Inhibitory Killer Immunoglobulin-like Receptors Research Articles

Increased donor inhibitory KIR are associated with reduced GVHD and improved survival following HLA-matched unrelated donor HCT in paediatric acute leukaemia.

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated effects after haematopoietic stem cell transplantation (HCT). Previous work has shown that accounting for known KIR-KIRL interactions may identify donors with optimal NK cell-mediated alloreactivity in the adult transplant setting. Paediatric acute leukaemia patients were retrospectively analysed, and KIR-KIRL combinations and maximal inhibitory KIR ligand (IM-KIR) scores were determined. Clinical outcomes were examined using a series of graphs depicting clinical events and endpoints. The graph methodology demonstrated that prognostic variables significant in the occurrence of specific clinical endpoints remained significant for relevant downstream events. KIR-KIRL combinations were significantly predictive for reduced grade 3-4 aGVHD likelihood, in patients transplanted with increased inhibitory KIR gene content and IM-KIR = 5 scores. Improvements were also observed in associated outcomes for both ALL and AML patients, including relapse-free survival, GRFS and overall survival. This study demonstrates that NK cell KIR HLA interactions may be relevant to the paediatric acute leukaemia transplant setting. Reduction in aGVHD suggests KIR effects may extend beyond NK cells. Moving forward clinical trials utilizing donors with a higher iKIR should be considered for URD HCT in paediatric recipients with acute leukaemia to optimize clinical outcomes.

Impact of KIR-HLA Genotype on Natural-Killer-Cell-Based Immunotherapy for Preventing Hepatocellular Carcinoma after Living-Donor Liver Transplantation

Natural killer (NK) cells have immunosurveillance potential in hepatocellular carcinoma (HCC). We performed adaptive immunotherapy using donor-liver-derived natural killer (NK) cells after living-donor liver transplantation (LDLT) to prevent HCC recurrence. Dominant inhibitory signals tightly regulate NK cell activity via human leukocyte antigen (HLA)-specific inhibitory receptors, such as killer immunoglobulin-like receptors (KIRs). The functional recognition of HLA through KIR raises the NK cell capacity, which is a process termed “licensing.” Here, we investigated the effect of polymorphic KIR-HLA genotypes on the efficacy of NK-cell-based immunotherapy after LDLT. Seventy-seven Japanese recipients with HCC who underwent LDLT and their corresponding donors between 1996 and 2016 were enrolled in this study. The median follow-up period was 8.3 years. The HCC recurrence risk was stratified using radiological and pathological assessments according to the Milan criteria. Of the 77 recipients, 38 received immunotherapy. Immunotherapy improves early post-transplantation survival and lowers the recurrence rate in the intermediate-risk recipients. We analyzed the genotypes of five inhibitory KIRs and HLA using sequence-specific polymorphism-based typing. The polymorphic KIR-HLA genotype revealed that genetically vulnerable liver transplant recipients with a poorly licensed NK genotype have an improved prognosis by immunotherapy with donor-liver-derived NK cells. Thus, the combination of recipient and donor KIR-HLA genotypes is worthy of attention for further investigation, especially considering the clinical application of NK-cell-based immunotherapy.

Structural basis for the activity and specificity of the immune checkpoint inhibitor lirilumab

The clinical success of immune checkpoint inhibitors has underscored the key role of the immune system in controlling cancer. Current FDA-approved immune checkpoint inhibitors target the regulatory receptor pathways of cytotoxic T-cells to enhance their anticancer responses. Despite an abundance of evidence that natural killer (NK) cells can also mediate potent anticancer activities, there are no FDA-approved inhibitors targeting NK cell specific checkpoint pathways. Lirilumab, the most clinically advanced NK cell checkpoint inhibitor, targets inhibitory killer immunoglobulin-like receptors (KIRs), however it has yet to conclusively demonstrate clinical efficacy. Here we describe the crystal structure of lirilumab in complex with the inhibitory KIR2DL3, revealing the precise epitope of lirilumab and the molecular mechanisms underlying KIR checkpoint blockade. Notably, the epitope includes several key amino acids that vary across the human population, and binding studies demonstrate the importance of these amino acids for lirilumab binding. These studies reveal how KIR variations in patients could influence the clinical efficacy of lirilumab and reveal general concepts for the development of immune checkpoint inhibitors targeting NK cells.

Immunological Aspects of Infertility-The Role of KIR Receptors and HLA-C Antigen.

The mechanisms of immune tolerance of a mother against an antigenically foreign fetus without a concomitant loss of defense capabilities against pathogens are the factors underlying the success of a pregnancy. A significant role in human defense is played by killer immunoglobulin-like receptor (KIR) receptors, which regulate the function of the natural killer (NK) cells capable of destroying antigenically foreign cells, virus-infected cells, or tumor-lesioned cells. A special subpopulation of NK cells called uterine NK cells (uNK) is found in the uterus. Disruption of the tolerance process or overactivity of immune-competent cells can lead to immune infertility, a situation in which a woman's immune system attacks her own reproductive cells, making it impossible to conceive or maintain a pregnancy. Since the prominent role of the inflammatory response in infertility, including KIR receptors and NK cells, has been postulated, the process of antigen presentation involving major histocompatibility complex (MHC) molecules (HLA) appears to be crucial for a successful pregnancy. Proper interactions between KIR receptors on female uNK cells and HLA class I molecules, with a predominant role for HLA-C, found on the surface of germ cells, are strategically important during embryo implantation. In addition, maintaining a functional balance between activating and inhibitory KIR receptors is essential for proper placenta formation and embryo implantation in the uterus. A disruption of this balance can lead to complications during pregnancy. The discovery of links between KIR and HLA-C has provided valuable information about the complexity of maternal-fetal immune interactions that determine the success of a pregnancy. The great diversity of maternal KIR and fetal HLA-C ligands is associated with the occurrence of KIR/HLA-C combinations that are more or less favorable for reproductive success.

Negativity begets longevity in T cells.

Killer immunoglobulin-like receptors (KIRs) are polymorphic receptors for human leukocyte antigens (HLAs) that provide positive or negative signals controlling lymphocyte activation. Expression of inhibitory KIRs by CD8+ T cells affects their survival and function, which is linked to improved antiviral immunity and prevention of autoimmunity. In this issue of the JCI, Zhang, Yan, and co-authors demonstrate that increased numbers of functional inhibitory KIR-HLA pairs equating to greater negative regulation promoted longer lifespans of human T cells. This effect was independent of direct signals provided to KIR-expressing T cells and was instead driven by indirect mechanisms. Since the long-term maintenance of CD8+ T cells is critical for immune readiness against cancer and infection, this discovery has implications for immunotherapy and the preservation of immune function during aging.

Combined flow cytometry natural killer immunophenotyping and KIR/HLA-C genotyping reveal remarkable differences in acute myeloid leukemia patients, but suggest an overall impairment of the natural killer response.

Natural killer (NK) cells are key anti-tumor effectors of the innate immunity. Phenotypic differences allow us to discriminate in between three functional stages of maturation, named immature, mature and hypermature that are distinctive in terms of receptor expression, cytokine secretion, cytotoxic properties and organ trafficking. NKs display an impressive repertoire of highly polymorphic germline encoded receptors that can be either activating, triggering the effector's function, or inhibitory, limiting the immune response. In our study, we have investigated peripheral blood NK cells of acute myeloid leukemia (AML) patients. The Killer Immunoglobulin-like receptors (KIRs) and the HLA-C genotypes were assessed, as HLA-C molecules are cognate antigens for inhibitory KIRs. The AA mainly inhibitory KIR haplotype was found in a higher proportion in AML, while a striking low frequency of the 2DS3 characterized the mainly activating Bx haplotype. Flow cytometry immunophenotyping evidenced a lower overall count of NK cells in AML versus healthy controls, with lower percentages of the immature and mature subpopulations, but with a markedly increase of the hypermature NKs. The analysis of the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A inhibitory receptors surface expression revealed a remarkable heterogeneity. However, an overall trend for a higher expression in AML patients could be noticed in all maturation subpopulations. Some of the AML patients with complex karyotypes or displaying a FLT3 gene mutation proved to be extreme outliers in terms of NK cells percentages or inhibitory receptors expression. We conclude that while the genetic background investigation in AML offers important pieces of information regarding susceptibility to disease or prognosis, it is flow cytometry that is able to offer details of finesse in terms of NK numbers and phenotypes, necessary for an adequate individual evaluation of these patients.

The role of the HLA allelic repertoire on the clinical severity of COVID-19 in Canadians, living in the Saskatchewan province

AimsThe HLA system has been implicated as an underlying determinant for modulating the immune response to SARS-CoV-2. In this study, we aimed to determine the association of patients’ HLA genetic profiles with the disease severity of COVID-19 infection. MethodsProspective study was conducted on COVID-19 patients (n = 40) admitted to hospitals in Saskatoon, Canada, between March and December 2020. Next-generation sequencing was performed on the patient samples to obtain high-resolution HLA typing profiles. The statistical association between HLA allelic frequency and disease severity was examined. The disease severity was categorized based on the length of hospital stay and intensive care needs or demise during the hospital stay. ResultsHLA allelic frequencies of the high and low-severity cohorts were normalized against corresponding background allelic frequencies. In the high-severity cohort, A*02:06 (11.8-fold), B*51:01 (2.4-fold), B*15:01(3.1-fold), C*01:02 (3.3-fold), DRB1*08:02 (31.2-fold), DQ*06:09 (11-fold), and DPB1*04:02(4-fold) were significantly overrepresented (p < 0.05) making these deleterious alleles. In the low-severity cohort, A*24:02 (2.8-fold), B*35:01 (2.8-fold), DRB1*04:07 (5.3-fold), and DRB1*08:11 (22-fold) were found to be significantly overrepresented (p < 0.05) making these protective alleles. These above alleles interact with NK cell antiviral activity via the killer immunoglobulin-like receptors (KIR). The high-severity cohort had a higher predilection for HLA alleles associated with KIR subgroups; Bw4-80I (1.1-fold), and C1 (1.6-fold) which promotes NK cell inhibition, while the low-severity cohort had a higher predilection for Bw4-80T (1.6-fold), and C2 (1.6-fold) which promote NK cell activation. ConclusionIn this study, the HLA allelic repository with the distribution of deleterious and protective alleles was found to correlate with the severity of the clinical course in COVID-19. Moreover, the interaction of specific HLA alleles with the KIR-associated subfamily modulates the NK cell-mediated surveillance of SARS-CoV-2. Both deleterious HLA alleles and inhibitory KIR appear prominently in the severe COVID-19 group focusing on the importance of NK cells in the convalescence of COVID-19.

Matched Unrelated Donor Hematopoietic Cell Transplant with Increased Donor Inhibitory KIR Improves Survival in a Pediatric Acute Leukemia: A CIBMTR Cohort Analysis

Natural killer (NK) cells potentially mediate innate immunity early after hematopoietic cell transplantation (HCT). NK cells posses an array of activating and inhibitory receptors on their surface, including killer immunoglobulin-like receptor (KIR) and which interact with KIR-ligands (KIRL) on target cells. The impact of NK cell mediated alloreactivity in pediatric HCT is not well characterized. In this study, we explored a mathematical approach to quantify KIR-KIRL combinations to assess clinical outcomes in a cohort of pediatric patients who underwent HLA-matched unrelated donor (MUD) HCT for acute leukemia including AML and ALL. This retrospective study was performed on deidentified donor and recipient demographic and clinical outcomes data provided by the Center for International Blood and Marrow Transplant Research (CIBMTR). KIR-KIRL interaction values were assigned as previously (Krieger et. al 2021, Bone Marrow Transplantation). Briefly donor inhibitory (iKIR) and activating (aKIR) KIR- recipient KIRL interactions, accounting for missing KIR-L (mKIR), were summed up, using a system of matrices. The study cohort was comprised of 686 pediatric donor-recipient pairs (DRP) who underwent MUD HCT for AML (N=355) and ALL (N=333) and had neutrophil engraftment (Table 1). This cohort of pediatric patients was aged 0-18 years (average 11). All patients received T cell replete grafts; 26% received in vivo T cell depletion, with either anti-thymocyte globulin (ATG) or alemtuzumab. The majority, 77% received a bone marrow graft and 90% received myeloablative conditioning. The effect of all 3 KIR-KIRL interaction scores (absolute values of iKIR, mKIR and aKIR) on clinical outcomes were evaluated. Higher donor iKIR score was associated with improvement in overall survival (OS) SHR 0.73 (multivariate P=0.003) and relapse-free survival (RFS), SHR 0.8 (P=0.019); grade 3-4 acute GVHD was lower in patients transplanted with higher iKIR or mKIR scores (iKIR; SHR 0.58 (P=0.013); mKIR SHR 0.6 (P=0.041)). Higher aKIR scores, were not significantly associated with any clinical outcomes examined. As in previous analysis, the iKIR and mKIR scores were added to give an IM-KIR score, which was dichotomized for analysis into those DRP with an IM KIR score of 5 (IM = 5), and those with an IM-KIR score of 2, 3 or 4 (IM < 5). Of note, KIR haplotype A patients always have an IM KIR score of 4, and are thus categorized among IM<5 donors. In this cohort of pediatric patients transplanted for both AML and ALL, those transplanted with IM=5 donors demonstrated improved OS, SHR 0.75 (P = 0.027), RFS, SHR 0.79 (P=0.05) and grade 3-4 aGVHD incidence, SHR 0.37 (P= 0.003) in multivariate analysis. We next examined outcomes in patients with ALL and AML to understand disease specific effects. Patients transplanted for AML with IM=5 donors had an improvement in OS, SHR 0.62 (P=0.01), as well as RFS, SHR 0.70 (p=0.033) (Fig. 1). There was a trend towards relapse risk reduction in this cohort SHR 0.67 (univariate P=0.048). Patients transplanted for ALL with IM=5 donors demonstrated a marked decrease in grade 3-4 acute GVHD incidence SHR, 0.31 (P=0.017). This large pediatric study confirms that unrelated DRPs with donor haplotype B genotypes and a higher inhibitory KIR content (regardless of KIR ligand status), confer significant improvement in overall survival and severe aGVHD after MUD HCT. Those patients transplanted for AML experienced an improvement in OS and RFS despite the lower event rate observed in pediatric-age patients, the majority of whom receive myeloablative preparatory regimens without the use of in vivo T cell depletion. This challenges the notion that KIR are irrelevant to pediatric donor selection. Future clinical trials evaluating donor selection for URD HCT should include this measure to evaluate its value prospectively. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Coexistence of HLA and KIR ligand mismatches as a risk factor for viral infection early after cord blood transplantation.

Viral infection is one of the lethal adverse events after cord blood transplantation (CBT). Human leukocyte antigen (HLA) and killer immunoglobulin-like receptor (KIR) ligand divergences can increase the risk of viral infection due to conflicting interactions between virus-infected cells and immune cells. However, the relationship between these disparities and the frequency of viral infection after CBT remains to be evaluated. Herein, we have conducted a retrospective multicenter study to assess the effect of HLA and KIR ligand mismatches on viral infections after CBT. The study included 429 patients, among which 126 viral infections occurred before day 100. Viral infection was significantly associated with poorer overall survival (OS; hazard ratio [HR] 1.74, p < 0.01). Patients harboring ≥3 mismatches in the HLA allele and inhibitory KIR ligand mismatches (HLA & KIR mismatches) had a significantly greater prevalence of viral infection (HR 1.66, p = 0.04). Thus, patients with HLA & KIR mismatches had poorer outcomes in terms of non-relapse mortality (HR 1.61, p = 0.05). Our study demonstrates the unfavorable impacts of HLA & KIR mismatches on viral infections and non-relapse mortality after CBT. Evaluating the viral infection risk and performance of an appropriate and early intervention in high-risk patients and optimizing the graft selection algorithm could improve the outcome of CBTs.

Echinococcus multilocularis induces surface high expression of inhibitory killer immunoglobulin-like receptor on natural killer cells.

Alveolar echinococcosis (AE) is a malignant and fatal parasitic disease caused by the larvae of Echinococcus multilocularis (E. multilocularis), which inhibits the activity and proliferation of natural killer (NK) cells. In this study, the functional alteration of hepatic NK cells and their related molecules were studied. The AE-infected patient's tissue was fixed with formalin, embedded in paraffin, and stained with Masson's trichrome or hematoxylin and eosin (H&E). Single cells from AE-infected patient or E. multilocularis-infected mice were blocked with Fc-receptor (FcR), and stained with monoclonal antibodies, including CD16, CD56, CD3, KIR2DL1, granzyme B, perforin, Interferon gamma (IFN-γ), and tumor necrosis factor-α (TNFα) or isotype control, to measure molecules and cytokines of NK cells and analyzed by flow cytometry. The Sirius red staining was used to quantitate hepatic fibrosis by calculating quantitative collagen deposition. AE can adjust both the number of hepatic CD56+ NK cells and its KIR2DL1 expression processes. Moreover, the overexpression of KIR2DL1 in NK cells could downregulate the functioning of immune cells in the liver area close to parasitic lesions. The number and dysfunction of NK cells in E. multilocularis infection could be related to the molecule dynamics of cell surface inhibitory receptor Ly49A, leading to hepatic damage and progression of fibrosis. This study illustrated significant increase in hepatic fibrogenesis and apparent upregulation of hepatic CD56+ NK cell population and its KIR2DL1 expression in AE-infected patients. This opposite variation might be related to the impaired NK cells functioning, such as granzyme B, IFN-γ, and TNF-α secretion. In addition, the cell surface inhibitory receptor Ly49A was related to the intracellular cytokine secretion functions of NK cells.

Increased donor inhibitory KIR with known HLA interactions provide protection from relapse following HLA matched unrelated donor HCT for AML.

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated graft versus leukemia effect (GVL) after hematopoietic cell transplant (HCT) for AML. Accounting for known KIR-KIRL interactions may identify donors with optimal NK cell-mediated alloreactivity and GVL. A retrospective study of 2359 donor-recipient pairs (DRP) who underwent unrelated donor (URD) HCT for AML was performed. KIR-KIRL combinations were determined and associations with clinical outcomes examined. Relapse risk was reduced in DRP with both higher inhibitory KIR-KIRL (iKIR) and missing KIRL (mKIR) scores, with HR 0.86 (P = 0.01) & HR 0.84 (P = 0.02) respectively. The iKIR and mKIR score components were summed to give a maximal inhibitory KIR ligand (IM-KIR) score for each donor, which if it was 5, as opposed to <5, was also associated with a lower relapse risk, SHR 0.8 (P = 0.004). All IM = 5 donors possess KIR Haplotype B/x. Transplant-related mortality was increased among those with IM-KIR = 5, HR, 1.32 (P = 0.01). In a subset analysis of those transplanted with 8/8 HLA-matched DRP, anti-thymocyte globulin recipients with IM-KIR = 5, had a lower relapse rate HR, 0.61 (p = 0.001). This study demonstrates that HLA-matched unrelated donors with the highest inhibitory KIR content confer relapse protection, albeit with increased TRM. These donors all have KIR haplotype B. Clinical trials utilizing donors with a higher iKIR content in conjunction with novel strategies to reduce TRM should be considered for URD HCT in recipients with AML to optimize clinical outcomes.

Killer Immunoglobulin-like Receptors (KIR) in Low-Risk Myelodysplastic Syndrome: Genotyping and Gene Expression Evaluation

Introduction: MDS are a group of myeloid malignancies characterized by ineffective hematopoiesis, peripheral cytopenias, and 30% chance of transformation into AML. MDS pathogenesis involves, in some cases, a phenomenon of autoimmunity which can suppress normal hematopoiesis by direct cytotoxicity as well as by release of cytokines. Dysplasia-associated antigens released from dying cells might be responsible for evoking an adaptive immune response. NK cells appear activated, with high expression of perforin and granzyme, and mediate the cytotoxicity of bone marrow (BM) precursor cells in lower risk disease. Their activity is modulated by killer immunoglobulin-like receptors (KIR). Prior experience suggests that Tipifarnib, a farnesyl transferase inhibitor, may suppress abnormal NK cell function and autoimmunity that contributes to MDS. It is hypothesized that Tipifarnib could be especially of benefit in lower risk MDS patients where autoimmunity is known to play a role and immunosuppression is of clinical benefit in some cases.KIR genes are polymorphic and two broad haplotypes exist: KIR-haplotype A mainly encode for inhibitory receptors and only for one activating (KIR2DS4), whereas group B haplotype encodes more for activating KIRs (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 und KIR3DS1). We hypothesized that KIR molecular profile could be correlated with response to Tipifarnib and molecular characteristics of patients with MDS. With this goal in mind, the key objective of the present study was to develop a methodology and a reportable range of KIR2DS2, KIR2DS5, KIR2DL2 and KIR2DL5 RNA expression and their ratios by RT-PCR in samples from low/INT-1 risk MDS patients.Methods: We evaluated the KIR genotyping and gene expression from DNA and RNA samples, respectively, in BM mononuclear cells of 50 lower risk MDS patients followed at CUMC from 2010 to 2015. KIR typing was performed by conventional PCR using commercial Miltenyi KIR genotyping kit for all known 15 human KIR genes. Gene expression was accessed by qRT-PCR for two activating (2DS2, and 2DS5), two inhibitory (2DL2, and 2DL5) KIR genes, and five leukocyte markers: CD56, CD4, GZMM, CD8A, and CD8B . Correlation between clinical and molecular data of the patients was sought.Results: Performance specifications (precision, limit of detection, linearity, and reportable range) were determined for all assays used in this study. All 50 MDS patients were positive for KIR2DL4, 3DL2, 3DL3, 2DP1 and 3DP1, whereas 2DS3 [7/55; 13%], 2DS5 [16/55; 29%], and 2DL5 [19/55; 35%] were less frequent (Figure 1). These results corroborate the existing literature showing similar distribution in the normal population. Patients with normal karyotype had higher frequency of 2DL5 [51% vs 33.3%], 2DS1 [46% vs 33.3%], 2DS3 [17% vs 6%], 2DS5 [37% vs 20%], and 3DS1 genes [43% vs 27%] than patients with abnormal cytogenetics, however none of these results was statistically significant. KIR2DS2 and 2DL2, and 2DS5 and 2DL5, co-occurred in the same patients, as already described in normal American population. All patients that presented KIR2DS2 [22/55; 44%], 2DL2 [22/55; 44%], 2DS5 [23/55; 46%], or 2DL5 [23/55; 46%] had their samples analyzed by qRT-PCR to the respective targets besides some leukocytes markers. Correlation between KIR copy number and MDS subtype, BM cellularity, WBC, RBC, Hg, and platelets was not observed.Survival analysis showed poor survival for the patients carrying KIR2DL5, 2DS1, and 3DS1 genes [p<0.05] (Fig. B,C, & D). 42% (21/50) of the patients were positive for all three genes, showing the same ratio already identified in the normal population (36%). Additionally, survival was different between the four haplotypes groups CenA/TelA, CenA/TelB, CenB/TelA, and CenB/TelB (P=0.0628; Fig. E) but a subset analysis of CenA-TelA and CenB-TelB haplotypes showed a significantly different survival between two groups (P= 0.0145; Fig 1E). There was no correlation between gene expression data and survival.Conclusions: We report on the methodology and its use to define a reportable range of KIR2DS2, KIR2DS5, KIR2DL2 and KIR2DL5 RNA expression and their ratios by RT-PCR in 50 lower risk MDS patients. Our findings suggest that KIR genes might be associated with some clinical features in patients, mainly, survival and cytogenetics profile. KIR expression will be correlated with clinical response to Tipifarnib as part of an ongoing clinical trial. [Display omitted] DisclosuresCoutinho:Kura Oncology: Consultancy. Ali:Onconova Therapeutics: Consultancy; Kura Oncology: Consultancy. Gualberto:Kura Oncology: Employment, Equity Ownership, Other: Chief Medical Officer, Patents & Royalties. Scholz:Kura Oncology: Employment, Equity Ownership, Patents & Royalties. Bracken:Kura Oncology: Employment. Jurcic:Forma Therapeutics: Research Funding; Celgene: Research Funding; Alexion Pharmaceuticals: Consultancy; Genentech: Research Funding; Incyte: Consultancy; Actinium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas Pharma, Inc: Research Funding; Daiichi-Sankyo: Research Funding; Kura Oncology: Research Funding; Merck: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Syros Pharmaceuticals: Research Funding; Amgen: Consultancy. Raza:Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Celgene Inc.: Research Funding; Kura Oncology: Research Funding; Janssen R&D: Research Funding; Syros Pharmaceuticals: Research Funding; Onconova Therapeutics: Research Funding, Speakers Bureau.

Increased Inhibitory KIR-Ligand Interactions Confer Relapse Protection Following HLA Matched Unrelated Donor HCT for AML

Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated graft versus leukemia effect (GVL) after hematopoietic stem cell transplant (HCT) for AML. There is considerable heterogeneity in the KIR gene and KIRL content of individuals, making it difficult to estimate the full clinical impact of NK cell alloreactivity following HCT. Herein, we validate a mathematical model accounting for KIR-KIRL interactions identifying donors with optimal NK cell-mediated alloreactivity and GVL. This retrospective study was performed on de-identified donor and recipient demographic and clinical outcomes data provided by the Center for International Blood and Marrow Transplant Research (CIBMTR). Donor recipient pairs (DRP) who underwent unrelated donor (URD) HCT for early and intermediate AML were included. KIR-KIRL interaction values were assigned as follows; if an inhibitory KIR (iKIR) on the NK cell encounters a ligand on its target, this will give the NK cell an inhibitory signal and this is scored as a single interaction(Figure 1b), as is the case, if there is no ligand for an inhibitory KIR, i.e., missing KIRL (mKIRL) (Figure 1c). Finally, activating KIR (aKIR) interacting with its ligands is similarly scored(Figure 1a). The absolute values of the iKIR and mKIR scores were summed to calculate the composite inhibitory-missing ligand (IM)-KIR score (Figure 1d). The study cohort was comprised of 2365 donor-recipient pairs (DRP) who underwent URD HCT for early or intermediate AML. Mean age was 53 years; 85% of DRPs were high-resolution 8/8 HLA-matched for HLA-A, -B, -C, and -DRB1. All patients received T cell replete grafts; 42% (n=996) received in vivo T cell depletion, 937 (94%) with anti-thymocyte globulin (ATG); 86% received a graft of mobilized peripheral blood stem cells (PBSC), 59% received myeloablative conditioning. This cohort was primarily of Caucasian descent (89%). When adjusted for recipient age, donor age, CMV status, KPS, GVHD prophylaxis, cytogenetics, disease status, conditioning regimen, in vivo T cell depletion, graft source, and sex match, relapse risk was significantly reduced in donor-recipient pairs (DRP) with higher inhibitory KIR-KIRL interaction and missing KIRL (mKIR) scores, with HR=0.86 (P=0.01) &amp; HR=0.84 (P=0.02) respectively. This effect was not observed with activating KIR-KIRL interactions. Chronic GVHD and TRM were not significantly affected by iKIR, mKIR or aKIR. Given the significant individual impact of iKIR and mKIR, the summed inhibitory-missing ligand (IM-KIR) score was next assessed, and when this score was 5 (as opposed to &amp;lt;5), the IM-KIR score was also associated with lower relapse risk, HR 0.8 (P=0.004) (Figure 2a). Acute and chronic graft vs. host disease (GVHD), survival, GRFS, and relapse-free survival were not significantly different, likely due to increased TRM among these patients, HR 1.32 (P=0.01). Interaction analysis indicated that amongst the HLA matched DRP, ATG recipients with IM-KIR=5, had a lower relapse rate compared to those with an IM-KIR&amp;lt;5, HR 0.61 (p=0.001) (Figure 2b), among the cohort who did not receive ATG there was no significant difference in relapse among IM-KIR=5 and IM-KIR&amp;lt;5; thus, the use of ATG significantly modified the effect of IM KIR score in an interaction analysis (p=0.049), suggesting higher NK cell magnitude of KIR-KIRL interaction may compensate for the general increase of relapse in those who receive in vivo T cell depletion. Nevertheless, TRM was also increased in these patients, HR 1.49 (p=0.034), likely abrogating survival advantage from a lower relapse risk. This large international study confirms that unrelated DRPs with greater magnitude of inhibitory KIR-KIRL interactions confer significant relapse protection after MUD HCT in standard-risk AML. This challenges the notion that KIR are irrelevant to donor selection. Future clinical trials evaluating donor selection for URD HCT should include this measure to evaluate its value prospectively in uniformly treated patient cohorts, with adequate GVHD and antiviral prophylaxis to mitigate TRM. Disclosures No relevant conflicts of interest to declare.

Concordance between peripheral and decidual NK cell subsets and killer immunoglobulin-like receptors in women with recurrent spontaneous miscarriages

BackgroundThe role of decidual natural killer (dNK) cells in normal and complicated pregnancy and their relation with peripheral NK (pNK) cells remains unclear. The study aim was phenotypic analysis of pNK and dNK cells at time of miscarriage in recurrent spontaneous miscarriage (RSM) patients to assess whether measuring levels of pNK cell populations can reflect changes in dNK cells or not. MethodsThis study included 40 middle aged pregnant women in the 1st trimester subjected to evacuation because of a current miscarriage. They had a history of previous ≥ two unexplained miscarriages. Frequencies of pNK and dNK cells, based on the expression of CD56, CD16, inhibitory (CD158b) and activating (CD161) Killer immunoglobulin-like receptors (KIRs), were detected by flow cytometry. ResultsPercentages of CD56+ NK cells in peripheral blood and decidua were 17.5 % and 17.3 %, respectively. In both blood and decidua, CD56dim NK cells were exceeding CD56bright NK cells. The CD56dim CD16− NK cells were the predominating subset of NK cells, followed by CD56dim CD16dim. No substantial differences were detected in the levels of KIRs expression by the different NK subsets between blood and decidua. Abnormal up-regulation of both CD161 and CD158b on NK cells was observed in blood and decidua. ConclusionAt the time of miscarriage, patients with RSM have an extremely active immune system and an increased number of toxic NK cells both in blood and decidua. The pNK cells reflect dNK cell changes during miscarriage and may be a useful non-invasive predicting tool in reproductive failure setting.

Rapid dose-dependent Natural Killer (NK) cell modulation and cytokine responses following human rVSV-ZEBOV Ebolavirus vaccination

The rVSV-ZEBOV Ebolavirus vaccine confers protection within days after immunization, suggesting the contribution of innate immune responses. We report modulation of rVSV-ZEBOV vaccinee blood CD56+ NK cell numbers, NKG2D or NKp30 surface receptor expression, Killer Immunoglobulin-like Receptor (KIR)+ cell percentages and NK-cell-related genes on day 1 post immunization. Inverse correlations existed between the concentration of several plasma cytokines and inhibitory KIR+ CD56dim or cytokine-responsive CD56bright NK cells. Thus, NK cells may contribute to the early protective efficacy of rVSV-ZEBOV in humans.

Killer immunoglobulin-like receptors/human leukocyte antigen class-I, a crucial immune pathway in cancer

Natural killer cells (NK cells) play a crucial role in tumor immunity. The function of the NK cells is regulated by various receptors expressed on the surface. Among them, the killer immunoglobulin-like receptor (KIR) is one of the most important. The ligand of KIR is major histocompatibility complex class-I (MHC class-I), which is also called human leukocyte antigen class-I (HLA class-I). The combination of HLA class-I and inhibitory KIRs could inhibit NK cells and induce autoimmune tolerance. Inhibitory KIRs were highly expressed on malignant tumor patients, which were related to poor prognosis. KIR/HLA class-I pathway affected the clinical outcomes of cancer through several mechanisms, and inhibitory KIRs could be an ideal target of immunotherapy strategy.

KIR B donors improve the outcome for AML patients given reduced intensity conditioning and unrelated donor transplantation

AbstractNatural killer (NK) cell recognition and killing of target cells are enhanced when inhibitory killer immunoglobulin-like receptors (KIR) are unable to engage their cognate HLA class I ligands. The genes of the KIR locus are organized into either KIR B haplotypes, containing 1 or more activating KIR genes or KIR A haplotypes, which lack those genes. Analysis of unrelated donor (URD) hematopoietic cell transplants (HCT), given to acute myeloid leukemia (AML) patients between 1988 and 2009, showed that KIR B haplotype donors were associated with better outcomes, primarily from relapse protection. Most of these transplants involved marrow grafts, fully myeloablative (MAC) preparative regimens, and significant HLA mismatch. Because the practice of HCT continues to evolve, with increasing use of reduced intensity conditioning (RIC), peripheral blood stem cell grafts, and better HLA match, we evaluated the impact of URD KIR genotype on HCT outcome for AML in the modern era (2010-2016). This analysis combined data from a prospective trial testing URD selection based on KIR genotypes (n = 243) with that from a larger contemporaneous cohort of transplants (n = 2419). We found that KIR B haplotype donors conferred a significantly reduced risk of leukemia relapse and improved disease-free survival after RIC, but not MAC HCT. All genes defining KIR B haplotypes were associated with relapse protection, which was significant only in transplant recipients expressing the C1 epitope of HLA-C. In the context of current HCT practice using RIC, selection of KIR B donors could reduce relapse and improve overall outcome for AML patients receiving an allogeneic HCT.

Subordinate Effect of -21M HLA-B Dimorphism on NK Cell Repertoire Diversity and Function in HIV-1 Infected Individuals of African Origin.

Natural Killer (NK) cells play an important role in antiviral defense and their potent effector function identifies them as key candidates for immunotherapeutic interventions in chronic viral infections. Their remarkable functional agility is achieved by virtue of a wide array of germline-encoded inhibitory and activating receptors ensuring a self-tolerant and tunable repertoire. NK cell diversity is generated by a combination of factors including genetic determinants and infections/environmental factors, which together shape the NK cell pool and functional potential. Recently a genetic polymorphism at position -21 of HLA-B, which influences the supply of HLA-E binding peptides and availability of HLA-E for recognition by the inhibitory NK cell receptor NKG2A, was shown to have a marked influence on NK cell functionality in healthy human cytomegalovirus (HCMV) seronegative Caucasian individuals. In this study, -21 methionine (M)-expressing alleles supplying HLA-E binding peptides were largely poor ligands for inhibitory killer immunoglobulin-like receptors (KIRs), and a bias to NKG2A-mediated education of functionally-potent NK cells was observed. Here, we investigated the effect of this polymorphism on the phenotype and functional capacity of peripheral blood NK cells in a cohort of 36 African individuals with human immunodeficiency virus type 1 (HIV-1)/HCMV co-infection. A similarly profound influence of dimorphism at position -21 of HLA-B on NK cells was not evident in these subjects. They predominantly expressed African specific HLA-B and -C alleles that contribute a distinct supply of NKG2A and KIR ligands, and these genetic differences were compounded by the marked effect of HIV-1/HCMV co-infection on NK cell differentiation. Together, these factors resulted in a lack of correlation of the HLA-B -21 polymorphism with surface abundance of HLA-E and loss of the NK cell functional advantage in subjects with -21M HLA-B alleles. Instead, our data suggest that during HIV/HCMV co-infection exposure of NK cells to an environment that displays altered HLA-E ligands drives adaptive NKG2C+ NK cell expansions influencing effector responses. Increased efforts to understand how NK cells are functionally calibrated to self-HLA during chronic viral infections will pave the way to developing targeted therapeutic interventions to overcome the current barriers to enhancing immune-based antiviral control.

KIR Genotype and KIR Ligand Phenotypes That Improve the in Vitro Cord Blood or Autologous NK Cell Mediated Cytotoxicity Against Myeloma Marrow Plasma Cells

<h3>Instruction</h3> Killer Immunoglobulin-like Receptor (KIR) inhibition is known to influence the cytotoxic effects of natural killer (NK) cells against cancer cells. KIRs can be protective against relapse in myeloma (MM) (Gabriel et al Blood 2010, Kröger et al leukemia 2011) by NK cell licencing <i>or through</i> blocking of the inhibitory KIR receptors such as NKG2A (Mahaweni et al, 2018). <h3>Objectives</h3> In this study, the role of KIR geno/phenotypes and their ligands on autologous and cord blood (CB) derived NK cell mediated cytotoxicity against MM plasma cells (PC) were investigated. <h3>Methods</h3> Peripheral blood NK cells and marrow PCs were isolated from 20 patients with relapsed MM. PC were isolated from fresh BM aspirates using EasySep Human CD138 Positive Selection Kit (StemCell Technologies) and were subjected to cytotoxicity assays with either autologous or CB derived NK cells isolated (Human NK cell Enrichment Cocktail, Stemcell Technologies) and cultured in the presence of IL-15 (10ng/ml) for 2-8 days. KIR/KIR ligand genotyping were performed by Olerup SSP KIR Genotyping Kit (Olerup, Stockholm, Sweden) in order to detect KIR genotypes; KIR2DL1, 2DL2, 2DL3, 2DL4, 2DL5A/B, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1, 2DP1, 3DP1, and KIR ligands; HLA-ABw4+, HLA-BBw4+Thr80, HLA-BBw4+Ile80, HLA-BBw4+Asp77,Thr80, HLA-CAsn80, and HLA-CLys80. Effector cells were stained with antibodies against NKG2A (CD159a) and CD158i (KIR2DS4) and analyzed using a Beckman Coulter Flow Cytometer. <i>Cytotoxicity assays</i>: Using <i>3,3</i> (DiO) as an indicator and 1:10 target:effector cell ratio PC death was evaluated after 120 mins of incubation, using flow cytometry. <h3>Results</h3> 20 MM patients were tested in vitro. Following incubation of PC wo NK cells, spontaneous PC death ratio was found to be 22.0% (11.4%-51.2%). While autologous NK mediated cytotoxicity was median 17.3% (11.4%-43.6%), CB-NK cell mediated cytotoxicity was 23.2% (range: 11.5%-51.2%). Highest cytotoxicity was achieved with CB-NK cells (CB7) with a KIR AB haplotype, carrying 8 inhibitory KIR genes against PC from MM12. Effector cells with the iKIR gene number above 6 were found to cause higher cytotoxicity (13/19; p=0.031). Additionally, higher (>30%) expression of NKG2A on CB-NK cells were found to be associated with lack of cytotoxicity. <h3>Conclusion</h3> CB-derived NK cells were found to be able to induce higher PC death than autologous PB-NK cells. Also higher number of iKIR genotypes and lacking KIR2DS4 receptor was found to be associated with higher cytotoxicity. In our current study we are reporting KIR NKG2A which binds with HLA-E to play an additional role. Allogeneic NK cell mediated immunotherapies are promising due their clinical advantages ie no need for HLA matching. Furthermore certain KIR haplotypes (ie KIR2DS4⁺ or NKG2A⁻) may offer better therapeutic efficacy if confirmed by others as well.

The Education of NK Cells Determines Their Responsiveness to Autologous HIV-Infected CD4 T Cells.

Several studies support a role for specific killer immunoglobulin-like receptor (KIR)-HLA combinations in protection from HIV infection and slower progression to AIDS. Natural killer (NK) cells acquire effector functions through education, a process that requires the interaction of inhibitory NK cell receptors with their major histocompatibility complex (MHC) class I (or HLA class I [HLA-I]) ligands. HLA-C allotypes are ligands for the inhibitory KIRs (iKIRs) KIR2DL1, KIR2DL2, and KIR2DL3, whereas the ligand for KIR3DL1 is HLA-Bw4. HIV infection reduces the expression of HLA-A, -B, and -C on the surfaces of infected CD4 (iCD4) T cells. Here we investigated whether education through iKIR-HLA interactions influenced NK cell responses to autologous iCD4 cells. Enriched NK cells were stimulated with autologous iCD4 cells or with uninfected CD4 cells as controls. The capacities of single-positive (sp) KIR2DL1, KIR2DL2, KIR2DL3, and KIR3DL1 NK cells to produce CCL4, gamma interferon (IFN-γ), and/or CD107a were assessed by flow cytometry. Overall, we observed that the potency of NK cell education was directly related to the frequency of each spiKIR+ NK cell's ability to respond to the reduction of its cognate HLA ligand on autologous iCD4 cells, as measured by the frequency of production by spiKIR+ NK cells of CCL4, IFN-γ, and/or CD107a. Both NK cell education and HIV-mediated changes in HLA expression influenced NK cell responses to iCD4 cells.IMPORTANCE Epidemiological studies show that natural killer (NK) cells have anti-HIV activity: they are able to reduce the risk of HIV infection and/or slow HIV disease progression. How NK cells contribute to these outcomes is not fully characterized. We used primary NK cells and autologous HIV-infected cells to examine the role of education through four inhibitory killer immunoglobulin-like receptors (iKIRs) from persons with HLA types that are able to educate NK cells bearing one of these iKIRs. HIV-infected cells activated NK cells through missing-self mechanisms due to the downmodulation of cell surface HLA expression mediated by HIV Nef and Vpu. A higher frequency of educated than uneducated NK cells expressing each of these iKIRs responded to autologous HIV-infected cells by producing CCL4, IFN-γ, and CD107a. Since NK cells were from non-HIV-infected individuals, they model the consequences of healthy NK cell-HIV-infected cell interactions occurring in the HIV eclipse phase, when new infections are susceptible to extinction.