1 doses (0 nM, 1 nM, 10 nM, or 100 nM) prior to RNA-seq. Data was evaluated through a bioinformatics pipeline including pathway analysis and post hoc false discovery rate (FDR)-correction. RESULTS: We observed significant differential expression of 50 genes between T2D and healthy neutrophils (p < 0.05), including decreased T2D gene expression in inflammatory- and lipid-related genes SLC9A4, NECTIN2, and PLPP3 (p < 0.003). RvE1 treatment induced dose-dependent differential gene expression (uncorrected p < 0.05) across groups, including 59 healthy and 216 T2D neutrophil genes. Comparing T2D to healthy neutrophils, 1097 genes were differentially expressed across RvE1 doses, including two significant genes, LILRB5 and AKR1C1, involved in inflammation (p < 0.05). CONCLUSIONS: The neutrophil transcriptomic database revealed novel chronic inflammatory- and lipid-related genes that were differentially expressed between T2D cells when compared to controls, and cells responded to RvE1 dose-dependently by gene expression changes. Unraveling the mechanisms regulating abnormalities in diabetic neutrophil responses could lead to better diagnostics and therapeutics targeting inflammation and inflammation resolution.

The effect of LILRB1 but not LILRA3 gene polymorphism in immunopathology of ankylosing spondylitis-A parallel to KIR genes.

LILR and KIR receptors recognize HLA-B27 and may influence immune response in ankylosing spondylitis (AS) development. Purpose of the study was to analyse LILRB1/LILRA3 polymorphisms in AS. We observed a possible protective effect of the T allele of LILRB1 rs1061680:T>C and no association with insertion/deletion polymorphisms of LILRA3 with AS.

The impact of HLA-G, LILRB1 and LILRB2 gene polymorphisms on susceptibility to and severity of endometriosis.

Endometriosis is a disease in which endometriotic tissue occurs outside the uterus. Its pathogenesis is still unknown. The most widespread hypothesis claims that ectopic endometrium appears as a result of retrograde menstruation and its insufficient elimination by immunocytes. Some reports have shown expression of non-classical HLA-G molecules on ectopic endometrium. HLA-G is recognized by KIR2DL4, LILRB1 and LILRB2 receptors on natural killer (NK) and other cells. These receptors are polymorphic, which may affect their activity. In this study we investigated whether HLA-G, KIR2DL4, LILRB1 and LILRB2 polymorphisms may influence susceptibility to endometriosis and disease progression. We used polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and allelic discrimination methods with TaqMan SNP Genotyping Assays for typing of 276 patients with endometriosis and 314 healthy fertile women. The HLA-G rs1632947:GG genotype was associated with protection against the disease and its severe stages; HLA-G rs1233334:CT protected against progression; LILRB1 rs41308748:AA and LILRB2 rs383369:AG predisposed to the disease and its progression. No effect of KIR2DL4 polymorphism was observed. These results support the role of polymorphisms of HLA-G and its receptors LILRB1 and LILRB2 in susceptibility to endometriosis and its progression.

Effect of cytomegalovirus infection and leukocyte immunoglobulin like receptor B1 polymorphisms on receptor expression in peripheral blood mononuclear cells.

Leukocyte immunoglobulin like receptor B1 (LILRB1) plays a significant role in a number of infectious, autoimmune, cardiovascular, and oncologic disorders. LILRB1 expression varies between individuals and may be associated with polymorphisms on the regulatory region of the LILRB1 gene, as well as to previous cytomegalovirus infection. In this study, the contribution of these two factors to LILRB1 expression in peripheral blood mononuclear cells of healthy young adults was analyzed. LILRB1 expression in NK cells, T cells, B cells and monocytes was significantly stronger in individuals who had had cytomegalovirus infection than in those who had not (P < 0.001, P < 0.001, P < 0.01, and P < 0.001, respectively). Overall, no differences in LILRB1 expression were observed between individuals with and without GAA haplotypes of the LILRB1 regulatory region. However, when analyzed according to cytomegalovirus infection status, significant differences in LILRB1+ NK cells were observed. A higher proportion of LILRB1+ cells was found in GAA+ than in GAA- individuals who had not been infected (P < 0.01), whereas GAA- individuals had a larger proportion of LILRB1+ cells than GAA+ individuals who were cytomegalovirus positive (P < 0.01). In conclusion, cytomegalovirus infection has a major effect on LILRB1 expression in NK and other mononuclear cells and polymorphisms in the LILRB1 regulatory region appear to have a modulatory influence over this effect.

Autophagic Regulation of p62 is Critical for Cancer Therapy.

Sequestosome1 (p62/SQSTM 1) is a multidomain protein that interacts with the autophagy machinery as a key adaptor of target cargo. It interacts with phagophores through the LC3-interacting (LIR) domain and with the ubiquitinated protein aggregates through the ubiquitin-associated domain (UBA) domain. It sequesters the target cargo into inclusion bodies by its PB1 domain. This protein is further the central hub that interacts with several key signaling proteins. Emerging evidence implicates p62 in the induction of multiple cellular oncogenic transformations. Indeed, p62 upregulation and/or reduced degradation have been implicated in tumor formation, cancer promotion as well as in resistance to therapy. It has been established that the process of autophagy regulates the levels of p62. Autophagy-dependent apoptotic activity of p62 is recently being reported. It is evident that p62 plays a critical role in both autophagy and apoptosis. Therefore in this review we discuss the role of p62 in autophagy, apoptosis and cancer through its different domains and outline the importance of modulating cellular levels of p62 in cancer therapeutics.

Human Leukocyte Antigen-G Inhibits the Anti-Tumor Effect of Natural Killer Cells via Immunoglobulin-Like Transcript 2 in Gastric Cancer.

BACKGROUND/AIMS: Human leukocyte antigen-G (HLA-G) plays an important role in inhibiting natural killer (NK) cell function and promoting immune escape. However, the specific mechanism of HLA-G on NK in gastric cancer (GC) remains not well understood. This study investigated the expression of HLA-G in GC and the role of HLA-G-effected NK cells in GC progression. METHODS: HLA-G expression in GC tissues obtained from 49 patients with GC was analyzed by immunohistochemistry and western blot. The number of tumor-infiltrating NK cells and the expression of their surface receptors were analyzed by immunohistochemistry and flow cytometry, respectively. The effect of HLA-G on NK cell proliferation was examined by Cell Counting Kit-8 (CCK8) assay. LDH release assay was used to evaluate the effect of HLA-G on the cytotoxic activity of NK cells, and the levels of IFN-γ and TNF-α in the co-cultured supernatant were detected by ELISA. Mice bearing a xenograft tumor model were used to examine the effect of HLA-G on the anti-tumor effect of NK cells. RESULTS: HLA-G positive expression was detected in most of the GC tissues, and was correlated with the adverse prognosis of the disease. The expression of HLA-G was negatively associated with the number of tumor-infiltrating NK cells. Furthermore, GC cell lines with overexpressed HLA-G revealed their ability to inhibit the cell proliferation and cytotoxic activity of NK-92MI cells, and reduce the secretion of IFN-γ and TNF-α through immunoglobulin-like transcript 2 (ILT2). Finally, this in vivo experiment was able to prove that HLA-G can inhibit the anti-tumor effect of NK cells through ILT2. CONCLUSION: The expression of HLA-G was strongly correlated with the adverse prognosis of GC. The reason may be that it inhibits the proliferation and cytotoxic activity of infiltrating NK cells through ILT2.

Computational and atomistic studies applied to the understanding of the structural and behavioral features of the immune checkpoint HLA-G molecule and gene.

We took advantage of the increasingly evolving approaches for in silico studies concerning protein structures, protein molecular dynamics (MD), protein-protein and protein-DNA docking to evaluate: (i) the structure and MD characteristics of the HLA-G well-recognized isoforms, (ii) the impact of missense mutations at HLA-G receptor genes (LILRB1/2), and (iii) the differential binding of the hypoxia-inducible factor 1 (HIF1) to hypoxia-responsive elements (HRE) at the HLA-G gene. Besides reviewing these topics, they were revisited including the following novel results: (i) the HLA-G6 isoforms were unstable docked or not with ß2-microglobulin or peptide, (ii) missense mutations at LILRB1/2 genes, exchanging amino acids at the intracellular domain, particularly those located within and around the ITIM motifs, may impact the HLA-G binding strength, and (iii) HREs motifs at the HLA-G promoter or exon 2 regions exhibiting a guanine at their third position present a higher affinity for HIF1 when compared to an adenine at the same position. These data shed some light into the functional aspects of HLA-G, particularly how polymorphisms may influence the role of the molecule. Computational and atomistic studies have provided alternative tools for experimental physical methodologies, which are time-consuming, expensive, demanding large quantities of purified proteins, and exhibit low output.

Evidence for Epistatic Interaction between HLA-G and LILRB1 in the Pathogenesis of Nonsegmental Vitiligo.

Vitiligo is the most frequent cause of depigmentation worldwide. Genetic association studies have discovered about 50 loci associated with disease, many with immunological functions. Among them is HLA-G, which modulates immunity by interacting with specific inhibitory receptors, mainly LILRB1 and LILRB2. Here we investigated the LILRB1 and LILRB2 association with vitiligo risk and evaluated the possible role of interactions between HLA-G and its receptors in this pathogenesis. We tested the association of the polymorphisms of HLA-G, LILRB1, and LILRB2 with vitiligo using logistic regression along with adjustment by ancestry. Further, methods based on the multifactor dimensionality reduction (MDR) approach (MDR v.3.0.2, GMDR v.0.9, and MB-MDR) were used to detect potential epistatic interactions between polymorphisms from the three genes. An interaction involving rs9380142 and rs2114511 polymorphisms was identified by all methods used. The polymorphism rs9380142 is an HLA-G 3'UTR variant (+3187) with a well-established role in mRNA stability. The polymorphism rs2114511 is located in the exonic region of LILRB1. Although no association involving this SNP has been reported, ChIP-Seq experiments have identified this position as an EBF1 binding site. These results highlight the role of an epistatic interaction between HLA-G and LILRB1 in vitiligo pathogenesis.

Unveiling the key genes, environmental toxins, and drug exposures in modulating the severity of ulcerative colitis: a comprehensive analysis.

Background: As yet, the genetic abnormalities involved in the exacerbation of Ulcerative colitis (UC) have not been adequately explored based on bioinformatic methods. Materials and methods: The gene microarray data and clinical information were downloaded from Gene Expression Omnibus (GEO) repository. The scale-free gene co-expression networks were constructed by R package "WGCNA". Gene enrichment analysis was performed via Metascape database. Differential expression analysis was performed using "Limma" R package. The "randomForest" packages in R was used to construct the random forest model. Unsupervised clustering analysis performed by "ConsensusClusterPlus"R package was utilized to identify different subtypes of UC patients. Heat map was established using the R package "pheatmap". Diagnostic parameter capability was evaluated by ROC curve. The"XSum"packages in R was used to screen out small-molecule drugs for the exacerbation of UC based on cMap database. Molecular docking was performed with Schrodinger molecular docking software. Results: Via WGCNA, a total 77 high Mayo score-associated genes specific in UC were identified. Subsequently, the 9 gene signatures of the exacerbation of UC was screened out by random forest algorithm and Limma analysis, including BGN,CHST15,CYYR1,GPR137B,GPR4,ITGA5,LILRB1,SLFN11 and ST3GAL2. The ROC curve suggested good predictive performance of the signatures for exacerbation of UC in both the training set and the validation set. We generated a novel genotyping scheme based on the 9 signatures. The percentage of patients achieved remission after 4 weeks intravenous corticosteroids (CS-IV) treatment was higher in cluster C1 than that in cluster C2 (54% vs. 27%, Chi-square test, p=0.02). Energy metabolism-associated signaling pathways were significantly up-regulated in cluster C1, including the oxidative phosphorylation, pentose and glucuronate interconversions and citrate cycle TCA cycle pathways. The cluster C2 had a significant higher level of CD4+ T cells. The"XSum"algorithm revealed that Exisulind has a therapeutic potential for UC. Exisulind showed a good binding affinity for GPR4, ST3GAL2 and LILRB1 protein with the docking glide scores of -7.400 kcal/mol, -7.191 kcal/mol and -6.721 kcal/mol, respectively.We also provided a comprehensive review of the environmental toxins and drug exposures that potentially impact the progression of UC. Conclusion: Using WGCNA and random forest algorithm, we identified 9 gene signatures of the exacerbation of UC. A novel genotyping scheme was constructed to predict the severity of UC and screen UC patients suitable for CS-IV treatment. Subsequently, we identified a small molecule drug (Exisulind) with potential therapeutic effects for UC. Thus, our study provided new ideas and materials for the personalized clinical treatment plans for patients with UC.

Uterine NK cells underexpress KIR2DL1/S1 and LILRB1 in reproductive failure.

A significant proportion of recurrent miscarriage, recurrent implantation failure and infertility are unexplained, and these conditions have been proposed to have an etiology of immunological dysfunction at the maternal-fetal interface. Uterine Natural Killer cells (uNK) comprise three subsets and are the most numerous immune cells found in the uterine mucosa at the time of implantation. They are thought to play an important role in successful pregnancy by regulation of extravillous trophoblast (EVT) invasion and spiral artery remodelling. Here, we examine the frequency, phenotype and function of uNK1-3 from the uterine mucosa of 16 women with unexplained reproductive failure compared to 11 controls with no reproductive problems, during the window of implantation. We report that KIR2DL1/S1 and LILRB1 expression is lower in the reproductive failure group for both uNK (total uNK, uNK 2 and 3) and pNK. We also show that degranulation activity is significantly reduced in total uNK, and that TNF-α production is lower in all uNK subsets in the reproductive failure group. Taken together, our findings suggest that reproductive failure is associated with global reduction in expression of uNK receptors important for interaction with HLA-C and HLA-G on EVT during early pregnancy, leading to reduced uNK activation. This is the first study to examine uNK subsets during the window of implantation in women with reproductive failure and will serve as a platform to focus on particular aspects of phenotype and function of uNK subsets in future studies. Further understanding of uNK dysregulation is important to establish potential diagnostic and therapeutic targets in the population of women with unexplained reproductive failure.

Genetic diversity of the LILRB1 and LILRB2 coding regions in an admixed Brazilian population sample.

Leukocyte immunoglobulin (Ig)-like receptors (LILR) LILRB1 and LILRB2 may play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and nonclassical HLA molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LILRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes, and 41 LILRB2 SNVs arranged into 11 haplotypes. Although we may not exclude as a possible effect of population structure, we found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have revealed that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Overall, our findings demonstrate that LILRB1 and LILRB2 are as polymorphic as HLA class Ib genes and provide strong evidence supporting the directional selection regime hypothesis.

Placental Malaria is Associated with Higher LILRB2 Expression in Monocyte Subsets and Lower Anti-Malarial IgG Antibodies During Infancy.

Background: Placental malaria (PM) is associated with a higher susceptibility of infants to Plasmodium falciparum (Pf) malaria. A hypothesis of immune tolerance has been suggested but no clear explanation has been provided so far. Our goal was to investigate the involvement of inhibitory receptors LILRB1 and LILRB2, known to drive immune evasion upon ligation with pathogen and/or host ligands, in PM-induced immune tolerance. Method: Infants of women with or without PM were enrolled in Allada, southern Benin, and followed-up for 24 months. Antibodies with specificity for five blood stage parasite antigens were quantified by ELISA, and the frequency of immune cell subsets was quantified by flow cytometry. LILRB1 or LILRB2 expression was assessed on cells collected at 18 and 24 months of age. Findings: Infants born to women with PM had a higher risk of developing symptomatic malaria than those born to women without PM (IRR=1.53, p=0.040), and such infants displayed a lower frequency of non-classical monocytes (OR=0.74, p=0.01) that overexpressed LILRB2 (OR=1.36, p=0.002). Moreover, infants born to women with PM had lower levels of cytophilic IgG and higher levels of IL-10 during active infection. Interpretation: Modulation of IgG and IL-10 levels could impair monocyte functions (opsonisation/phagocytosis) in infants born to women with PM, possibly contributing to their higher susceptibility to malaria. The long-lasting effect of PM on infants' monocytes was notable, raising questions about the capacity of ligands such as Rifins or HLA-I molecules to bind to LILRB1 and LILRB2 and to modulate immune responses, and about the reprogramming of neonatal monocytes/macrophages.

Leukocyte immunoglobulin-like receptor B1 and B4 (LILRB1 and LILRB4): Highly sensitive and specific markers of acute myeloid leukemia with monocytic differentiation.

BACKGROUND: Acute myeloid leukemia (AML) with monocytic differentiation (M-AML) remains a diagnostic challenge largely due to lack of sensitive and specific markers for immature monocytes. The immunoglobulin-like inhibitory receptors, LILRB1 and LILRB4, are expressed on monocytes but have not yet been systematically evaluated in the clinical setting. METHODS: We evaluated the diagnostic performance of LILRB1 and LILRB4 as monocytic markers for both immature and mature monocytes in comparison to other myelomonocytic markers including CD14, CD15, CD33, CD36, and CD64 in eight cases of control bone marrow (BM, 5) and peripheral blood (PB, 3), 64 cases of (M-AML), and 57 cases of AML without monocytic differentiation (NM-AML) by flow cytometric immunophenotyping. RESULTS: In control BM, LILRB1 and LILRB4 were consistently expressed on monocytes at all stages of maturation, from CD34+ /CD14- monocytic precursors to CD14-/dim+ maturing and CD14+ mature monocytes. In M-AML, LILRB1 and LILRB4 were consistently expressed on monocytes, regardless of the degree of maturity, from CD14-/dim+ monoblasts/promonocytes to CD14+ mature monocytes but were not expressed on myeloblasts. The diagnostic performances as a monocytic marker assessed by sensitivity/specificity were 100%/100% for LILRB1/LILRB4, 100%/82% for CD11b, 80%/100% for CD14, 100%/81% for CD64, 100%/58% for CD15/CD33, and 89%/97% for CD36/CD64. CONCLUSION: The co-expression of LILRB1/LILRB4 outperformed other myelomonocytic markers as a highly sensitive and specific marker for monocytes at all stages of maturation and could reliably distinguish M-AML from NM-AML. LILRB4 additionally represents a novel therapeutic target for treating M-AML.

B Cells Control Mucosal-Associated Invariant T Cell Responses to Salmonella enterica Serovar Typhi Infection Through the CD85j HLA-G Receptor.

Mucosal-associated invariant T (MAIT) cells are an innate-like population of T cells that display a TCR Vα7.2+ CD161+ phenotype and are restricted by the nonclassical MHC-related molecule 1 (MR1). Although B cells control MAIT cell development and function, little is known about the mechanisms underlying their interaction(s). Here, we report, for the first time, that during Salmonella enterica serovar Typhi (S. Typhi) infection, HLA-G expression on B cells downregulates IFN-γ production by MAIT cells. In contrast, blocking HLA-G expression on S. Typhi-infected B cells increases IFN-γ production by MAIT cells. After interacting with MAIT cells, kinetic studies show that B cells upregulate HLA-G expression and downregulate the inhibitory HLA-G receptor CD85j on MAIT cells resulting in their loss. These results provide a new role for HLA-G as a negative feedback loop by which B cells control MAIT cell responses to antigens.