Killer Immunoglobulin-like Receptors Expression Research Articles

P-447 Dynamic changes in uterine natural killer cell (uNK) function across menstrual phases: Implications for implantation

Abstract Study question Does the function of uNK cells change from the proliferative stage of the menstrual cycle to the secretory phase in readiness for implantation? Summary answer During the secretory phase, uNK cells demonstrate an enhanced ability to produce cytokines indicating their importance for implantation. What is known already The abundance of uNK cells during implantation, and their secretion of trophoblast chemoattractant factors, suggests their pivotal role in placentation. uNK cells express Killer Immunoglobulin-like Receptors (KIR), which interact with Human Leukocyte Antigen-C (HLA-C) on trophoblasts, inducing cytokine production that aids trophoblast migration. It has recently been discovered that uNK cells consist of three distinct subsets, with different propensities to produce cytokines. However, the cytokine production abilities of each subset, and how this changes over the menstrual cycle, remains unexplored. Study design, size, duration A prospective cross-sectional study was conducted to compare uNK cell function between the proliferative and secretory phases of the menstrual cycle. The proliferative phase was defined as before day 14 of the menstrual cycle, and the secretory phase after this, in those with a regular 28-32 day menstrual cycle. The current data analysed is from 6 proliferative phase samples and 10 secretory phase samples however recruitment is ongoing. Participants/materials, setting, methods Endometrial samples were collected with a Pipelle biopsy from healthy fertile women attending the sexual health clinic for contraception. In the laboratory, endometrial lymphocytes were isolated and cultured with Anti-CD107a PerCP-eFlour710 (100ul/ml), Brefeldin (3ug/ml) and Monensin (2uM/ml). Cells were then stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (2ul/ml) and incubated for 4 hours at 37 °C. Flow cytometry was employed to analyse the samples after staining with viability dye, cell surface antibodies, and intracellular markers. Main results and the role of chance In initial experiments, degranulation declined during the proliferative phase in both uNK2 and uNK3 compared to the other two phases of the menstrual cycle. There was a significant increase in the production of TNFa by uNK1 and IL-8 by uNK3 in the secretory phase compared to the proliferative phase. There were also increases in IFNg and GM-CSF production across all subsets although this did not reach significance (Statistical testing was done using Kruskal Wallis with a post-hoc Dunn test, p < 0.05). Current work explores the production of more recently identified trophoblast chemoattractants, including XCL1 and CSF1, which we would expect to increase in a similar manner. Limitations, reasons for caution Limitations include the cross-sectional nature of the study, limiting the ability to establish causation. However another study, tracking pregnancy outcomes in those with recurrent implantation failure aims to address this. Additionally, variations within menstrual phases may exist. The sample size is also a limitation which I am working to expand. Wider implications of the findings A comprehensive understanding of uNK throughout various menstrual phases holds profound implications for reproductive health. The heightened cytokine production observed during the secretory phase indicates their pivotal role in the process of implantation. This finding not only underscores their significance but also serves as a catalyst for further research. Trial registration number not applicable

Cattle killer immunoglobulin-like receptor expression on leukocyte subsets suggests functional divergence compared to humans

Cattle, sheep, and goats are the only species outside primates known to have an expanded and diversified family of killer immunoglobulin-like receptors (KIR). Primate KIR are expressed on the surface of NK and T cells and bind MHC-I to control activation. However, the surface expression, ligands and function of bovid KIR remain unknown. Cattle botaKIR2DL1 is the only functional KIR of the same DL-lineage as the expanded KIR in primates and we examined if leukocyte expression patterns were consistent with human. We raised a specific mouse anti-botaKIR2DL1 monoclonal antibody and assessed its utility in flow cytometry, ELISA, and western blot. Unlike primates, cattle DL-lineage KIR (botaKIR2DL1) is present on B cells and monocytes in addition to T cells and low-level expression on NK cells. Expression decreases after in vitro PBMC stimulation with IL-2. This suggests that botaKIR2DL1 has different functions, and potentially ligands, compared to primate KIR.

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.

Myelodysplastic Syndrome associated TET2 mutations affect NK cell function and genome methylation

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders, representing high risk of progression to acute myeloid leukaemia, and frequently associated to somatic mutations, notably in the epigenetic regulator TET2. Natural Killer (NK) cells play a role in the anti-leukemic immune response via their cytolytic activity. Here we show that patients with MDS clones harbouring mutations in the TET2 gene are characterised by phenotypic defects in their circulating NK cells. Remarkably, NK cells and MDS clones from the same patient share the TET2 genotype, and the NK cells are characterised by increased methylation of genomic DNA and reduced expression of Killer Immunoglobulin-like receptors (KIR), perforin, and TNF-α. In vitro inhibition of TET2 in NK cells of healthy donors reduces their cytotoxicity, supporting its critical role in NK cell function. Conversely, NK cells from patients treated with azacytidine (#NCT02985190; https://clinicaltrials.gov/) show increased KIR and cytolytic protein expression, and IFN-γ production. Altogether, our findings show that, in addition to their oncogenic consequences in the myeloid cell subsets, TET2 mutations contribute to repressing NK-cell function in MDS patients.

Epitope characterization of a monoclonal antibody that selectively recognizes KIR2DL1 allotypes.

Killer immunoglobulin‐like receptor (KIR) genes code for a family of inhibitory and activating receptors, finely tuning NK cell function. Numerous studies reported the relevance of KIR allelic polymorphism on KIR expression, ligand affinity, and strength in signal transduction. Although KIR variability, including gene copy number and allelic polymorphism, in combination with HLA class I polymorphism, impacts both KIR expression and NK cell education, only a precise phenotypic analysis can define the size of the different KIRpos NK cell subsets. In this context, reagents recognizing a limited number of KIRs is essential. In this study, we have characterized the specificity of an anti‐KIR mAb termed HP‐DM1. Testing its binding to HEK‐293T cells transfected with plasmids coding for different KIRs, we demonstrated that HP‐DM1 mAb exclusively reacts with KIR2DL1. Using site‐directed mutagenesis, we identified the four amino acids relevant for HP‐DM1 recognition: M44, S67, R68, and T70. HP‐DM1 mAb binds to a conformational epitope including M44, the residue crucial for HLA‐C K80 recognition by KIR2DL1. Based on the HP‐DM1 epitope characterization, we could extend its reactivity to all KIR2DL1 allotypes identified except for KIR2DL1*022 and, most likely, KIR2DL1*020, predicting that it does not recognize any other KIR with the only exception of KIR2DS1*013. Moreover, by identifying the residues relevant for HP‐DM1 binding, continuously updating of its reactivity will be facilitated.

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 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.

Dynamic Changes of Inhibitory Killer-Immunoglobulin-Like Receptors on NK Cells after Allogeneic Hematopoietic Stem Cell Transplantation: An Initial Study.

Killer-immunoglobulin-like receptors (KIRs) are critical natural killer (NK) cell regulators. The expression of KIRs is a dynamic process influenced by many factors. Their ligands—HLA(Human Leukocyte Antigen) class I molecules—are expressed on all nucleated cells that keep NK cells under control. In hematopoietic stem cell transplantation (HSCT), NK cells play an essential role in relapse protection. In the presented pilot study, we characterized the dynamic expression of inhibitory KIRS (iKIRs), which protect cells against untoward lysis, in donors and patients during the first three months after HSCT using flow cytometry. The expression of all iKIRs was highly variable and sometimes correlated with patients’ clinical presentation and therapy regiment. Cyclophosphamide (Cy) in the graft-versus-host disease (GvHD) prevention protocol downregulated KIR2DL1 to just 25% of the original donor value, and the FEAM (Fludarabine + Etoposid + Ara-C + Melphalan) conditioning protocol reduced KIR2DL3. In lymphoid neoplasms, there was a slightly increased KIR2DL3 expression compared to myeloid malignancies. Additionally, we showed that the ex vivo activation of NK cells did not alter the level of iKIRs. Our study shows the influence of pre- and post-transplantation protocols on iKIR expression on the surface of NK cells and the importance of monitoring their cell surface.

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.

Variability in Killler Immunglobulin like Receptor Gene Expression As a Periodic Function of Gene Position on Chromosome 19

Killer Immunoglobulin Like Receptors (KIR) are expressed on natural killer (NK) and T cell surface. KIR interactions with KIR-ligands have been implicated in outcomes of hematopoietic stem cell transplantation, placental implantation, autoimmune disease and viral infections. While these KIR interactions are clearly important our understanding of what governs their expression is lacking. The KIR Locus is made up of a highly polymorphic and homologous set of genes located on Chromosome 19q13.4 within the leukocyte receptor complex. Unique to the KIR Gene Cluster, KIR haplotypes not only vary allelically, but they also differ in the number of KIR genes present in different individuals, ranging from 7 to 12 genes. KIR haplotypes have been divided into 2 broad groups; Haplotypes A &amp; B, based on the number of activating and inhibitory KIR genes they possess; where haplotype A contains only one activating allele KIR2DS4 and haplotype B contains various combinations of activating alleles. Expression of KIR on the NK cell surface stochastic, with some KIR found more commonly then others. In this abstract we aim to explore the relationship of KIR gene expression and the organization of the KIR gene locus using available genomic and tissue expression data. KIR gene coordinates on chromosome 19 were collected using the UCSC Genome Browser GRCh38/hg38 gene assembly and the NCBI gene website gene assembly NC_000019.10. Initial KIR gene nucleotide position along chromosome 19 were obtained and converted to angular distance (A.D. in radians) from reference. This was done to account for the double helical nature of DNA molecules, using the following equation A.D. = 2px/10.4; where x is the initial KIR gene nucleotide coordinate. Haplotype A was utilized as an initial test case as gene expression data are available for the genes in this haplotype. GTEX Portal was used to collect mRNA expression for each of the haplotype A genes, KIR-2DL1, -2DL3, -2DL4, -2DP1, -2DS4, -3DL1, -3DL2, -3DL3 &amp; -3DP1, including total (median), splenic and whole blood expression in Reads/Kilobase of transcript/million mapped reads (RPKM). Gene expression was determined via RNA-seq of 53 tissues from 570 donors. KIR haplotype A is located along chromosome 19 between nucleotide position 55,236,713 and 55,370,584 which corresponded with an A.D. of 33,354,476 to 33,435,314 radians. When the KIR gene expression was aligned with the coordinates of the corresponding gene on the KIR locus a distinct periodic pattern of variation in expression levels was observed across the KIR genes comprising Haplotype A (Figure 1). This was true for total KIR expression from across various organs, as well as in blood and splenic tissue. This is borne out by expression data on several Haplotype A KIR genes reported in the literature (Figure 1, yellow curve). These observations suggesting that KIR gene expression may be a periodic function of the gene coordinates on the chromosome are analogous to the periodicity observed in T cell receptor VDJ recombination (Meier et al, BBMT 2019 25(5);868). This observation supports the idea that the spiral structure of the helical DNA molecules coiled around the histone molecules and their arrangement on chromosomes may have a fundamental influence on the expression of different genes, perhaps in conjunction with known epigenetic influence of DNA methylation and histone acetylation. This may also point to a role for the non-coding DNA in regulation of gene expression. We posit that the helical DNA chromosome structure may have a fundamental role in determining gene expression, as exemplified by the KIR and T cell receptor gene loci. This knowledge may allow an improved understanding of variability in global gene expression. Disclosures No relevant conflicts of interest to declare.

Family-based Association Study of Killer Cell Immunoglobulin-Like Receptor Genes with Leukemia

Abstract NK cell function is controlled by the cell expression of killer immunoglobulin-like receptors (KIRs) and their ligation with the corresponding HLA ligands. Various malignancies have been associated with certain KIRs surface cell expression and various KIR/HLA ligand combinations. Prior research using case/control study design demonstrates the role of KIR and KIR HLA ligands as genetic factor involved in tumor susceptibility. The objective of this study was to investigate the family-based association of KIRs, HLA class I ligands and KIR/ligand combinations with leukemia diagnosis in families having a leukemia diagnosed child. Sixty-seven families that met the index leukemia case criteria (acute lymphoblastic leukemia, ALL, n = 45; acute myeloid leukemia, AML, n = 13; chronic myeloid leukemia, CML, n = 9; first degree healthy relatives n = 159) were examined. Our study consisted of two phases. In Phase1 case-control study, we primarily compared patients to their healthy siblings to asses if a marker or genotype may be associated with leukemia, excluding the impact of the environment. Phase 2 consisted of a secondary family-based association study. KIR genotyping was performed by PCR-SSP method. KIR HLA ligands were defined by direct method using PCR-SSP method and/or indirect base on high resolution typing of HLA-A, -B, -C alleles. Results of phase 1 showed an increase in the frequency of KIR genotype (with a ratio = 0.57; higher frequency for inhibitory KIRs vs. activating KIRs) among leukemia patients compared to healthy siblings. Results of the phase 2 familial study observed an association between HLA-C1+/BBw4+/ABw4+ haplotype (a mediator of inhibitory signals) and leukemia. Also, we concluded that the absence of HLA-ABw4 alleles was related to leukemia development.

Certain Killer immunoglobulin like receptor (KIR)/ KIR HLA class I ligand genotypes influence NK antitumor activity in acute myelogenous leukemia but not in acute lymphoblastic leukemia. A case control leukemia association study

Objective:Natural killers (NK) cell function is mainly controlled by the expression of killer immunoglobulin-like receptors (KIRs) and their ligation with the corresponding ligands. The objective of this study was to investigate the putative association of KIRs, HLA class I ligands, and KIR/ligand combinations with rates of development of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).Materials and Methods:The KIR/HLA I genotypes of 82 patients with leukemia (ALL, n=52; AML, n=17; and CML, n=13) were determined by PCR-SSP method and compared with genotypes of healthy controls (n=126).Results:KIR genotype frequency differed significantly between myelogenous leukemia patients and healthy controls for KIR2DL5A (17.6% vs. 47.7%, p=0.02), KIR3DS1 (17.6% vs. 47.6%, p=0.02), and KIR2DS4*001 (36.6% vs. 20.2%, p=0.017). The incidence of homozygous HLA-BBw4 (31.0% vs. 12.5%, p=0.042) and HLA-Bw4Thr80 Thr80 (13.0% vs. 1.2%, p=0.01) was significantly elevated in myeloid leukemia patients compared to healthy controls. KIR/HLA class I ligand profile KIR3DS1(+)/L (-) was decreased and KIR3DL2(+)/HLA-A3/11(-) was increased among myeloid leukemia cases compared to controls.Conclusion:These data suggest that the activity of NK cells as determined by inherited KIR/HLA class I ligand polymorphisms influences the susceptibility to myelogenous leukemia, but not to lymphoblastic leukemia. Additionally, the KIR genotype characterized by the absence of the inhibitory KIR2DL2 and the activating KIR2DS2 and KIR2DS3 (ID2) was found at a lower frequency in patients compared to controls, which confirmed the need for complex analysis based on all possible KIR/HLA class I ligand polymorphism combinations.

Tuning Natural Killer Cell Anti-multiple Myeloma Reactivity by Targeting Inhibitory Signaling via KIR and NKG2A.

Natural killer (NK) cells are attractive candidates for allogeneic cell-based immunotherapy due to their potent antitumor effector function and good safety profile. NK cells express killer immunoglobulin-like receptors (KIRs) and the NKG2A receptor important for NK cells education as well as providing inhibitory signals upon encountering HLA-expressing target cells. Multiple myeloma (MM) is an example of a tumor expressing relatively high levels of HLA molecules. In this review, we discuss the functional relevance of inhibitory KIRs and NKG2A for NK cells anti-MM response and strategies to lower these inhibitory signaling to enhance clinical efficacy of allogeneic NK cells in MM.

Abstract LB-048: Impact of KIR/KIR ligand genotype for neuroblastoma patients in a Phase 3 COG immunotherapy trial

Abstract Introduction: High-risk neuroblastoma (NBL) patients (pts) enrolled in a COG Phase 3 clinical trial (ANBL0032) were randomized to isotretinoin (RA) alone or Immunotherapy: dinutuximab (anti-GD2 mAb) + IL2 + GMCSF + RA (Yu et al., NEJM, 2010). Dinutuximab acts via antibody-dependent cell-mediated cytotoxicity by innate immune cells, including NK cells. NK cells express Killer Immunoglobulin-like Receptors (KIRs); most of the inhibitory KIRs have KIR-ligands that belong to the HLA class I family. Specifically, KIR2DL1 is a receptor for HLA-C2, KIR2DL2 and KIR2DL3 are receptors for HLA-C1, and KIR3DL1 is a receptor for HLA-Bw4. Some prior studies of anti-GD2 mAb immunotherapies have shown associations with outcome based on the genotypes of these inhibitory KIR/KIR ligand relationships. We investigated whether certain KIR/KIR-ligand genotypes were associated with event-free survival (EFS) and overall survival (OS) in this trial. Methods: Of the 226 pts randomized, 174 pts had DNA allowing evaluation of genotype correlations with outcome (RA: n=86; Immunotherapy: n=88; &amp;gt;5yr follow-up if no event). We looked for associations of inhibitory KIRs with their respective KIR-ligands and clinical outcome. Log-rank tests and Cox proportional hazards regression models were used to compare EFS/OS by genotype group; adjustment was made for non-proportional hazards as needed using time-dependent covariates. Results: We found that certain, hypothesis-identified, inhibitory KIR/KIR-ligand combinations were associated with improved clinical outcome. Namely, pts that were both KIR2DL2+/HLA-C1+ and KIR3DL1+/HLA-Bw4+ had improved EFS and OS if treated with Immunotherapy (n=23) vs. RA (n=26) (5-yr EFS: 61% vs. 27%, p=0.02; 5-yr OS: 91% vs. 34%, p=0.007). Conversely, for pts that were not both KIR2DL2+/HLA-C1+ and KIR3DL1+/HLA-Bw4+, we found insufficient evidence to support an improvement in EFS or OS with Immunotherapy (n=65) vs. RA (n=60) (5-yr EFS: 57% vs. 53%, p=0.76; 5-yr OS: 68% vs. 68%, p=0.66). Conclusions: Our data suggest that KIR/KIR-ligand genotype may be predictive of benefit from Immunotherapy. The impact of immunotherapy appears different for pts that were both KIR2DL2+/HLA-C1+ and KIR3DL1+/HLA-Bw4+ vs. those that were not both KIR2DL2+/HLA-C1+ and KIR3DL1+/HLA-Bw4+. Validation of these KIR/KIR-ligand associations in similarly treated high-risk NBL patients would be required prior to proposing their prospective use in the aid of clinical treatment decisions. Further investigation of KIR/KIR-ligand genotypes may also clarify their role and the role of NK cells in the activity of this form of cancer immunotherapy. Citation Format: Amy K. Erbe, Wei Wang, Lakeesha Carmichael, KyungMann Kim, Patrick K. Reville, Wendy B. London, Jacquelyn A. Hank, Mitchell B. Diccianni, Arlene Naranjo, Michael Hogarty, Julie R. Park, Alice L. Yu, Paul M. Sondel. Impact of KIR/KIR ligand genotype for neuroblastoma patients in a Phase 3 COG immunotherapy trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-048. doi:10.1158/1538-7445.AM2017-LB-048

Modulation of T-bet and Eomes during Maturation of Peripheral Blood NK Cells Does Not Depend on Licensing/Educating KIR.

Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94negCD56dimNK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive.

Human NK cells: From surface receptors to clinical applications

Natural killer (NK) cells play a major role in innate defenses against pathogens, primarily viruses, and are also thought to be part of the immunosurveillance against tumors. They express an array of surface receptors that mediate NK cell function. The human leukocytes antigen (HLA) class I-specific inhibitory receptors allow NK cells to detect and kill cells that have lost or under-express HLA class I antigens, a typical feature of tumor or virally infected cells. However, NK cell activation and induction of cytolytic activity and cytokine production depends on another important checkpoint, namely the expression on target cells of ligands recognized by activating NK receptors. Despite their potent cytolytic activity, NK cells frequently fail to eliminate tumors. This is due to mechanisms of tumor escape, determined by the tumor cells themselves or by tumor-associated cells (i.e. the tumor microenvironment) via the release of soluble suppressive factors or the induction of inhibitory loops involving induction of regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells. The most important clinical application involving NK cells is the cure of high-risk leukemias in the haplo-identical hematopoietic stem cell transplant (HSCT) setting. NK cells originated from hematopoietic stem cells (HSC) of HLA-haploidentical donors may express Killer Immunoglobulin-like receptors (KIRs) that are mismatched with the HLA class I alleles of the recipient. This allows NK cells to kill leukemia blasts residual after the conditioning regimen, while sparing normal cells (that do not express ligands for activating NK receptors). More recent approaches based on the specific removal of TCR α/β+ T cells and of CD19+ B cells, allow the infusion, together with CD34+ HSC, of mature KIR+ NK cells and of TCR γ/δ+ T cells, both characterized by a potent anti-leukemia activity. This greatly reduces the time interval necessary to obtain alloreactive, KIR+ NK cells derived from donor HSC. Another promising approach is based on the use of anti-KIR blocking monoclonal antibodies (mAbs), rendering alloreactive any KIR+ NK cells.

KIR genotype distribution among patients with chronic lymphocytic leukemia: Is there a role for KIR 2DS4 and KIR 2DS5 genes?

IntroductionThe function of natural killer (NK) cells is regulated by different antigen receptors including killer immunoglobulin-like receptors (KIR). In addition to their important role in fighting infection, natural killer cells produce cytotoxicity against some cancer cells. Studies demonstrated that NK cells have a reduced function in chronic lymphocytic leukemia (CLL). AimThe aim of this study is to investigate KIR expression of NK cells in CLL patients to check for any association between KIR genotypes and this disease. MethodsKIR genotype was analyzed for 120 healthy Lebanese patients and 56 CLL patients using the KIR Genotyping SSP kit. ResultsAmong the 56 CLL patients, the AA, AB, and BB genotypes frequencies were, respectively, 38%, 46%, and 16% with an A:B ratio of 1.55:1. As for the controls, the AA, AB, and BB genotypes frequencies were, respectively, 39.17%, 50%, and 10.83% with an A:B ratio of 1.79:1. KIR 2DS4*001/002 and KIR 2DS5 were found to be significantly more prevalent among CLL patients as compared to controls. ConclusionThis is the first study that reports such an interesting prevalence of KIR genes in CLL necessitating further clinical and translational research pertaining to the pathophysiology of this disease.

Post-Transplant Donor Lymphocyte Infusion with Methotrexate Prophylaxis Is Associated with Low Incidence of Relapse after CD34-Selected, T Cell-Depleted Haploidentical Donor Stem Cell Transplantation Regardless of NK Alloreactivity

▪Background: NK alloreactivity based on KIR (Killer Immunoglobulin-like Receptor) expression by natural killer cells has been reported to decrease the risk of leukemia relapse after HLA-haploidentical stem cell transplantation (SCT). The use of donors with high KIR B-content score has been correlated with a decreased risk of relapse.Objective: We examined the outcome of leukemia relapse following CD34-positive selected T-cell depleted haploidentical donor peripheral blood SCT followed by donor lymphocyte infusion with methotrexate prophylaxis.Methods: Between 2009-2015, twenty patients underwent haploidentical donor CD34-selected, T cell-depleted peripheral blood SCT on an IRB-approved protocol. Nine patients had acute lymphoblastic leukemia (ALL), and eleven had myeloid leukemia (10 AML, 1 JMML). Among ALL patients, remission status was CR1 in 5, with either induction failure or MRD+ after consolidation, 3 were in CR2 and 1 was CR3. Four patients had T-cell disease. Among AML patients, 5 were in CR1, one had refractory disease and 5 were CR2 or beyond. Ligand-ligand NK alloreactivity based on HLA typing was available for all patients. Receptor-ligand NK alloreactivity based on immunophenotyping of KIR was available in 16 patients and KIR B-content score was available in twelve patients. Patients were considered to have receptor-ligand NK alloreactivity if immunophenotyping of the donor had >5 (cells/uL) expressing KIR2DL1 (recognizes C2 group) or KIR2DL1/2DL13 (recognizes C1 group). Patients with alloreactive KIR only recognizing BW4 were not considered alloreactive due to predicted weak alloreactivity. All patients received a uniform conditioning regimen including TBI 1200cGy, thiotepa 10mg/kg, fludarabine 200mg/m2, and rabbit-anti-thymocyte globulin (rATG) 6mg/kg, with no post-transplant immunosuppression. All patients received a planned donor lymphocyte infusion (DLI) 3-5 x 104 CD3+ cells/kg between days 30- 42 after transplant and received weekly IV methotrexate prophylaxis following DLI. The incidence of relapse and GVHD was evaluated.Results: Based on a ligand-ligand model of NK alloreactivity, only 4/20 patients had NK alloreactivity predicted. Seven patients expressed all 3 ligands and blocked all possible NK alloreactivity. Median follow-up was 306 days (range 45 days-5 years). Four of 20 patients relapsed (1/4 with predicted NK alloreactivity and 3/16 without). Median time to relapse was 110 days (range 124-455 days). NK alloreactivity based on receptor-ligand as defined above was present in 5/16 patients and relapse occurred in 2/5 with and 1/11 without. Donor KIR B-content was >=2 in 4 patients and 0-1 in 8 patients. Relapse occurred in 1/4 with 2 and 2/8 with 0-1. No donors had KIR B-content score of 3-4. The incidence of grade II-IV acute graft-versus-host disease after prophylactic DLI was 25%, with 2 grade II, 3 grade III and no grade IV GVHD. Three patients developed cGVHD, two mild and one severe by NIH consensus criteria.Conclusion: An approach using a DLI followed by methotrexate prophylaxis after a CD34-selected, T-cell depleted SCT for pediatric leukemia diminishes the effect of NK alloreactivity and KIR B-content. DisclosuresNo relevant conflicts of interest to declare.

Decreased expression of pSTAT, IRF-1 and DAP10 signalling molecules in peripheral blood lymphocytes of patients with metastatic melanoma

AimsAs numerous signalling molecules regulate effector functions of peripheral blood lymphocytes (PBLs) that have an important anti-tumour activity, the aim of this study was to analyse their level in patients...

KIR2DS5 in the presence of HLA-C C2 protects against endometriosis.

Endometriosis is defined as the presence of functional endometrial tissue outside the uterine cavity. Several hypotheses have attempted to explain the etiology and pathogenesis of endometriosis. Recently, it has been suggested that a defect of the natural killer (NK) activity in the recognition and lysis of endometrial cells is one of the crucial points in the development of this disease. Natural killer cells can express killer immunoglobulin-like receptors (KIR), which recognize class I human leukocyte antigens on target cells. We asked whether polymorphisms in KIR, HLA-C, and HLA-B genes are risk factors for endometriosis. We tested 153 women with endometriosis diagnosed on the basis of laparoscopic and histological examination, and 213 control healthy women, who gave birth to at least one child. The frequency of KIR genes in patients was similar to that in controls except for KIR2DS5, which exerted a protective effect only in HLA-C C2-positive individuals. Moreover, KIR2DS5-positive women with endometriosis had 13 times lower chance that the disease would occupy the peritoneum than KIR2DS5- and KIR2DS4del-negative ones (OR = 0.077, P = 0.0061). Similarly, KIR2DS4del-positive endometriotic persons had 11 times lower chance for peritoneal disease (OR = 0.094, P < 0.001). Negative linkage disequilibrium between KIR2DS5 and KIR2DS4del indicates that these genes are mutually exclusive. Our data suggest that KIR2DS5 may be associated with protection from endometriosis, whereas KIR2DS4del seems to be associated with higher disease stages, possibly by exclusion of protective KIR2DS5.Electronic supplementary materialThe online version of this article (doi:10.1007/s00251-015-0828-3) contains supplementary material, which is available to authorized users.