Bioinformatic Analysis of the Significance of the KIR2DL4 Gene in Recurrent Implantation Failure.

Related studies have pointed out that Killer immunoglobulin-like receptor 2DL4 (KIR2DL4) was associated with vascular remodeling in early pregnancy, and it might play an important role in immunity. In this study, recurrent implantation failure (RIF)-related GSE58144 dataset was extracted from the Gene Expression Omnibus (GEO) database. Firstly, the immune micro-environment analyses were conducted to analyze the pathogenesis of KIR2DL4 in RIF. Then, the gene set enrichment analysis (GSEA) was performed to investigate the function of KIR2DL4. Moreover, the TF-mRNA-miRNA and the co-expression networks were constructed to reveal the potential regulation of KIR2DL4. Furthermore, the genes that were associated with KIR2DL4 and differentially expressed in RIF were obtained and defined as key genes, and the functions of these genes were further explored. KIR2DL4 could be used for clinical diagnosis of RIF, and it was correlated with the changes in the immune micro-environment in RIF. From the perspective of function, KIR2DL4 was associated with complement and coagulation cascades, natural killer cell-mediated cytotoxicity, etc. Moreover, the TF-mRNA-miRNA regulatory network was constructed with KIR2DL4, 9 TFs, and 29 miRNAs. Furthermore, KIR2DL4, ACSM1, IL2RB, and PTPN11 were screened as key genes, which were associated with immune-related functions. This study deeply analyzed the function of KIR2DL4 and its role in RIF, and we found that STAT1 might up-regulate KIR2DL4 by INF-γ/JAK2/STAT1 signaling pathway. Besides, over-expressed KIR2DL4 in the mid-luteal endometrium might influence embryo implantation by affecting the embryo implantation microenvironment, which might help deepen the understanding of the molecular mechanism of RIF.

Clinical significance and immune landscape of KIR2DL4 and the senescence-based signature in cutaneous melanoma.

Senescence is an effective barrier to tumor progression. Mutations that inhibit senescence and promote cell division are mandatory for the development of cancer. Therefore, it is particularly important to explore the differences between cutaneous melanoma (CM) patients with severe and mild degrees of senescence. We clustered all the patients with CM in the Cancer Genome Atlas (TCGA) database based on all the genes of the senescence pathway in the CellAge and MSigDB database. The prognosis, immunotherapy effect, tumor microenvironment score, NRAS mutation rate, expression of CD274, CTLA4, and PDCD1, and abundance of CD8+ T and natural killer (NK) cell infiltration in the younger group of patients (YG) were higher than those in the older group (OG). Compared with the American Joint Committee on Cancer (AJCC) stage, the risk scoring system stratified the risk of CM patients and guided immunotherapy more accurately. The nomogram model, which combined the AJCC stage and risk score, greatly improved the ability and accuracy of prognosis prediction. As KIR2DL4 is the core molecule in the risk scoring system (RSS), knocking down the KIR2DL4 of human NK cells in vitro can inhibit the cytotoxicity of NK cells and can also inhibit the secretion of tumor necrosis factor-α and interferon-γ by NK cells. In contrast, upregulation of KIR2DL4 can activate the MEK/ERK signaling pathway, which is the activation pathway of NK cells. Our RSS and nomogram model can accurately stratify the risk of CM patients and effectively predict the effect of immunotherapy and prognosis in CM patients.

KIR2DL4 genetic diversity in a Brazilian population sample: implications for transcription regulation and protein diversity in samples with different ancestry backgrounds.

KIR2DL4 is an important immune modulator expressed in natural killer cells; HLA-G is its main ligand. We have characterized the KIR2DL4 genetic diversity by considering the promoter, all exons, and all introns in a highly admixed Brazilian population sample and by using massively parallel sequencing. We introduce a molecular method to amplify and to sequence the complete KIR2DL4 gene. To avoid the mapping bias and genotype errors commonly observed in gene families, we have developed and validated a bioinformatic pipeline designed to minimize these errors and applied it to survey the variability of 220 individuals from the State of São Paulo, southeastern Brazil. We have also compared the KIR2DL4 genetic diversity in the Brazilian cohort with the diversity previously reported by the 1000Genomes consortium. KIR2DL4 presents high linkage disequilibrium throughout the gene, with coding sequences associated with specific promoters. There are few but divergent promoter haplotypes. We have also detected many new KIR2DL4 sequences, all bearing nucleotide exchanges in introns and encoding previously described proteins. Exons 3 and 4, which encode the external domains, are the most variable. The ancestry background influences the KIR2DL4 allele frequencies and must be considered for association studies regarding KIR2DL4.

Expression of HLA-G and KIR2DL4 receptor in chorionic villous in missed abortion.

OBJECTIVE: Human leukocyte antigen-G (HLA-G) is a molecule that was first known to confer protection to the fetus from destruction by the immune system of its mother. HLA-G expression is mainly restricted to the fetal-maternal interface on the extravillous cytotrophoblast, placenta, amnion.Methods: The purpose of this study is to investigate the HLA-G and KIR2DL4 expression in chorionic villous among 2 groups with missed abortion: group 1 - 27cases with normal karyotype and group 2 - 22 with fetal polyploidy. Criteria of inclusion: abortive material from two groups of women with missed abortion; 6-12 weeks gestational age, singleton pregnancy, cytogenetic of chorionic villous was obligatory - normal fetal karyotype and polyploidy of fetus.Results: During IHC investigations the average relative area of HLA-G expression in trophoblast was counted (in 1st group 33.9 ± 3.5 and in 2nd group 38.6 ± 2.8). Expression of HLA-G the most verified in extravillous chorion stroma. The average relative area of KIR2DL4 receptor was not statistically different among two groups (31.6 ± 2.4 and 32.2 ± 1.7). CONCLUSIONS: These results suggest the role of HLA-G for the progression in early reproductive losses. Low expression of HLA-G is associated with pregnancy complications and can be one of the reasons for spontaneous abortion.

Killer Immunoglobulin-Like Receptor 2DL4 (CD158d) Regulates Human Mast Cells both Positively and Negatively: Possible Roles in Pregnancy and Cancer Metastasis.

Killer immunoglobulin-like receptor (KIR) 2DL4 (CD158d) was previously thought to be a human NK cell-specific protein. Mast cells are involved in allergic reactions via their KIT-mediated and FcɛRI-mediated responses. We recently detected the expression of KIR2DL4 in human cultured mast cells established from peripheral blood of healthy volunteers (PB-mast), in the human mast cell line LAD2, and in human tissue mast cells. Agonistic antibodies against KIR2DL4 negatively regulate the KIT-mediated and FcɛRI-mediated responses of PB-mast and LAD2 cells. In addition, agonistic antibodies and human leukocyte antigen (HLA)-G, a natural ligand for KIR2DL4, induce the secretion of leukemia inhibitory factor and serine proteases from human mast cells, which have been implicated in pregnancy establishment and cancer metastasis. Therefore, KIR2DL4 stimulation with agonistic antibodies and recombinant HLA-G protein may enhance both processes, in addition to suppressing mast-cell-mediated allergic reactions.

[The Role of Soluble HLA-G in the Vertical Transmission of Toxoplasma gondii].

Soluble human leukocyte antigen G (sHLA-G) plays a key role in pregnancy through interaction with decidual natural killer (dNK) cell inhibitory receptors at the maternal-fetal interface. To demonstrate the possible role of sHLA-G during the pregnancy with Toxoplasma gondii infection, we compared the concentration of a murine functional homolog of sHLA-G, Qa-2, in T. gondii infected and non-infected pregnant C57BL/6 mice, and that of sHLA-G in BeWo culture supernatant. In addition, the levels of KIR2DL4 expressed on human dNK cells and NKG2A in pregnant mice were evaluated. We showed that T. gondii infection result in significant increase in the level of Qa-2 and NKG2A in pregnant mice. sHLA-G and KIR2DL4 in human samples were also significantly upregulated under the condition of T. gondii infection. The further treatment with sHLA-G antibody could reduce the expression level of KIR2DL4 which was upregulated by T. gondii infection. In summary, sHLA-G could upregulate the expression level of KIR2DL4 which lead to excessive immunological tolerance, and further contributed to T. gondii immunity escaping and affecting fetus via vertical transmission which may lead to adverse outcomes.

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.

Adaptive evolution at immune system genes and deep pregnancy implantation in primates.

A major evolutionary change in the lineage ancestral to humans, chimpanzee and gorilla (HCG) has been the embedding of the embryo into maternal tissue. Thus, the first layer of cells (trophoblast) to differentiate after fertilization must adapt to invade the uterus. Such event would likely leave signatures of positive selection at genes with roles in embryo implantation. Here, 163 pregnancy implantation genes are tested for evidence of adaptive diversification in the ancestral lineage to HCG. Two immune system genes, HLA-E and KIR2DL4 showed evidence of positive selection. Some of the positive selected sites involve amino acid substitution with predicted damaging effects on protein function, thus highlighting the possibility of antagonistic pleiotropic effects. Selection at a gene coding for a receptor expressed in uterine cells (KIR) that interacts with trophoblast human leukocyte antigen (HLA) genes suggests a main role for immunological adaptations in embryo deep invasion of the maternal endometrium.

Genetic polymorphisms and expression of HLA-G and its receptors, KIR2DL4 and LILRB1, in non-small cell lung cancer.

Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class I molecule absent from most normal tissues but detected in many malignant tumors. It is recognized by cells of the immune system using LILRB1, KIR2DL4 and LILRB2 receptors. We attempted to find out whether some polymorphisms of HLA-G, LILRB1 and KIR2DL4 genes are associated with susceptibility to nonsmall cell lung cancer (NSCLC). Four polymorphisms in HLA-G, i.e. -964A>G (rs1632947), -725C>G>T (rs1233334), -716T>G (rs2249863) in the promoter, and a 14 base pair insertion/deletion (14 bp indel) in the 3'-untranslated region (3'UTR), and five in LILRB1 - 5651G>A (rs41308748) in intron 14, 5717C>T L622L (rs1061684), 5724G>A E625K (rs16985478), 5774 C>A P641P (rs41548213) in exon 15, and 5806C>T (rs8101240) in 3'UTR - as well as 9620 9A/10A (rs11410751) polymorphism in exon 7 of KIR2DL4 were typed using different laboratory techniques. Only one single nucleotide polymorphism (SNP) in HLA-G (-964A>G) and one in LILRB1 (5724G>A) were found to influence the risk of NSCLC. In addition, 5724G>A was associated with protection from tumor cell infiltration of regional lymph nodes. Most importantly, we detected HLA-G and LILRB1 expression in tumor specimens, but no correlation with genetic polymorphisms was observed. HLA-G and LILRB1 protein expression levels in tumor tissue were significantly correlated with tumor stage.

Integrated bioinformatics analysis and validation identify KIR2DL4 as a novel biomarker for predicting chemotherapy resistance and prognosis in colorectal cancer.

Background: Chemotherapy and immunotherapy have improved the cure rate and survival period for colorectal cancer (CRC), but genetic heterogeneity among patients leads to chemotherapy resistance and disease progression. Identifying new molecular markers is crucial for improving prognosis for CRC patients. KIR2DL4, a transmembrane glycoprotein expressed by immune cells, has shown potential therapeutic and prognostic value in other cancers, but in CRC remains unclear. Methods: This study validated the expression levels of KIR2DL4 in CRC by integrating multiple public databases and assessed through immunohistochemistry (IHC). We further evaluated the diagnostic and prognostic value of KIR2DL4 and explored correlation with immune cell infiltration and chemotherapy sensitivity. The role of KIR2DL4 was further validated through functional enrichment analysis. Cellular assays were conducted using CCK8, colony-formation assay and scratch wound assay. Results: The study found that KIR2DL4 is significantly downregulated in CRC and closely associated with poor prognosis. The low expression of KIR2DL4 is associated with decreased immune cell infiltration and reduced chemotherapy sensitivity. Functional enrichment analysis suggests that KIR2DL4 may inhibit development of CRC by affecting immune cell infiltration and modulating chemotherapy sensitivity. Cellular assays have confirmed that inhibiting KIR2DL4 significantly promotes the proliferation and migration of CRC. Inhibition of KIR2DL4 expression significantly decreased the chemosensitivity of CRC cells to oxaliplatin and 5-FU. Conclusion: The significant downregulation of KIR2DL4 in CRC, associated with CRC metastasis and poor prognosis, highlights its importance as a potential new biomarker for treatment and prognosis assessment of CRC. Future research should delve into the molecular mechanisms of KIR2DL4 and potential applications in regulating immunotherapy and chemotherapy sensitivity.

Combined maternal KIR2DL4 and fetal HLA-G polymorphisms were associated with preeclampsia in a Han Chinese population.

Preeclampsia is the main cause of maternal and infant mortality and morbidity during pregnancy. Killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4) and human leukocyte antigen G (HLA-G) play crucial roles in immune tolerance at the maternal-fetal interface. In this case‒control study, 154 maternal-fetal pairs were recruited, including 74 pairs with preeclampsia (56 of 74 pairs from family triads) and 80 pairs with a normal pregnancy (78 of 80 pairs from family triads). SNaPshot technology was used to detect genetic polymorphisms for 7 TagSNPs in the KIR2DL4 and HLA-G genes. Among the fetal HLA-G gene polymorphisms, rs9380142 (A vs. G: OR = 2.802, 95% CI = 1.761-4.458) and rs1063320 (G vs. C: OR = 1.807, 95% CI = 1.144-2.852) differed between the preeclampsia group and the control group. The transmission disequilibrium test (TDT) suggested that the differences in the rs9380142G/A polymorphism in foetuses between preeclampsia triads and control triads were due to differences in transmission from the parents (P = 0.001). There was no significant difference in the distribution of maternal KIR2DL4 alleles or genotype frequency between the preeclampsia group and the control group. Gene‒gene interaction analysis revealed that the combined genotypes of maternal rs649216-CC and fetal rs9380142-GG, maternal rs1051456-CG/GG and fetal rs9380142-GG, maternal rs34785252-CC and fetal rs9380142-AA/GA, and maternal rs34785252-CC/AA and fetal rs9380142-GG were associated with a significantly lower risk of preeclampsia. Therefore, this study suggested that the combination of maternal KIR2DL4 and fetal HLA-G polymorphisms was associated with preeclampsia in a Han Chinese population.

HLA and KIR genetic association and NK cells in anti-NMDAR encephalitis.

Introduction: Genetic predisposition to autoimmune encephalitis with antibodies against N-methyl-D-aspartate receptor (NMDAR) is poorly understood. Given the diversity of associated environmental factors (tumors, infections), we hypothesized that human leukocyte antigen (HLA) and killer-cell immunoglobulin-like receptors (KIR), two extremely polymorphic gene complexes key to the immune system, might be relevant for the genetic predisposition to anti-NMDAR encephalitis. Notably, KIR are chiefly expressed by Natural Killer (NK) cells, recognize distinct HLA class I allotypes and play a major role in anti-tumor and anti-infection responses. Methods: We conducted a Genome Wide Association Study (GWAS) with subsequent control-matching using Principal Component Analysis (PCA) and HLA imputation, in a multi-ethnic cohort of anti-NMDAR encephalitis (n=479); KIR and HLA were further sequenced in a large subsample (n=323). PCA-controlled logistic regression was then conducted for carrier frequencies (HLA and KIR) and copy number variation (KIR). HLA-KIR interaction associations were also modeled. Additionally, single cell sequencing was conducted in peripheral blood mononuclear cells from 16 cases and 16 controls, NK cells were sorted and phenotyped. Results: Anti-NMDAR encephalitis showed a weak HLA association with DRB1*01:01~DQA1*01:01~DQB1*05:01 (OR=1.57, 1.51, 1.45; respectively), and DRB1*11:01 (OR=1.60); these effects were stronger in European descendants and in patients without an underlying ovarian teratoma. More interestingly, we found increased copy number variation of KIR2DL5B (OR=1.72), principally due to an overrepresentation of KIR2DL5B*00201. Further, we identified two allele associations in framework genes, KIR2DL4*00103 (25.4% vs. 12.5% in controls, OR=1.98) and KIR3DL3*00302 (5.3% vs. 1.3%, OR=4.44). Notably, the ligands of these KIR2DL4 and KIR3DL3, respectively, HLA-G and HHLA2, are known to act as immune checkpoint with immunosuppressive functions. However, we did not find differences in specific KIR-HLA ligand interactions or HLA-G polymorphisms between cases and controls. Similarly, gene expression of CD56dim or CD56bright NK cells did not differ between cases and controls. Discussion: Our observations for the first time suggest that the HLA-KIR axis might be involved in anti-NMDAR encephalitis. While the genetic risk conferred by the identified polymorphisms appears small, a role of this axis in the pathophysiology of this disease appears highly plausible and should be analyzed in future studies.

Genetic polymorphism of KIR2DL4 (CD158d), a putative NK cell receptor for HLA-G, does not influence susceptibility to asthma.

Human leukocyte antigen (HLA)-G is upregulated on the bronchial epithelium of asthma patients and genetic polymorphism affecting expression of HLA-G has been reported to influence susceptibility to asthma. As the NK cell receptor KIR2DL4 has been reported to induce interferon gamma (IFNγ) secretion when ligated with HLA-G, we postulated that the 9A/10A genetic polymorphism of KIR2DL4 which influences receptor structure may influence susceptibility to asthma. KIR2DL4 genotypes were determined in two cohorts of children (n = 219 and n = 1356) in whom total serum IgE, allergen-specific IgE, atopy, bronchial reactivity and asthma symptoms had been studied between birth and 14 years. No reproducible associations with KIR2DL4 genotype were identified, leading us to conclude that the KIR2DL4 9A/10A polymorphism has no influence on susceptibility to asthma.

The effects of KIR2DL4 stimulated NK-92 cells on the apoptotic pathways of HER2 + /HER-breast cancer cells.

Natural killer (NK) cells are immune cells that have attracted significant attention due to their cytotoxic properties. They are believed to be highly effective in cancer therapy. In this study, anti-KIR2DL4 (Killer cell Immunoglobulin like Receptor, 2 Ig Domains and Long cytoplasmic tail 4) was used to stimulate the NK-92 activator receptor to increase their cytotoxicity on breast cancer cell lines. Unstimulated and stimulated NK-92 cells (sNK-92) were cocultured with breast cancer (MCF-7 and SK-BR-3) and normal breast (MCF-12A) cell lines at 1:1, 1:5, and 1:10 (Target:Effector) ratios. The most effective cell cytotoxicity ratio (1:10) was used in the immunostaining and western blot assays to evaluate apoptosis pathway proteins. The sNK-92 cells showed higher cytotoxic activity on breast cancer cells than NK-92 cells. sNK-92 cells had a selective significant cytotoxicity effect on MCF-7 and SK-BR-3 cells but not MCF-12A cells. While sNK-92 cells were effective at all cell concentrations, they were most effective at a 1:10 ratio. Immunostaining and western blots showed significantly higher BAX, caspase 3, and caspase 9 protein levels in all breast cancer cell groups cocultured with sNK-92 than with NK-92 cells. NK-92 cells stimulated with KIR2DL4 showed elevated cytotoxic activity. The cytotoxic activity of sNK-92 cells on breast cancer cells is via apoptosis pathways. However, their effect on normal breast cells is limited. While the obtained data contains only basic information, additional clinical studies are needed to provide a basis for a new treatment model.

The novel KIR2DL4*00108 allele identified by sequencing-based typing in a Chinese Han individual.

The novel KIR2DL4*00108 allele differs from the closest allele KIR2DL4*00102 by a single synonymous mutation.

Roles of HLA-G/KIR2DL4 in Breast Cancer Immune Microenvironment.

Human leukocyte antigen (HLA)-G is a nonclassical MHC Class I molecule, which was initially reported as a mediator of immune tolerance when expressed in extravillous trophoblast cells at the maternal-fetal interface. HLA-G is the only known ligand of killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), an atypical family molecule that is widely expressed on the surface of NK cells. Unlike other KIR receptors, KIR2DL4 contains both an arginine-tyrosine activation motif in its transmembrane region and an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, suggesting that KIR2DL4 may function as an activating or inhibitory receptor. The immunosuppressive microenvironment exemplified by a rewired cytokine network and upregulated immune checkpoint proteins is a hallmark of advanced and therapy-refractory tumors. Accumulating evidence has shown that HLA-G is an immune checkpoint molecule with specific relevance in cancer immune escape, although the role of HLA-G/KIR2DL4 in antitumor immunity is still uncharacterized. Our previous study had shown that HLA-G was a pivotal mediator of breast cancer resistance to trastuzumab, and blockade of the HLA-G/KIR2DL4 interaction can resensitize breast cancer to trastuzumab treatment. In this review, we aim to summarize and discuss the role of HLA-G/KIR2DL4 in the immune microenvironment of breast cancer. A better understanding of HLA-G is beneficial to identifying novel biomarker(s) for breast cancer, which is important for precision diagnosis and prognostic assessment. In addition, it is also necessary to unravel the mechanisms underlying HLA-G/KIR2DL4 regulation of the immune microenvironment in breast cancer, hopefully providing a rationale for combined HLA-G and immune checkpoints targeting for the effective treatment of breast cancer.

Characterization of the novel KIR2DL4*037 allele identified in a Chinese Hani individual.

The novel KIR2DL4*037 allele differs from the closest allele KIR2DL4*00501 by a single missense mutation.

Description of the novel KIR2DL4*00603 allele identified in a Chinese Hani individual.

The novel KIR2DL4*00603 allele differs from the closest allele KIR2DL4*00602 by a silent mutation.

Identification of the novel KIR2DL4*036 allele in a Chinese Hani individual.

The novel KIR2DL4*036 allele differs from the closest allele KIR2DL4*00102 by a single missense mutation.

The novel KIR2DL4*038 allele identified by sequencing-based typing in a Chinese Naxi individual.

The novel KIR2DL4*038 allele differs from the closest allele KIR2DL4*00102 by a single missense mutation.