Killer Cell Immunoglobulin-like Receptors Receptors Research Articles

Immunomodulatory Treatment Impact on IVF Outcomes in KIR AA Genotype: Personalized Fertility Insights.

Background and Objectives: Recurrent implantation failure (RIF) affects 10% of couples undergoing in vitro fertilization (IVF), spurring exploration into tailored treatments to enhance implantation rates. Maternal immune tolerance towards embryos, particularly killer-cell immunoglobulin-like receptors (KIRs) on natural killer (NK) cells, is a focal point in RIF research. Materials and Methods: This retrospective cohort study, conducted at fertility clinic in Oradea, Romania, involved 65 infertile couples undergoing IVF treatment between January 2022 and December 2023. Couples were divided into two groups: KIR AA (Group A) and KIR Bx (Group B). Results: Factors such as age, type of infertility, oocytes retrieved, embryos produced, pregnancy rates in Group A without and with immunomodulatory treatment were documented. Group A, receiving immunomodulatory treatment, achieved a pregnancy rate of 47.8%, significantly higher than the 23.73% rate without treatment (p = 0.008). Group B had a higher mean patient age than Group A. However, miscarriage rates did not significantly differ between Group A with treatment and Group B (p = 0.2457), suggesting comparable outcomes with immunomodulation. Conclusions: The impact of immunological factors on recurrent implantation failure is being more and more emphasized and warrants the attention of specialists in human reproduction. Uterine natural killers and their function though KIR receptors deserve particular attention as immunomodulatory treatment may improve pregnancy rates in patients with KIR AA haplotype.

KIR2DS4 and Its Variant KIR1D in KIR-AA Genotype Donors Showed Differential Survival Impact in Patients with Lymphoid Disease after HLA-Matched Unrelated Hematopoietic Stem Cell Transplantation

Previous studies illustrated associations between presence of activating killer cell immunoglobulin-like receptor (KIR) genes and lower susceptibility to hematologic malignancies in humans. In addition, favorable hematopoietic stem cell transplantation (HSCT) outcomes were reported in patients, who received transplants from donors with KIR genotypes dominant for activating KIR receptors. However, the association of activating KIR genes on an allelic level with disease and their impact on HSCT outcome has been only scarcely investigated. To this end, we genotyped a large transplantation cohort for KIR two Ig domains and short cytoplasmic tail 4 (KIR2DS4) polymorphisms and investigated their association with disease. We next investigated the impact of KIR-AA genotype donor KIR2DS4 polymorphisms (AA/KIR2DS4 versus (vs.) AA/KIR1D), on clinical outcome of HSCT in patient subgroups (myeloid vs. lymphoid). Among 2810 transplant pairs, 68.8% (n=1934) were 10/10 human leukocyte antigen (HLA) matched and 31.2% (n=876) were 9/10 HLA-matched. The distribution of KIR one Ig domain (KIR1D) in patients vs. donors was equal (P value = 0.205). Multivariate analysis in 10/10 HLA-matched patients with lymphoid disease showed improved HSCT outcome (overall survival (OS): hazard ratio (HR) 0.62, P=0.002; disease free survival (DFS): HR 0.70, P=0.011; graft-versus-host disease free and relapse-free survival (GRFS): HR 0.67, P=0.002; non-relapse mortality (NRM): HR 0.55, P<0.001) when they received grafts from AA/KIR1D donors. This effect was not seen in either 9/10 HLA-matched patients with lymphoid disease or patients with myeloid disease. Our study indicates that the presence of KIR1D alleles is not associated with disease in patients and interestingly, using grafts from AA/KIR1D donors translated into a beneficial survival outcome in 10/10 HLA-matched patients with lymphoid disease.

Lack of association of the KIR and HLA class I ligands with ZIKV infection in south and southeast of Brazil.

BACKGROUND Zika virus (ZIKV) is an emerging arbovirus associated with foetal malformations and neurological complications. The infection is usually associated with mild symptoms. The comparison between the allelic frequency of polymorphic genes in symptomatic infected individuals in the population can clarify the pathogenic mechanisms of ZIKV. During ZIKV infection, cytokines are produced and natural killer (NK) cells are recruited, whose activation depends on signaling pathways activated by specific receptors, such as killer cell immunoglobulin-like receptors (KIR). These molecules interact with human leukocyte antigen (HLA) class I ligands and are encoded by polymorphic genes.OBJECTIVES This study aimed to evaluate the frequency of allelic variants of the genes encoding the KIR receptors and their HLA class I ligands in 139 symptomatic ZIKV-patients and 170 controls negative for the virus, and to evaluate the role of these variants for ZIKV susceptibility.METHODS KIR and HLA class I genes were genotyped using the polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) technique.FINDINGS No significant differences in the frequency distribution of KIRs and KIR-HLA in patients compared to controls were observed.MAIN CONCLUSIONS KIR and its HLA ligands might play a minor role in ZIKV infection in the south and southeast Brazilian individuals.

The New Kid on the Block: HLA-C, a Key Regulator of Natural Killer Cells in Viral Immunity.

The human leukocyte antigen system (HLA) is a cluster of highly polymorphic genes essential for the proper function of the immune system, and it has been associated with a wide range of diseases. HLA class I molecules present intracellular host- and pathogen-derived peptides to effector cells of the immune system, inducing immune tolerance in healthy conditions or triggering effective immune responses in pathological situations. HLA-C is the most recently evolved HLA class I molecule, only present in humans and great apes. Differentiating from its older siblings, HLA-A and HLA-B, HLA-C exhibits distinctive features in its expression and interaction partners. HLA-C serves as a natural ligand for multiple members of the killer-cell immunoglobulin-like receptor (KIR) family, which are predominately expressed by natural killer (NK) cells. NK cells are crucial for the early control of viral infections and accumulating evidence indicates that interactions between HLA-C and its respective KIR receptors determine the outcome and progression of viral infections. In this review, we focus on the unique role of HLA-C in regulating NK cell functions and its consequences in the setting of viral infections.

Natural killer-cell immunoglobulin-like receptors trigger differences in immune response to SARS-CoV-2 infection.

BackgroundThe diversity in the clinical course of COVID-19 has been related to differences in innate and adaptative immune response mechanisms. Natural killer (NK) lymphocytes are critical protagonists of human host defense against viral infections. It would seem that reduced circulating levels of these cells have an impact on COVID-19 progression and severity. Their activity is strongly regulated by killer-cell immuno-globulin-like receptors (KIRs) expressed on the NK cell surface. The present study’s focus was to investigate the impact of KIRs and their HLA Class I ligands on SARS-CoV-2 infection.MethodsKIR gene frequencies, KIR haplotypes, KIR ligands and combinations of KIRs and their HLA Class I ligands were investigated in 396 Sardinian patients with SARS-CoV-2 infection. Comparisons were made between 2 groups of patients divided according to disease severity: 240 patients were symptomatic or paucisymptomatic (Group A), 156 hospitalized patients had severe disease (Group S). The immunogenetic characteristics of patients were also compared to a population group of 400 individuals from the same geographical areas.ResultsSubstantial differences were obtained for KIR genes, KIR haplotypes and KIR-HLA ligand combinations when comparing patients of Group S to those of Group A. Patients in Group S had a statistically significant higher frequency of the KIR A/A haplotype compared to patients in Group A [34.6% vs 23.8%, OR = 1.7 (95% CI 1.1–2.6); P = 0.02, Pc = 0.04]. Moreover, the KIR2DS2/HLA C1 combination was poorly represented in the group of patients with severe symptoms compared to those of the asymptomatic-paucisymptomatic group [33.3% vs 50.0%, OR = 0.5 (95% CI 0.3–0.8), P = 0.001, Pc = 0.002]. Multivariate analysis confirmed that, regardless of the sex and age of the patients, the latter genetic variable correlated with a less severe disease course [ORM = 0.4 (95% CI 0.3–0.7), PM = 0.0005, PMC = 0.005].ConclusionsThe KIR2DS2/HLA C1 functional unit resulted to have a strong protective effect against the adverse outcomes of COVID-19. Combined to other well known factors such as advanced age, male sex and concomitant autoimmune diseases, this marker could prove to be highly informative of the disease course and thus enable the timely intervention needed to reduce the mortality associated with the severe forms of SARS-CoV-2 infection. However, larger studies in other populations as well as experimental functional studies will be needed to confirm our findings and further pursue the effect of KIR receptors on NK cell immune-mediated response to SARS-Cov-2 infection.

Differences in the Expression of KIR, ILT Inhibitory Receptors, and VEGF Production in the Induced Decidual NK Cell Cultures of Fertile and RPL Women.

Results KIR2DL1 and ILT-2 expression on idNK cells was higher in healthy women than in RPL patients. Sildenafil enhanced NKG2A expression in RPL patients. VEGF concentration was higher in fertile woman idNK cell cultures. idNK cells were more sensitive for necrosis in RPL than in fertile women. SC did not influence VEGF production or idNK cell apoptosis. Conclusions A combination of hypoxia, IL-15, and AZA promotes the conversion of pbNK into idNK cells CD56+CD16−-expressing KIR receptors and produces VEGF. Alterations in KIR2DL1 and ILT-2 expression as well as impaired VEGF production were associated with RPL. SC affects NKG2A expression on RPL idNK cells. SC had no effect on VEGF release or idNK cell apoptosis.

IFNL4 and donor selection for matched unrelated donor haematopoietic stem-cell transplantation

IFNL4 and donor selection for matched unrelated donor haematopoietic stem-cell transplantation

Maternal killer-cell immunoglobulin-like receptors and paternal human leukocyte antigen ligands in recurrent pregnancy loss cases in Turkey.

Objective The survival of a semi-allogeneic fetus depends on several immunological mechanisms, and it has been suggested that recurrent pregnancy loss (RPL) could develop as a result of one or more immunological abnormalities.Methods Compatibility between partners for human leukocyte antigen (HLA) genotypes and the relationships between maternal killer-cell immunoglobulin-like receptor (KIR) and paternal HLA-Bw4/Bw6 and HLA-C1/C2 supra-groups were investigated in 25 couples with RPL in comparison to healthy couples with children. HLA and KIR genotyping was performed using polymerase chain reaction with sequence-specific primers and/or sequence-specific oligonucleotides.Results HLA class I incompatibility between partners, especially in HLA-B alleles, was more common in the RPL group (p= 0.01). HLA-C2 homozygosity was more frequent in the male partners of RPL couples than in other groups (p= 0.03). The KIR2DL5 gene frequency was significantly higher in both the female and male partners of RPL couples, whereas the KIR2DS3 gene frequency in male partners of RPL couples was significantly reduced (p= 0.03). The presence of KIR2DL3 in women with RPL was correlated with the presence of HLA-C2 alleles in their spouses (p= 0.03).Conclusion Our data from a Turkish population suggest that male HLA-C2 homozygosity may play an important role in RPL. Additionally, an incidental match between male HLA-C2 and female HLA-C1 ligand KIR receptors might perturb the balance between activatory and inhibitory KIR-ligand interactions during pregnancy in couples affected by RPL. The roles of orphan KIR2DL5 and orphan KIR2DS3 in RPL remain obscure.

The impact of KIR/HLA genes on the risk of developing multibacillary leprosy.

BackgroundKiller-cell immunoglobulin-like receptors (KIRs) are a group of regulatory molecules able to activate or inhibit natural killer cells upon interaction with human leukocyte antigen (HLA) class I molecules. Combinations of KIR and HLA may contribute to the occurrence of different immunological and clinical responses to infectious diseases. Leprosy is a chronic neglected disease, both disabling and disfiguring, caused mainly by Mycobacterium leprae. In this case–control study, we examined the influence of KIRs and HLA ligands on the development of multibacillary leprosy.Methodology/Principal findingsGenotyping of KIR and HLA genes was performed in 264 multibacillary leprosy patients and 518 healthy unrelated controls (238 healthy household contacts and 280 healthy subjects). These are unprecedented results in which KIR2DL2/KIR2DL2/C1/C2 and KIR2DL3/2DL3/C1/C1 indicated a risk for developing lepromatous and borderline leprosy, respectively. Concerning to 3DL2/A3/A11+, our study demonstrated that independent of control group (contacts or healthy subjects), this KIR receptor and its ligand act as a risk factor for the borderline clinical form.Conclusions/SignificanceOur finding suggests that synergetic associations of activating and inhibitory KIR genes may alter the balance between these receptors and thus interfere in the progression of multibacillary leprosy.

Association study between KIR polymorphisms and rheumatoid arthritis disease: an updated meta-analysis

BackgroundCurrently published studies investigating association between the killer cell immunoglobulin-like receptor (KIR) gene polymorphisms and rheumatoid arthritis (RA) reported inconsistent and contradictory results. Hence, we aim to carry out this comprehensive meta-analysis of all eligible studies meeting the inclusion criteria to achieve precise and comprehensive relationships between genetic variations in KIR gene cluster and risk of RA.MethodsDatabases of Medline/PubMed and Scopus were searched to investigate case-control studies prior to May 2018. The associations between KIR gene polymorphisms and RA susceptibility were analyzed by computing the odds ratio (OR) and 95% confidence interval (95% CI) for each study.ResultsA total of 11 comparative case-control studies involving 1847 RA patients and 2409 healthy individuals were included in this meta-analysis. Four significant associations of 2DL3 (OR = 0.591, 95% CI = 0.351–0.994; P = 0.047), 2DL5 (OR = 0.716, 95% CI = 0.601–0.853; P < 0.001), 2DS5 (OR = 0.623, 95% CI = 0.393–0.988; P = 0.045), and 3DL3 (OR = 0.324, 95% CI = 0.129–0.814; P = 0.016) genes with decreased RA risk were discovered in this meta-analysis. Although, other KIR receptors including 2DL1, 2DL2, 2DL4, 3DL1, 3DL2, 3DS1, 2DS1-2DS4, and two pseudo gens of 2DP1 and 3DP1 displayed no significant association with predisposition to RA.ConclusionsThese findings provide reliable evidence that 2DL3, 2DL5, 3DL3, and 2DS5 might have a potential protective role for RA.

Donor KIR Genotype Impacts on Clinical Outcome after T Cell–Depleted HLA Matched Related Allogeneic Transplantation for High-Risk Pediatric Leukemia Patients

Preliminary data suggest that ex vivo T cell-depleted matched related donor (MRD) hematopoietic stem cell transplantation (HSCT) is feasible and confers a clinically beneficial reduction in graft-versus-host disease. Classically, T cell-depleted grafts were associated with reduction of the graft-versus-leukemia (GVL) effect because of delayed T cell immune reconstitution. However, natural killer (NK) cell alloreactivity is also critical for an early GVL effect as well as for lymphocyte immune reconstitution. Here, we study the role of NK cells in MRD T cell-depleted HSCT, which is still poorly understood. Given that MRD ligands for inhibitory killer cell immunoglobulin-like receptors (KIRs) are matched, we focused on activating KIR receptors. We retrospectively analyzed KIR genotyping in patients and MRDs in 40 ex vivo T cell-depleted pediatric HSCTs. The log-rank test and Cox proportional risk test were performed to correlate genotype with clinical outcome (relapse rate, disease-free survival, and overall survival) and immune reconstitution. The statistical analysis revealed poorer overall survival when donors have a KIR-B content score of ≥2, a best/better subtype, or present the KIR2DS1 gene. The patient's relapse rate was higher when donors present the KIR2DL5A gene, as well as a poorer probability of disease-free survival when the donor is classified with a best/better subtype. Regarding immune reconstitution, donor KIR haplotype A or the presence of inhibitory KIR genes promote best recovery of T lymphocytes, whereas donor KIR haplotype B or the presence of activating KIR genes confer better expansion of NK cells. These findings suggest that the selection of MRDs with an inhibitory KIR phenotype improve T cell expansion as well as the clinical outcome after pediatric ex vivo T cell-depleted HSCT.

Влияние аллореактивности естественных киллерных клеток на эффективность аллогенной трансплантации гемопоэтических стволовых клеток с деплецией α/β TCR/CD19+ лимфоцитов у педиатрических пациентов с острыми лейкозами

Natural killer (NK) cells constitute the first lymphocyte population to recover after an allogeneic hematopoietic stem cell transplantation (HSCT). It is believed that NK cell alloreactivity is regulated by the difference between activating and inhibitory signals from activating and inhibitory killer cell immunoglobulin-like receptors (KIRs). Human Leukocyte Antigen (HLA) molecules serve as ligands for KIR receptors. NK cell alloreactivity is determined by a KIR-HLA mismatch between donor and patient as well as the presence of specific activating KIR genes. Numerous recent studies show that several factors influence the effectiveness of the graft-versus-leukemia reaction. In particular, these factors include donor’s KIR genotype and the transplant protocol. The aim of the study is to evaluate the influence of KIR genotype of both HLA-haploidentical related donors and HLA-identical unrelated donors on the relapse risk and survival of acute leukemia patients after allogeneic HSCT. The study includes 142 patients (the median age 11) with acute myeloid leukemia (n = 70) and acute lymphoid leukemia (ALL) (n = 72) received first HLA-identical unrelated (n = 64) and HLA-haploidentical (n = 78) with α/βTCR-depleted allogeneic HSCT. All patients were in complete hematologic remission at the moment of HSCT. Peripheral blood mononuclear cells were collected after the administration of granulocyte colony-stimulating factor and used as the source of hematopoietic stem cells. Results of the study are consistent with the conception that potential NK alloreactivity depends on the transplant protocol – conditioning regimen, transplant processing and post-transplant graft-versus-host-disease prophylaxis. The key finding of this study is that more number of activating KIR genes in donor’s KIR genotype is associated with better overall survival of ALL patients (0,67 (95% CI: 0,53–0,81) vs 0,31 (95% CI: 0,07–0,55); р = 0,016). A similar effect was observed for the event-free survival. We failed to find a beneficial effect of NK alloreactivity based on the «ligand-ligand» model in the HLA-haploidentical related HSCT. NK alloreactivity for the same patient group, based on receptor-ligand» model also showed no significant beneficial effect, however there was a tendency for better overall survival for patients transplanted from potentially NK alloreactive donors (0,76 (95% CI: 0,63–0,88) vs 0,56 (95% CI: 0,32–0,81); р = 0,486). In summary, it is recommended to consider donor’s KIR genotype as additional criteria for donor selection prior to allogeneic HSCT for all patients.

Next-generation sequencing technology a new tool for killer cell immunoglobulin-like receptor allele typing in hematopoietic stem cell transplantation

Killer cell Immunoglobulin-like Receptor (KIR) genes are a family of genes located together within the leukocyte receptor cluster on human chromosome 19q13.4. To date, 17 KIR genes have been identified including nine inhibitory genes (2DL1/L2/L3/L4/L5A/L5B, 3DL1/L2/L3), six activating genes (2DS1/S2/S3/S4/S5, 3DS1) and two pseudogenes (2DP1, 3DP1) classified into group A (KIR A) and group B (KIR B) haplotypes. The number and the nature of KIR genes vary between the individuals. In addition, these KIR genes are known to be polymorphic at allelic level (907 alleles described in July 2017). KIR genes encode for receptors which are predominantly expressed by Natural Killer (NK) cells. KIR receptors recognize HLA class I molecules and are able to kill residual recipient leukemia cells, and thus reduce the likelihood of relapse. KIR alleles of Hematopoietic Stem Cell (HSC) donor would require to be known (Alicata et al. Eur J Immunol 2016) because the KIR allele polymorphism may affect both the KIR+ NK cell phenotype and function (Gagne et al. Eur J Immunol 2013; Bari R, et al. Sci Rep 2016) as well as HSCT outcome (Boudreau et al. JCO 2017). The introduction of the Next Generation Sequencing (NGS) has overcome current conventional DNA sequencing method limitations, known to be time consuming. Recently, a novel NGS KIR allele typing approach of all KIR genes was developed by our team in Nantes from 30 reference DNAs (Maniangou et al. Front in Immunol 2017). This NGS KIR allele typing approach is simple, fast, reliable, specific and showed a concordance rate of 95% for centromeric and telomeric KIR genes in comparison with high-resolution KIR typing obtained to those published data using exome capture (Norman PJ et al. Am J Hum Genet 2016). This NGS KIR allele typing approach may also be used in reproduction and to better study KIR+ NK cell implication in the control of viral infections.

HIV-1 adaptation to NK cell-mediated immune pressure.

The observation, by Alter et al., of the enrichment of NK cell “escape” variants in individuals carrying certain Killer-cell Immunoglobulin-like Receptor (KIR) genes is compelling evidence that natural killer (NK) cells exert selection pressure on HIV-1. Alter et al hypothesise that variant peptide, in complex with HLA class I molecules binds KIR receptors and either increases NK cell inhibition or decreases NK cell activation compared to wild type peptide thus leading to virus escape from the NK cell response. According to this hypothesis, in order for NK cells to select for an escape variant, an individual must carry both the KIR and an HLA ligand that binds the variant peptide. In this study we estimate the proportion of the population that is capable of selecting for escape variants and use both epidemiological modelling and a model-free approach to investigate whether this proportion explains the observed variant enrichment. We found that the fraction of individuals within whom the variant would have a selective advantage was low and was unable to explain the high degree of enrichment observed. We conclude that whilst Alter et al’s data is consistent with selection pressure, the mechanism that they postulate is unlikely. The importance of this work is two-fold. Firstly, it forces a re-evaluation of some of the clearest evidence that NK cells exert a protective effect in HIV-1 infection. Secondly, it implies that there is a significant aspect of immunology that is not understood: it is possible that KIRs bind much more widely than was previously appreciated; that a gene in linkage with the KIR genes is responsible for considerable peptide-dependent selection or that variant peptides are indirectly impacting KIR ligation.

The impact of KIR2DS4 gene on clinical outcome after hematopoietic stem cell transplantation

Killer cell immunoglobulin-like receptors (KIR) are a family of inhibitory/activating receptors expressed on NK cells. Interactions of KIR receptors with KIR ligands have been shown to modify hematopoietic stem cell transplantation (HSCT) outcome. The aim of this research was to determine the KIR2DS4 allele variants distribution among 111 patients with different hematological malignancy who underwent HSCT and their donors, and to evaluate KIR2DS4 alleles' impact on HSCT outcome. The KIR gene frequency analysis showed a significantly higher incidence of full-length KIR2DS4 alleles among patients. The impact of KIR2DS4 alleles on transplantation outcomes revealed that donors' full-length KIR2DS4 alleles is associated with lower overall survival rates, higher risk of GVHD and higher relapse incidence. The expression of full-length KIR2DS4 allele variants may contribute to a worse clinical outcome after HSCT. KIR typing for KIR2DS4 could be used as an additional criterion for selecting suitable donors in cases when more than one HLA identical donor is identified for a specific patient.

Changes in Killer-Cell Immunoglobulin-like Receptor (KIR) Expression on NK Cells Following DNA Demethylation By Azacitidine in Relapsed and Refractory Lymphomas

Abstract Introduction We conducted a study to see the activity of hypomethylating agent (HMA) in relapsed refractory lymphoma. In parallel, we studied KIR expression and promoter methylation before and after treatment with HMA. Allele-specific stochastic KIR expression is maintained in mature natural killer (NK) cells by a combination of DNA methylation and histone modification, with DNA methylation being dominant. The aim of this part of the study is to understand how KIR gene expression changes in response to epigenetic therapy, so that we then can manipulate NK cells to optimize the effect of chemo-immunotherapy of neoplastic diseases. Methods Blood samples were collected from patients enrolled in an open-labeled phase I trial of the combination of azacitidine with cyclophosphamide, vincristine and rituximab in relapsed/refractory lymphoma. Regimen: Azacitidine on days one through five (starting at 25mg/m2), followed by oral cyclophosphamide at 300 mg/m2 on days six through nine, vincristine 1.4 mg/m2 day eight, and rituximab 375 mg/m2 day eight. Each cycle was to be repeated every 21 days, up to eight cycles if participants continued to benefit from therapy. Sampling: Between 10-20uL whole blood was collected from eligible patients before commencement of therapy and on Cycle 1 Day 5, Cycle 2 Day 1 and Cycle 3 Day 1. Peripheral blood mononuclear cells (PBMC) were isolated immediately ex vivo using Lymphocyte Separation Media (Lonza), and were divided into two aliquots: one for flowcytometry and the other for pyrosequencing (Epigendx, Hopkinton, MA). Samples from each time point were stained with fluorescently-labelled antibodies to CD3, CD16, CD56, CD14, CD20 and four different KIRs. For calculating the number of KIRs per NK cell, we used a gating strategy in which CD56dim NK cells are identified, and then the number of KIR expression is counted on those by sequential gating. The percentages of monocytes and lymphocytes among the PBMC population were also determined using CD14 and CD20 antibodies. Monocytes were further characterized according to their CD16 expression into Classical (CD14highCD16negative), Intermediate (CD14highCD16low), and Non-classical (CD14lowCD16high). Results PBMC from 10 patients were available for flow cytometric analysis and pyrosequencing. There were statistically significant differences (p=0.0089) in percent of DNA methylation determined by PBMC pyrosequencing, with significant decrease from Cycle 1 Day 1 to Cycle 1 Day 5 and then an increase from Cycle 1 day 5 to Cycle 2 Day 1. The decline in the percent of DNA methylation in PBMC at day 5 of cycle 1 of the chemotherapy was also found to be statistically significant (Figure 1; p=0.0202). There was a large variation in the types and numbers of KIR molecules expressed at baseline (Table 1) on CD56dim NK cells among the patients in the cohort studied, with no significant differences between the various time points and response to treatment. Despite an apparent trend of increasing percentages of non-classical and intermediate monocytes (at the expense of classical monocytes), the data was not statistically significant (Figure 2; p=0.06). We also noticed a trend of decreasing percentage of monocytes and increasing percentage of lymphocytes in the PBMC population with increasing treatment cycles. However, this was not statistically significant either. Conclusions KIR expression may play role in HMA induced tumor cell killing. In our study, no significant changes were seen in the number of KIR receptors on NK cells post-treatment with hypomethylating agent despite observing a reduction in global DNA methylation in PBMC. This could be due to a number of reasons, including the small size of the study, the rapid turnover of NK cells, or that the reduced methylation was not sufficient to induce changes in KIR expression. The significance of the increase in the number of non-classical and intermediate monocytes following treatment with HMA is not well understood and needs further investigation. (The study was supported by Celgene) Disclosures No relevant conflicts of interest to declare.

Analysis of killer cell immunoglobulin-like receptors and their human leukocyte antigen-ligands gene polymorphisms in Iranian patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease. Natural killer (NK) cells play a critical role in the pathogenesis of autoimmune disorders that mainly express killer cell immunoglobulin-like receptors (KIRs). The present study was undertaken to determine the association of the KIR alleles, genotypes, and KIR-human leukocyte antigen (HLA) ligand gene combinations with the susceptibility to SLE. The genotyping of 17 KIR and 5 HLA loci was performed using the polymerase chain reaction-sequence specific primer (PCR-SSP) method. The study population consisted of 230 SLE patients and 273 ethnical-, age-, and sex-matched healthy controls. The association of the polymorphisms with the prevalence of 11 clinical criteria in patients was analyzed. The carrier frequency of HLA-A-Bw4 was modestly decreased in the SLE patients. The prevalence of hematological and renal disorders was significantly increased in patients with combination of KIR3DL1(+); HLA-B-Bw4(Thr80+) and KIR2DS1(+); HLA-C2(+) genes, respectively. Female patients with combination of KIR2DL2(+); HLA-C1(-) genes were more likely to develop serositis. In addition the prevalence of renal disorders, oral ulcer and serositis was significantly increased in male patients with KIR3DP1(+), KIR2DS1(+), and KIR2DS3(+) genotypes respectively. Our results showed that the presence of activating KIR receptors alone or in combination with their HLA ligands and the absence of inhibitory KIRs in combination with their HLA ligands may activate NK cells and are significantly correlated with the prevalence of renal disease, hematologic disorders, serositis, and oral ulcer in SLE patients.

2DL1, 2DL2 and 2DL3 all contribute to KIR phenotype variability on human NK cells.

Natural killer (NK) cells are lymphocytes that function as part of the innate immune system. Their activity is controlled by a range of inhibitory and activating receptors, including the important killer-cell immunoglobulin-like receptors (KIR). The KIR are a multi-gene family of receptors that interact with the human leukocyte antigen (HLA) class I family of molecules and are characterised by extensive allelic polymorphism. Their expression on the cell surface of NK cells is highly variable, but the factors responsible for this variability are not yet clearly understood. In the current study, we investigated KIR expression in a healthy human cohort that we had previously characterised in depth at a genetic level, with KIR allele typing and HLA class I ligand genotypes available for all donors (n=198). Allelic polymorphism significantly affected the phenotypic expression of all KIR analysed, whereas HLA ligand background influenced the expression levels of 2DL1 and 2DL3. In particular, we found that although 2DL2 may influence 2DL1 expression, this appears to be owing to variation in 2DL1 copy number. Finally, the inhibitory receptor LILRB1 had higher expression levels in individuals with B/B KIR genotypes, suggesting a possible relationship between KIR and non-KIR receptors, which serves to balance NK cell activation potential.

Natural Killer Cells Enhance Transplantation Outcomes

Natural killer (NK) cells, the first lymphocyte subset to reconstitute after hematopoietic cell transplantation (HCT), may enhance transplant outcomes by killing virally-infected or malignant cells to reduce relapse and treatment-related mortality by promoting engraftment and by reducing graft vs. host disease. The function of NK cells is regulated by the net balance of signaling via several families of activating or inhibitory receptors. The killer-cell immunoglobulin-like receptor (KIR) family is of particular importance in HCT because of its interactions with class I human leukocyte antigen (HLA) molecules. HLA-C, HLA-Bw4 and some HLA-A allotypes function as ligands for the inhibitory KIR receptors, thus mediating tolerance to self. HLA-mismatched HCT may generate alloreactive NK cells in the recipient when there is a KIR-ligand mismatch at HLA-C, B or A. The proof of concept for this principle was established by the Perugia group in haploidentical transplants, where KIR-ligand mismatch was associated with reduced relapse in patients with acute myeloid leukemia. Subsequent groups have studied this and other algorithms, including KIR ligand absence or KIR-KIR ligand genotyping to evaluate the role of NK cells in mediating protection after allogeneic HCT. Human KIR haplotypes are defined by gene content, where the A haplotype contains mainly inhibitory KIR, and B haplotypes contain more activating KIR. Unrelated donors with more KIR B haplotype genes have been associated with protection from relapse and improved survival in AML, an effect which is enhanced in recipients expressing HLA-C1. Although NK cells mediate beneficial effects after HCT, compared to adult donor NK cells, engrafting NK cells are immature and hyporesponsive when exposed to tumor targets. Recently human cytomegalovirus (CMV) has been shown to drive the expansion of a population of long-lived CD57+NKG2C+ NK cells with attributes of immunologic memory. These cells have heightened capacity for cytokine production or cytolytic response to tumor targets. Several groups have shown that CMV reactivation correlates with reduced relapse after allogeneic HCT, suggesting that these CMV “adaptive” NK cells may contribute to the graft vs. leukemia effect. Strategies to isolate and expand “adaptive” NK cells without clinical CMV reactivation are being explored. Lastly, adult donor NK cells are an appealing population to exploit for adoptive cellular therapy. Donors can be chosen based on predicted NK cell alloreactivity or based on KIR gene content. Adoptively transferred NK cells which expand and proliferate in vivo in response to cytokine stimulation (IL-2 or IL-15) are potent anti-tumor effectors which do not mediate graft vs. host disease. Several groups are using haploidentical adult NK cell products to treat acute myeloid leukemia, multiple myeloma, lymphoma, myelodysplasic syndrome and a variety of solid tumors. Various approaches using adoptive transfer of NK cells, together with HCT, targeting antibodies which mediate antibody dependent cellular cytotoxicity, or cytokine stimulation are being tested clinically. In summary, we are just beginning to define the complexity of NK cell interactions with HLA and other ligands and to describe different properties of various NK cell subsets to develop more sophisticated strategies to exploit NK cells to treat cancer. Disclosures No relevant conflicts of interest to declare.

Sequence variations in HIV-1 p24 Gag-derived epitopes can alter binding of KIR2DL2 to HLA-C*03:04 and modulate primary natural killer cell function.

The aim of this study was to assess the consequence of sequence variations in HLA-C03:04-presented HIV-1 p24 Gag epitopes on binding of the inhibitory natural killer (NK) cell receptor KIR2DL2 to HLA-C03:04. HIV-1 may possibly evade recognition by KIR+ NK cells through selection of sequence variants that interfere with the interactions of inhibitory killer cell immunoglobulin-like receptors (KIRs) and their target ligands on HIV-1 infected cells. KIR2DL2 is an inhibitory NK cell receptor that binds to a family of HLA-C ligands. Here, we investigated whether HIV-1 encodes for HLA-C03:04-restricted epitopes that alter KIR2DL2 binding. Tapasin-deficient 721.220 cells expressing HLA-C03:04 were pulsed with overlapping peptides (10mers overlapped by nine amino acids, spanning the entire HIV-1 p24 Gag sequence) to identify peptides that stabilized HLA-C expression. The impact that sequence variation in HLA-C03:04-binding HIV-1 epitopes has on KIR2DL2 binding and KIR2DL2+ NK cell function was determined using KIR2DL2-Fc constructs and NK cell degranulation assays. Several novel HLA-C03:04 binding epitopes were identified within the HIV-1 p24 Gag consensus sequence. Three of these consensus sequence peptides (Gag144-152, Gag163-171 and Gag295-304) enabled binding of KIR2DL2 to HLA-C03:04 and resulted in inhibition of KIR2DL2+ primary NK cells. Furthermore, naturally occurring minor variants of epitope Gag295-304 enhanced KIR2DL2 binding to HLA-C03:04. Our data show that naturally occurring sequence variations within HLA-C03:04-restricted HIV-1 p24 Gag epitopes can have a significant impact on the binding of inhibitory KIR receptors and primary NK cell function.