Killer Immunoglobulin-like Receptors Genotypes Research Articles

The role of Killer immunoglobulin-like receptors (KIRs) in the genetic susceptibility to non-celiac wheat sensitivity (NCWS).

Non-celiac wheat sensitivity (NCWS) is an emerging clinical condition characterized by gastrointestinal and extraintestinal symptoms following the ingestion of gluten-containing foods in patients without celiac disease (CD) or wheat allergy. Despite the great interest for NCWS, the genetic risk factors still need to be fully clarified. In this study, we first assessed the possible contribution of KIR genes and KIR haplotypes on the genetic predisposition to NCWS. Fifty patients with NCWS, 50 patients with CD, and 50 healthy controls (HC) were included in this study. KIR genes and KIR genotyping were investigated in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method using Luminex technology. We found a statistically different distribution of some KIR genes among NCWS, CD, and HC. Specifically, NCWS showed a decreased frequency of KIR2DL1, -2DL3, -2DL5, -2DS2, -2DS3, -2DS4, -2DS5, and -3DS1 genes, and an increased frequency of -3DL1 gene respect to both CD and HC. No difference was detected in the KIR haplotype expression. At the multivariate analysis, KIR2DL5, -2DS4, and -2DS5 were independent predictors of NCWS. Our findings suggest a role of KIR genes in NCWS susceptibility, with KIR2DL5, -2DS4, and -2DS5 having a protective effect. Further large-scale multicentric studies are required to validate these preliminary findings.

Centromeric AA motif in KIR as an optimal surrogate marker for precision definition of alloimmune reproductive failure

Throughout pregnancy, the decidua is predominantly populated by NK lymphocytes expressing Killer immunoglobulin-like receptors (KIR) that recognize human leukocyte antigen-C (HLA-C) ligands from trophoblast cells. This study aims to investigate the association of KIR-HLA-C phenotypes in couples facing infertility, particularly recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF), in comparison to a reference population and fertile controls. This observational, non-interventional retrospective case–control study included patients consecutively referred to our Reproductive Immunology Unit from 2015 to 2019. We analyzed the frequencies of KIR and HLA-C genes. As control groups, we analyzed a reference Spanish population for KIR analysis and 29 fertile controls and their male partners for KIR and HLA-C combinations. We studied 397 consecutively referred women with infertility and their male partners. Among women with unexplained RPL (133 women) and RIF (176 women), the centromeric (cen)AA KIR genotype was significantly more prevalent compared to the reference Spanish population (p = 0.001 and 0.02, respectively). Furthermore, cenAA was associated with a 1.51-fold risk of RPL and a 1.2-fold risk of RIF. Conversely, the presence of BB KIR showed a lower risk of reproductive failure compared to non-BB KIR (OR: 0.12, p < 0.001). Women and their partners with HLA-C1C1/C1C1 were significantly less common in the RPL-Group (p < 0.001) and RIF-Group (p = 0.002) compared to the control group. Moreover, the combination of cenAA/C1C1 in women with C1C1 partners was significantly higher in the control group than in the RPL (p = 0.009) and RIF (p = 0.04) groups, associated with a 5-fold increase in successful pregnancy outcomes. In our cohort, the cenAA KIR haplotype proved to be a more accurate biomarker than the classic AA KIR haplotype for assessing the risk of RPL and RIF, and might be particularly useful to identify women at increased risk among the heterogeneous KIR AB or Bx population. The classification of centromeric KIR haplotypes outperforms classical KIR haplotypes, making it a better indicator of potential maternal–fetal KIR-HLA-C mismatch in patients.

Systematic evaluation of donor-KIR/recipient-HLA interactions in HLA-matched hematopoietic cell transplantation for AML

AbstractIn acute myeloid leukemia (AML), donor natural killer cell killer immunoglobulin–like receptors (KIR) and recipient HLA interactions may contribute to the graft-versus-leukemia effect of allogeneic hematopoietic cell transplantation (HCT). Analyses of individual KIR/HLA interactions, however, have yielded conflicting findings, and their importance in the HLA-matched unrelated donor (MUD) setting remains controversial. We systematically studied outcomes of individual donor-KIR/recipient-HLA interactions for HCT outcomes and empirically evaluated prevalent KIR genotypes for clinical benefit. Adult patients with AML (n = 2025) who received HCT with MUD grafts in complete remission reported to the Center for International Blood and Marrow Transplantation were evaluated. Only the donor-2DL2+/recipient-HLA-C1+ pair was associated with reduced relapse (hazard ratio [HR], 0.79; 95% confidence interval [CI], 0.67-0.93; P = .006) compared with donor-2DL2–/recipient-HLA-C1+ pair. However, no association was found when comparing HLA-C groups among KIR-2DL2+–graft recipients. We identified 9 prevalent donor KIR genotypes in our cohort and screened them for association with relapse risk. Genotype 5 (G5) in all recipients and G3 in Bw4+ recipients were associated with decreased relapse risk (HR, 0.52; 95% CI, 0.35-0.78; P = .002; and HR, 0.32; 95% CI, 0.14-0.72; P = .006; respectively) and G2 (HR 1.63, 95% CI, 1.15-2.29; P = .005) with increased relapse risk in C1-homozygous recipients, compared with other patients with the same ligand. However, we could not validate these findings in an external data set of 796 AML transplants from the German transplantation registry. Neither a systematic evaluation of known HLA-KIR interactions nor an empiric assessment of prevalent KIR genotypes demonstrated clinically actionable associations; therefore, these data do not support these KIR-driven strategies for MUD selection in AML.

The Impact of KIR-HLA Interplay in T-LGL Leukemia

T-Cell large granulocyte lymphocyte leukemia (T-LGLL), characterized by clonal expansions of cytotoxic T cells (CTLs), arises from polyclonal lymphoproliferative responses. Various triggers have been stipulated, ranging from autoimmune, viral or tumor antigens. However, one could also posit that an inherent propensity to overshooting CTL responses contributing to the initial clonal expansions or affecting normal termination of immune response might be at play. For instance, specific HLA risk alleles may be overrepresented or even diagnostic for certain autoimmune conditions. In contrast to the risk allele theory, which enriches haplotypes within the patient population, the concept of HLA evolutionary diversity (HED) posits that the structural divergence between alleles widen the recognition spectrum. HLA alleles also serve as ligands for killer immunoglobulin-like receptors (KIRs). Indeed, the KIR gene family, consisting of 15 genes and 2 pseudogenes, is responsible for regulating the activity of NK and a subset of T cells and thus may play a role in T-LGLL. The main KIR genes are classified as either activating or inhibiting, based on their ability to up or downregulate NK and T cells. As the KIR genes and their HLA ligands genetically assort independently of each other, KIR-HLA “mismatches” are possible. We analyzed HLA alleles in 91 T-LGLL patients vs 130 controls and investigated whether specific presentations of T-LGLL, namely neutropenia and anemia enrich for certain HLA alleles. We found that HLA-A*02 was more frequent in cases with neutropenia (59 vs 47%, p=0.045) and HLA-A*68 in patients with anemia (17 vs 8%, p=0.02) as compared to controls. Interestingly, HLA-A*02/HLA-C*05 was observed typically in STAT3 MT vs WT cases (22 vs 11%). Using the HLA calls, we calculated the HED for HLA class 1/2, but no increased HED could be asserted (consistent with the presence of risk alleles as described above). We then explored whether specific KIR-HLA interactions enhanced an individual's risk for clonal CTL expansion and subsequent T-LGLL occurrence. As KIR gene frequencies can vary based on ethnicity, controls were chosen to match T-LGLL cohort (European ancestry). We applied T1K as a computational method for KIR genotyping and validated the calls by the 4 framework KIR genes (always present): 3DL2, 3DL3, 3DP1, and 2DL4. Along with the framework KIRs, there were no differences in the frequency of KIR genotype between T-LGLL and ethnicity matched controls. However, we found that the frequency of individuals with the 3DL2 gene and either HLA A*03 or HLA A*11 was significantly higher in the T-LGLL compared to controls (26 vs 8%, p = .006). Moreover, we found a higher frequency of 2DL3 combined with HLA C2/C2, (31 vs 14%, p=0.05) in T-LGLL patients with neutropenia. As this KIR-ligand (KIRL) configuration is a mismatch, the normally inhibiting receptor will have no effect on cell activity. Stratified by the presence of anemia, the LGLL cohort had instead a significantly higher proportion of 2DS2 co-expressed with the HLA C1 allele (75.68 vs 35.39%, p=.001), indicating activating genotype. We also found that the number of cases with the co-occurrence of the 3DL2 receptor and absent HLA A*03/A*11 ligands paired with 2DL3 and the HLA C1/C1 allotype was higher in STAT3 MT compared to WT (39.1 vs 16.7% p=0.06). Finally, we attempted to estimate and compare the net KIR activity of T-LGLL patients and controls by creating a formula in which, for each individual, we subtracted the sum of the number of KIR/KIRL inhibitory matches from that of activating KIR/KIRL matches. Mismatches were also included either as activating or inhibiting depending on the KIR/KIRL configuration. Using this formula, we found that the net KIR average value for the control cohort was 0.85 compared to 0.69 for the T-LGLL cohort, indicating that the control cohort had slightly more activating KIR-ligand configurations. Even when mismatches were treated as neutral, the preexcitation balance was not changed in T-LGLL. These findings suggest that other factors may contribute to the baseline preexcitation levels of clonal CTLs in this disease. In conclusion, our study of HLA and KIR-HLA interactions shows modest impact of KIR and KIR-ligand genotype on the propensity for overshooting clonal CTL proliferation and points towards other possible mechanisms such as hypersecretion of high affinity IL6R or other stimulatory factors.

Association of killer cell immunoglobulin-like receptors and their cognate HLA class I ligands with susceptibility to acute myeloid leukemia in Iranian patients

Acute myeloid leukemia (AML) is one of the most prevalent leukemia in adults. Among the various NK receptors, killer immunoglobulin-like receptors (KIRs) carry out indispensable roles in NK cell development and function through engaging with class I human leukocyte antigens (HLA-I) as their ligands. Besides divergent KIR and HLA loci, KIR/HLA-I combinations have a significant effect on NK cell response. In this case–control study, we aimed to verify the association of KIR/HLA-I combinations with susceptibility to AML in the Southwestern Iranian population. KIR and HLA genotyping was performed with PCR-SSP by some novel primers for 181 patients with AML and 181 healthy controls. According to our results, the frequencies of KIR3DS1 (p = 0.0001, OR = 2.32, 95% CI 1.51–3.58), KIR2DS4fl (p = 0.02, OR = 1.53, 95% CI 1.05–2.21), CxT4 genotypes (p = 0.03, OR = 2.0, 95% CI 1.05–3.82), and T4 gene cluster (p = 0.01, OR = 1.99, 95% CI 1.17–3.41) were significantly higher in patients than controls, while C1/C2 genotype (p = 0.00002, OR = 0.39, 95% CI 0.25–0.61), HLA-A Bw4 (p = 0.02, OR = 0.6, 95% CI 0.38–0.94), and HLA-A*11 (p = 0.03, OR = 0.57, 95% CI 0.34–0.95) alleles were more frequent in controls. In addition, inhibitory (i)KIR/HLA-I combinations analysis revealed higher frequencies of KIR2DL1( +)/HLA-C2( +), KIR2DL2/3( +)/HLA-C1( +), KIR3DL1( +)/HLA-A Bw4( +), and KIR3DL2( +)/HLA-A*03/11( +) in the control group (p = 0.002, OR = 0.49, 95% CI 0.3–0.78; p = 0.04, OR = 0.62, 95% CI 0.39–0.99; p = 0.04, OR = 0.63, 95% CI 0.4–0.99; and p = 0.03, OR = 0.62, 95% CI 0.4–0.95, respectively). Overall, the number of iKIR/HLA-I combinations was more in the control group. Moreover, KIR3DS1( +)/HLA-B Bw4Ile80( +) and the sum of HLA-B Bw4/A Bw4 combined with KIR3DS1 as activating KIR/HLA-I combinations were more frequent among patients than controls (p = 0.01, OR = 1.99, 95% CI 1.14–3.49 and p = 0.005, OR = 1.97, 95% CI 1.22–3.19, respectively). In conclusion, our results postulate that inhibitory combinations play a protective role against AML by developing potent NK cells during education. It is noteworthy that KIR/HLA-I combination studies can be applicable in donor selection for allogeneic NK cell therapy in hematological malignancies.

Large case-control study indicates no association of KIR genotype and risk of developing acute myeloid leukemia.

Immunogenetic association studies may give rise to new hypotheses on the immune surveillance of cancer. We hypothesized that certain combinations of killer immunoglobulin-like receptor (KIR) and HLA genotypes may enhance natural killer (NK) cell immunity against nascent acute myeloid leukemia (AML) and, thereby, lead to a skewed genotype distribution among patients. For this purpose, we analyzed KIR and HLA genotypes of 1767 German patients with AML and compared the results with that of the data of 51 890 German volunteers who had registered with German bone marrow donor file (DKMS). Patient samples were retrieved from the Collaborative Biobank and the biorepository of the Study Alliance Leukemia. All samples were genotyped with high-resolution amplicon-based next-generation sequencing. Because of the large number of controls, this study was very sensitive to detect the impact of KIR genotype. Knowledge on KIRs and their cognate HLA ligands allowed for testing of several hypotheses of NK cell-mediated endogenous leukemia surveillance. We did not find significant differences between the 2 cohorts in regard to the presence or absence of single KIR genes. When grouped based on telomeric or centromeric gene content, the major haplotypes A/A, A/B, and B/B were equally distributed among patients and control subjects. Using information on KIRs and their HLA ligands, we further tested receptor-ligand models and summation models without revealing markedly significant differences between patients and controls, albeit we observed a trend pointing at a minor protective effect of a low number of inhibitory KIR/KIR-ligand pairs. The results suggest that the KIR/KIR-ligand genotype has no effect on the susceptibility for the development of de novo AML.

P-498 Towards precision medicine: maternal genetic profiles as determinant of adequate decidualization and reproductive success; a prospective study in oocyte donation cycles.

Abstract Study question To determine the impact of genes related to decidualization and immune cell activation as predictors for reproductive success. Summary answer Women with unfavorable genetic profiles for Human Leukocyte Antigen (HLA)-F and Killer Immunoglobulin-like Receptor (KIR) genes showed decreased pregnancy rates. What is known already KIR genes encode receptors present on uterine Natural Killer (uNK) cells that are essential during placentation and early gestation. It has been established that women of KIR AA genotype show lower reproductive success than KIR BX, due to the lack of activation of these immune cells. Additionally, another gene, HLA-F, expressed in endometrial cells, also seems to be involved in the recruitment and supplementary activation of uNKs. Certain alleles of this gene appear to decrease its expression in the uterus and hinder the processes in which it is involved during the decidualization process, decreasing implantation rates and increasing time-to-pregnancy. Study design, size, duration We conducted a prospective cohort study with 75 patients, with no history of recurrent miscarriage or implantation failure, who attended our institution to undergo assisted reproduction treatments (ART) with donated oocytes, between January 2021 and December 2022. By using the egg donation model, we were able to investigate the endometrial cell population avoiding the oocyte factor bias Participants/materials, setting, methods All patients included were under 45 years of age (40.5 ± 2.9 years), had a normal BMI (21.6 ± 1.9 kg/m2) and had not been diagnosed with uterine pathologies or immunological conditions. All recipients were genotyped for the KIR and HLA-F genes and pregnancy, miscarriage (MR) and live birth rates (LBR) obtained in their egg donation cycles were recorded. Fisher's test was used to determine statistically significant differences (p &amp;lt; 0.05) between groups Main results and the role of chance Patients were divided into two groups according to their KIR genotype: 29 KIR AA (inhibitor) and 46 KIR BX (activator). In each group, allelic load was determined for the three HLA-F regions studied (RS1362126, RS2523405 and RS2523393). For all these regions, patients with favorable HLA-F alleles and KIR BX genotype had higher pregnancy rates than KIR AA patients with unfavorable HLA-F alleles (85.42 % vs 54.17% for RS1362126 region, OR = 4.956, p = 0.008; 83.33% vs 58.62% for RS2523405 region, OR = 3.529, p = 0.0295; 82.5% vs 57.69% for RS2523393 region, OR = 3.457, p = 0.0462). No significant differences were obtained for MR or LBR. This demonstrates the involvement of KIR and HLA-F in embryo implantation and that the coincidence of unfavorable alleles for both decreases reproductive success considerably, probably due to impaired decidualization and lack of uNK recruitment and activation. No significant differences were obtained for the rest of the genetic profiles, except when comparing pregnancy rate of KIR AA women with favorable HLA-F alleles to KIR AA women with unfavorable HLA-F alleles for the RS1362126 region (79.41% vs 54.17%, OR = 3.264, p = 0.04). The latter finding suggests that the lack of uNK cell activation, so evident in KIR AA patients, could be compensated by expressing favorable HLA-F alleles. Limitations, reasons for caution The low sample size of this study does not allow us to compare groups taking into account other genes, such as embryonic HLA-C, which also, and in combination with KIR genotypes, has been shown to play a role in proper implantation and placentation. Wider implications of the findings uNKs activation is required to promote embryo implantation. Combination of unfavorable genetic profiles involved in these immune mechanism leads to lower pregnancy rate. The study of these genetic profiles can help to predict reproductive success, thus identifying patients susceptible to receive immunological treatment before embryo transfer, possibly decreasing their time-to-pregnancy. Trial registration number Not applicable

A Novel Hypothesis: Certain KIR/Cognate Ligand Containing Genotypes Differ in Frequency Among Patients With Myeloma and Have an Effect on Age of Disease Onset.

Natural killer (NK) cells are known to have cytotoxic effects mediated through killer immunoglobulin-like receptors (KIRs) and their cognate ligands. Role of KIRs in myeloma is yet unresolved. KIR genotypes and ligands of 204 newly diagnosed MM patients are compared with 424 healthy subjects. Statistical analysis included t-test, chi-square and binary logistic regression. KIR ligands were significantly more (C2C2: 27.5% vs 15.1%; OR 2.128; 95% CI, 1.417-3.196; P < .001) or less (C1C2: 40.2% vs 51.9%; OR 0.623; 95% CI, 0.444-0.874; P=.006) frequent among MM. Co-occurrence of genotype AA with C2C2 was also higher in frequency among MM (OR 2.509; 95% CI, 1.171-5.378; P=.015) likewise cAB1 with C1C2 was less frequent (OR 0.553; 95% CI, 0.333-0.919; P=.021). Genotypes AA with C1C1, cAB1 with C1C2 or C1C2 alone were associated with a delay (median age: 61 [48-73]; P=.044; 62 [31-81]; P=.030 or 59 [31-85]; P=.028), but AA with C2C2 with an earlier age of onset (48 [29-77]; P=.042). In multivariate analysis including R-ISS, light chain, KIR genotype/ligands; ligand C1C2 (P=.02) and genotype AA-C1C1 (P=.037) were independently associated with age of onset ≥60. C1C2 and C2C2 alone or in combination with KIR genotype (cAB1 and AA, respectively), is observed in less or higher frequency among MM cases and associated with delayed/earlier age of onset, respectively. Genotype AA-C1C1 although in similar frequency between patients and healthy subjects, is also associated with delay. To our knowledge, this is the first study demonstrating an association between KIR and MM onset age, independent from R-ISS or light chain type.

Probability of allogeneic hematopoietic stem cell transplant failure depending on the recipient's killer immunoglobulin-like receptor genotype

Introduction. Natural killers are the "first line" of antitumor and antiviral protection in the early stages after аllogeneic hematopoietic stem cell transplantation. Quantitative characteristics reach normal values already in the first month after the infusion of blood stem cells to the recipient. Self-tolerance of natural killers is achieved due to many receptors on their surface, but killer immunoglobulin-like receptors play a key role. Their role is to recognize "self" cells and block signals aimed at destroying their own cells. Knowledge of the functional activity of natural killers urged to studying the impact of mismatches between the inhibitory receptor gene and the ligand on the development of allogeneic hematopoietic stem cell transplant failure.The aim of research was to study the probability of the graft failure development in allogeneic hematopoietic stem cell transplantation depending on the recipient's killer immunoglobulin-like receptor genotype.Material and methods. Genotyping of killer-cell immunoglobulin-like receptors in 66 recipients of blood stem cells by the polymerase chain reaction method was performed in the study. Using an online calculator, receptors were classified as "best", "better" and "neutral" depending on the genotype. The end point of the assessment was the development of graft failure in the presence of different genotypes of immunoglobulin-like receptors in the recipient.Results. According to the data obtained, the presence of the “best” and "better" killer-cell immunoglobulin-like receptor genotype in the recipient significantly increased the risks of developing various forms of graft failure.Conclusion. The presence of the KIR2DL3 genotype in a recipient of hematopoietic stem cells significantly (by 3 times) reduces the likelihood of primary graft failure. This result is of great prognostic significance, although at present no ways of influencing it have been developed. The presence of the “best” killer immunoglobulin-like receptors genotype in the recipient increases the likelihood of developing graft failure by more than 3 times compared to the best and neutral genotype (44.4% vs. 13.4%).

Clinical impact of KIR haplotypes in 10/10 HLA-matched unrelated donor-recipient pairs undergoing allogeneic hematopoietic stem cell transplantation

To evaluate the impact of killer immunoglobulin-like receptor (KIR) genotyping in allogeneic hematopoietic stem cell transplantation for myeloid disorders at our institution, retrospective KIR genotyping was performed on 77 patients and their 10/10 matched unrelated donors. In a multivariate model including donor age, HLA-DPB1 permissiveness, and presence of donor KIR B/x, an association with overall survival was observed (p = .047). Within the model, increasing donor age increased risk (RR 1.03 [1.00–1.06]/year, p = .046), while donor KIR and HLA-DPB1 permissiveness were not associated with risk (RR 0.51 [0.26–1.03] and RR 0.68 [0.34–1.36]). Grouping recipients by conditioning regimen or limiting the analysis to recipients of peripheral blood stem cells, no association between donor KIR and survival or relapse was identified. No significant associations were observed between overall survival, relapse, grade III-IV acute, or chronic graft versus host disease and presence of KIR B (B/x), quantity of donor KIR B haplotype motifs, or centromeric KIR type (all p > .05).

Killer-Cell Immunoglobulin-like Receptor Diversity in an Admixed South American Population.

Natural Killer (NK) cells are innate immune cells that mediate antiviral and antitumor responses. NK cell activation and induction of effector functions are tightly regulated by the integration of activating and inhibitory receptors such as killer immunoglobulin-like receptors (KIR). KIR genes are characterized by a high degree of diversity due to presence or absence, gene copy number and allelic polymorphism. The aim of this study was to establish the distribution of KIR genes and genotypes, to infer the most common haplotypes in an admixed Colombian population and to compare these KIR gene frequencies with some Central and South American populations and worldwide. A total of 161 individuals from Medellin, Colombia were included in the study. Genomic DNA was used for KIR and HLA genotyping. We analyzed only KIR gene-content (presence or absence) based on PCR-SSO. The KIR genotype, most common haplotypes and combinations of KIR and HLA ligands frequencies were estimated according to the presence or absence of KIR and HLA genes. Dendrograms, principal component (PC) analysis and Heatmap analysis based on genetic distance were constructed to compare KIR gene frequencies among Central and South American, worldwide and Amerindian populations. The 16 KIR genes analyzed were distributed in 37 different genotypes and the 7 most frequent KIR inferred haplotypes. Importantly, we found three new genotypes not previously reported in any other ethnic group. Our genetic distance, PC and Heatmap analysis revealed marked differences in the distribution of KIR gene frequencies in the Medellin population compared to worldwide populations. These differences occurred mainly in the activating KIR isoforms, which are more frequent in our population, particularly KIR3DS1. Finally, we observed unique structural patterns of genotypes, which evidences the potential diversity and variability of this gene family in our population, and the need for exhaustive genetic studies to expand our understanding of the KIR gene complex in Colombian populations.

KIR-HLA Interactions Lack Clinical Utility in Matched Unrelated Donor Transplantation for AML: An Analysis of the CIBMTR and DRST Registries

KIR-HLA Interactions Lack Clinical Utility in Matched Unrelated Donor Transplantation for AML: An Analysis of the CIBMTR and DRST Registries

59 Associations between KIR/KIR-ligand genotypes and clinical outcome for patients with advanced solid tumors receiving BEMPEG plus nivolumab combination therapy in the PIVOT-02 trial

BackgroundHigh-dose IL2 therapy has shown clinical benefit in metastatic melanoma and renal cell carcinoma, however only in a select subset of patients. Bempegaldesleukin (BEMPEG), a novel CD122-preferential IL2 pathway agonist, preferentially binds the heterodimeric IL2βγR predominantly expressed on NK and CD8 T cells and minimizes binding to the IL2αR expressed on immunosuppressive Tregs. BEMPEG monotherapy in Phase I clinical trials induced proliferation and activation of NK cells in the blood and tumor microenvironment.1 NK activation is dependent on the balance of inhibitory and excitatory signals transmitted by NK receptors, including Fc-gamma receptors (FCγRs) and killer immunoglobulin-like receptors (KIRs) along with their KIR-ligands. The repertoire of KIRs/KIR-ligands an individual inherits and the single-nucleotide polymorphisms (SNPs) of FCγRs can influence NK cell function and affect responses to immunotherapies.2–4 Thus, we sought to investigate whether distinct immunogenotypes are associated with clinical response of patients enrolled in PIVOT-02, a Phase II study of BEMPEG plus nivolumab.Methods200 patients with advanced solid tumors enrolled in PIVOT-02 who were not previously treated with immunotherapy (immunotherapy-naïve, ‘IO’) had DNA available for genotyping and clinical data for correlative analyses. All patients received 0.006mg/kg BEMPEG plus 360mg nivolumab every 3 weeks.5 KIR/KIR-ligand gene status was assessed by real-time SYBR green PCR melt-curve analyses and PCR-SSP. FCγR SNP status was determined using Taqman primers/probes for FCγR2A, and FCγR3A and FCγR2C were determined using RNaseH primers/probes.6 Clinical outcome parameters included objective response rate (ORR), progression-free survival (PFS), and tumor shrinkage (TS, defined as best objective response &lt;0% by RECIST). Preliminary statistical analyses were done using a chi-square test, log-rank test, and Wilcoxon rank-sum test, respectively.ResultsKIR2DL2+/C1+ IO patients observed significantly greater TS (p=0.015) and a trend towards improved ORR (p=0.060) and increased PFS (p=0.058) compared to IO patients who were not KIR2DL2+/C1+. IO patients who were KIR2DL2+/C1+/KIR3DL1+/Bw4+ showed significantly improved ORR (p=0.014) and greater TS (p=0.044) compared to IO patients who were not KIR2DL2+/C1+/KIR3DL1+/Bw4+, with no significance found for PFS in these patients. FCγR polymorphisms did not influence ORR/PFS/TS.ConclusionsNK cells may play an important role in the anti-tumor efficacy of BEMPEG plus nivolumab, and NK activation is influenced by certain immunogenotypes. Similar to previous findings,2–4 the status of KIR2DL2+/C1+ and KIR2DL2+/C1+/KIR3DL1+/Bw4+ was associated with the response to BEMPEG plus nivolumab treatment in the PIVOT-02 trial. The potential to utilize these genotypes in clinical decision-making for immunotherapy treatment requires further investigation.Trial RegistrationClinicalTrialsgov NCT02983045ReferencesBentebibel S-E, et al. A first-in-human study and biomarker analysis of NKTR-214, a Novel IL2βγ-Biased cytokine, in patients with advanced or metastatic solid tumors. Cancer Discov 2019;9:711–21.Erbe AK, et al. Follicular lymphoma patients with KIR2DL2 and KIR3DL1 and their ligands (HLA-C1 and HLA-Bw4) show improved outcome when receiving rituximab. J Immunother Cancer 2019;7(1):70.Erbe AK, et al. Neuroblastoma patients’ KIR and KIR-Ligand genotypes influence clinical outcome for dinutuximab-based immunotherapy: A report from the Children’s Oncology Group. Clin Cancer Res 2018;24(1):189–96.Erbe AK, et al. FCGR polymorphisms influence response to IL2 in metastatic renal cell carcinoma. Clin Cancer Res 2017;23(9):2159–68.Diab A, et al. Bempegaldesleukin (NKTR-214) plus nivolumab in patients with advanced solid tumors: Phase I dose-escalation study of safety, efficacy, and immune activation (PIVOT-02). Cancer Discov 2020;10:1–16.Erbe AK, et al. Genotyping single nucleotide polymorphisms and copy number variability of the FCGRs expressed on NK cells. Methods Mol Biol 2016;1441:43–56.Ethics ApprovalThe study was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. The Protocol (online only) was approved by independent ethics committees and the relevant institutional review board at each site. All patients provided written informed consent.

A model integrating Killer Immunoglobulin-like Receptor (KIR) haplotypes for risk prediction of COVID-19 clinical disease severity.

Associations between inherited Killer Immunoglobulin-like Receptor (KIR) genotypes and the severity of multiple RNA virus infections have been reported. This prospective study was initiated to investigate if such an association exists for COVID-19. In this cohort study performed at Ankara University, 132 COVID-19 patients (56 asymptomatic, 51 mild-intermediate, and 25 patients with severe disease) were genotyped for KIR and ligands. Ankara University Donor Registry (n:449) KIR data was used for comparison. Clinical parameters (age, gender, comorbidities, blood group antigens, inflammation biomarkers) and KIR genotypes across cohorts of asymptomatic, mild-intermediate, or severe disease were compared to construct a risk prediction model based on multivariate binary logistic regression analysis with backward elimination method. Age, blood group, number of comorbidities, CRP, D-dimer, and telomeric and centromeric KIR genotypes (tAA, tAB1, and cAB1) along with their cognate ligands were found to differ between cohorts. Two prediction models were constructed; both included age, number of comorbidities, and blood group. Inclusion of the KIR genotypes in the second prediction model exp (-3.52 + 1.56 age group - 2.74 blood group (type A vs others) + 1.26 number of comorbidities - 2.46 tAB1 with ligand + 3.17 tAA with ligand) increased the predictive performance with a 92.9% correct classification for asymptomatic and 76% for severe cases (AUC: 0.93; P < 0.0001, 95% CI 0.88, 0.99). This novel risk model, consisting of KIR genotypes with their cognate ligands, and clinical parameters but excluding earlier published inflammation-related biomarkers allow for the prediction of the severity of COVID-19 infection prior to the onset of infection. This study is listed in the National COVID-19 clinical research studies database.Graphical abstract

Killer immunoglobulin-like receptor 2DS5 is associated with recovery from coronavirus disease 2019

BackgroundDespite numerous advances in the identification of risk factors for the development of severe coronavirus disease 2019 (COVID-19), factors that promote recovery from COVID-19 remain unknown. Natural killer (NK) cells provide innate immune defense against viral infections and are known to be activated during moderate and severe COVID-19. Killer immunoglobulin-like receptors (KIR) mediate NK cell cytotoxicity through recognition of an altered MHC-I expression on infected target cells. However, the influence of KIR genotype on outcome of patients with COVID-19 has not been investigated so far. We retrospectively analyzed the outcome associations of NK cell count and KIR genotype of patients with COVID-19 related severe ARDS treated on our tertiary intensive care unit (ICU) between February and June 2020 and validated our findings in an independent validation cohort of patients with moderate COVID-19 admitted to our tertiary medical center.ResultsMedian age of all patients in the discovery cohort (n = 16) was 61 years (range 50–71 years). All patients received invasive mechanical ventilation; 11 patients (68%) required extracorporeal membrane oxygenation (ECMO). Patients who recovered from COVID-19 had significantly higher median NK cell counts during the whole observational period compared to patients who died (121 cells/µL, range 16–602 cells/µL vs 81 cells/µL, range 6–227 cells/µL, p-value = 0.01). KIR2DS5 positivity was significantly associated with shorter time to recovery (21.6 ± 2.8 days vs. 44.6 ± 2.2 days, p-value = 0.01). KIR2DS5 positivity was significantly associated with freedom from transfer to ICU (0% vs 9%, p-value = 0.04) in the validation cohort which consisted of 65 patients with moderate COVID-19.ConclusionNK cells and KIR genotype might have an impact on recovery from COVID-19.

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.

DONOR KIR3DL1/RECEPTOR HLA-BW4-80I COMBINATION REDUCES ACUTE LEUKEMIA RELAPSE AFTER UMBILICAL CORD BLOOD TRANSPLANTATION WITHOUT IN VITRO T-CELL DEPLETION

BackgroundDonor natural killer (NK) cell alloreactivity in umbilical cord bone marrow transplantation (UCBT) can lead to leukemic relapse. However, NK cell function is calibrated by interaction with human leukocyte antigens (HLAs). This study aimed to investigate graft-resistant leukemia after transplantation and compared specific genotypes of killer immunoglobulin-like receptors (KIRs) in donors and human leukocyte antigen ligands in patients.MethodsWe retrospectively analyzed 232 patients with acute leukemia from a single center. Patients had undergone UCBT with myeloablative conditioning and without anti-thymocyte globulin. We identified the KIR genotypes of cord blood donors using polymerase chain reaction with sequence-specific primers. All of the donors contained KIR3DL1.ResultsThe patients were divided into three groups according to the HLA-B locus. The donor KIR3DL1 and recipient HLA-Bw4-80I combination was predictive of being highly educated and was associated with a lower relapse (P=0.006) and better overall survival (probability of relapse=0.13, P < 0.001) than the uneducated group. We found no significant increase in the incidence of acute or chronic graft-versus-host disease.ConclusionsOur data suggest that the donor KIR3DL1/receptor and HLA-Bw4-80I combination in UCBT results in stronger graft-versus-leukemia effects and improved outcomes in patients with acute leukemia.

Donor KIR genotype has a major influence on the rate of cytomegalovirus reactivation following T-cell replete stem cell transplantation

AbstractReactivation of cytomegalovirus (CMV) is a common complication following allogeneic stem cell transplantation. Genetic determinants in the host and donor that may influence the rate of reactivation are currently unknown. Viral replication is controlled by T cells and natural killer (NK) cells and these share expression of killer immunoglobulin-like receptors (KIRs). We analyzed whether activatory KIRs carried by the donor influenced the subsequent rate of CMV reactivation in the patient. In transplantations involving siblings where both donor and recipient were CMV seropositive, donors with more than one activating KIR gene were associated with a 65% reduction in CMV reactivation. Multivariate analysis confirmed a significantly reduced risk of CMV reactivation in sibling transplantations where the donor had more than one activating KIR. Reduced-intensity transplantation and graft-versus-host disease grade 2 or higher were associated with an increased risk of CMV reactivation. This observation indicates that activating KIRs play an important role in the cellular control of CMV reactivation.

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.

Hematopoietic Cell Transplantation Donor Selection Reimagined: KIR-KIR Ligand Interactions and a Formalized Donor Risk Index Effective at Predicting Survival

When selecting a human leukocyte antigen (HLA) matched unrelated donor (URD) for hematopoietic cell transplantation (HCT) it is generally accepted that donor age, sex, ABO blood group and viral serologic status should be considered. However, the inter-relationship among these variables is not well established and a consensus on how strongly to consider each variable has not been reached. Selection of the optimal donor gets more complicated as new donor recipient pair (DRP) selection parameters, including killer immunoglobulin-like receptors (KIR) haplotypes are included. In this study we seek to develop a logic-based method to reduce the inconsistencies in donor selection in the HLA matched HCT. Retrospective review of eligible adults with known KIR genotyping receiving HLA-A, B, C & DRB1 allelically matched URD HCT for hematologic malignancy. Donor recipient pairs were selected based on donor age, sex match, CMV sero-status match, and ABO compatibility when possible; KIR genotype was not considered in DRP selection. KIR-KIR ligand interactions were calculated for each DR pair and interaction unit values were ascribed as previously described and each DRP IM KIR score (eq. 1) was calculated. IM KIR Score = |iKIR| + |mKIRL| [1] Weights of each donor risk variable (age, sex, CMV & ABO match) contribution were ascribed using Cox regression methods and summed up to determine donor risk parameter. Donor risk parameters and reciprocal-IM-KIR were finally combined into a donor risk index. Ninety-eight DRP with known HLA & KIR genotyping were studied. A higher IM-KIR score describes a DRP with increased iKIR-KIR ligand interactions and missing KIR ligand interactions; which was associated with a favorable survival after HCT, HR of 0.44 (95%CI: 0.26 to 0.73; P=0.002). Univariate analysis of overall survival for donor age, sex match, ABO compatibility and CMV status were all statistically insignificant (p>0.05). Donor risk parameter was predictive of mortality with a hazard ratio (HR) of 2.76 (95% CI: 1.22-6.18, p=0.014). Combining the two into a donor risk index was predictive of survival with a HR of 2.38 (1.44-3.92, p=0.001). ROC-AUC comparison of survival for IM-KIR score and donor risk parameter showed AUCs of 0.63 and 0.67 respectively. Further, the combined donor risk index shows improved sensitivity and specificity over the donor risk parameter with AUCs of 0.72 and 0.67 respectively. A novel IM-KIR score independently predicts survival in HLA matched DRP, as does a formalized donor risk parameter which includes non-HLA donor characteristics. Moreover, their addition enhanced the specificity and sensitivity of predicting survival in these patients. If validated in a larger exploration and validation cohort this method of donor selection may improve the donor selection process, decreasing variability in clinical outcomes and improve overall survival.