Inhibitory Killer Immunoglobulin-like Receptors Genes Research Articles

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.

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.

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.

Clinical Significance of KIR Genotype in Patients with Methotrexate-Associated Lymphoproliferative Disorders

Background: Methotrexate-associated lymphoproliferative disorders (MTX-LPD) develops in patients with rheumatoid arthritis (RA) or other autoimmune disorders during low-dose MTX treatment. MTX-LPD includes wide variety disease spectrum, ranging from polymorphic proliferation to aggressive lymphoma. Although etiology of MTX-LPD has not been fully understood, approximately half of the MTX-LPD cases showed association with EB virus (EBV), suggesting that MTX treatment causes reduced immune response to EBV-positive cells, and results in MTX-LPD development. Natural killer (NK) cells play important roles in eradicating tumor and virus-infected cells. NK cell function is modulated by multiple cell surface receptors, including Killer immunoglobulin-like receptor (KIR). There are multiple KIR genes (inhibitory or activated), which are various in number and/or composition among individuals, on chromosome 19q. Previous reports demonstrated that combination of KIR genes affects NK cell function, and is associated with the risk of development of certain types of cancers, viral infections and collagen disease. There is no report about the association of KIR genotype and MTX-LPD. We consider that NK cells play a significant role in suppression of MTX-LPD development. In this study, we focused on examining genotype KIR and KIR-ligand (HLA class I). Methods: We retrospectively analyzed 35 MTX-LPD cases diagnosed between 2009 and 2019. Genomic DNA was extracted from mononuclear cells that were isolated from the bone marrow or peripheral blood samples of patients with MTX-LPD. KIR genotypes were analyzed using the KIR genotyping sequence-specific primers kit. The variations of KIR content and haplotype and their relationship with progression to malignant lymphoma (ML) and response to chemotherapy were investigated. HLA was analyzed using PCR-Luminex assay. The frequency of each HLA allele and each combination was determined by referring to the data base of an HLA laboratory. Chi-squared (χ2) tests and Wilcoxon rank sum tests were used to test associations between the variables. Results: Among the 35 patients, 25 were diagnosed with ML and 10 with polymorphic LPD. Diffuse large B cell lymphoma (DLBCL) was most common type in ML (57.7%). Table 1 showed characteristics of patients and summary of the results. All patients underwent MTX treatment for RA. The median duration of MTX administration at the time of MTX-LPD diagnosis is 11.5 years (range=0.8-27.2), and median MTX dose was 10mg/week (range=4-17.6). The duration and dose of MTX had no effect between ML and polymorphic LPD. Twenty-three patients required chemotherapy, and 12 patients had tumor regression after stopping MTX treatment. Relative patient populations requiring chemotherapy in ML or polymorphic LPD were 85% or 11%, respectively (P=0.0001). EBV-positive patients tended to regress tumors with MTX discontinuation alone (P=0.16). In KIR genotype analysis, patterns of number and combination of the KIR genes are mainly classified as haplotype "A" containing multiple inhibitory KIR genes with a KIR 2DS4 (an activated KIR [aKIR]) and haplotype "B" (other than haplotype "A"). Patients were classified in haplotype A (13 cases, 37%) and haplotype B (22 cases, 63%), respectively. ML patients showed higher ratio in haplotype A (ML 46.2% vs LPD 11.1% P=0.045). There was no difference in number of aKIR or iKIR between ML and polymorphic LPD patients. In HLA Class I analyses, there was significant difference in frequencies of HLA-C haplotype between lymphoma and polymorphic LPD patients (P=0.026). Furthermore, HLA- C1 / C1 patients were more relapsed or refractory to chemotherapy than C1 / C2 patients (P = 0.17). Conclusion: This is the first report showing clinical significance of KIR genotypes in MTX-LPD. Patients with haplotype A, a suppressive haplotype, seems to be at high risk for developing lymphomas that require chemotherapy during MTX treatment. HLA- C1/C1 patients are more likely to develop lymphomas that respond poorly to treatment, suggesting that the activity of NK cells may be lower because ligands can match with KIRs that are more restricted than C1/C2. Considering the potential NK functions with KIR genotype would improve the understanding of the prognosis and lead to prevention for MTX-LPD. Disclosures Hagiwara: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees. Harigai:Bristol Myers Squibb Co: Other: personal fees, Research Funding; Eisai Co: Other: personal fees, Research Funding; Ayumi Pharmaceutical Co: Other: personal fees, Research Funding; AbbVie Japan GK,: Other: personal fees, Research Funding; Eli Lilly Japan K.K: Other: personal fees; Kissei Pharmaceutical Co.: Other: personal fees; Teijin Pharma Ltd: Other: personal fees, Research Funding; Mitsubishi Tanabe Pharma Co: Research Funding; Nippon Kayaku Co.: Research Funding; Pfizer Japan Inc.: Other: personal fees; Chugai Pharmaceutical Co., Ltd.: Other: personal fees; Japan College of Rheumatology: Other: personal fees; Boehringer Ingelheim Japan, Inc: Other: personal fees; GlaxoSmithKline K.K: Other: personal fees; Oxford Immuotec,: Other: personal fees. Tanaka:Bristol-Myers Squibb: Research Funding.

Variation of maternal KIR and fetal HLA-C genes in reproductive failure: too early for clinical intervention

A distinctive type of (uterine) natural killer (NK) cell is present in the uterine decidua during the period of placental formation. Uterine NK cells express members of the killer immunoglobulin-like receptor (KIR) family that bind to parental HLA-C molecules on the invading placental trophoblast cells. The maternal KIR genes and their fetal ligands are highly variable, so different KIR/HLA-C genetic combinations occur in each pregnancy. Some women only possess inhibitory KIR genes, whereas other women also express activating KIR genes. The overall signal that NK cells receive from paternal HLA-C on trophoblast depends on the ratio of activating and inhibitory KIR genes expressed by them. Therefore, NK cells provide a balance during placentation to ensure maternal survival and an adequately nourished fetus. Because inhibitory KIRs are found more frequently in women with defective placentation, e.g. pre-eclampsia, fetal growth restriction or recurrent spontaneous abortion, some fertility clinics suggest that women should be ‘tissue typed’ for their KIR genotypes. We explain why, presently, it is premature to introduce KIR and HLA-C typing to predict pregnancy outcome. In future, however, selecting for certain combinations of KIR and HLA-C variants in surrogacy, egg or sperm donation may prove useful to reduce disorders of pregnancy.

Killer Cell Immunoglobulin-Like Receptor Genes and their HLA-C Ligands in Hashimoto Thyroiditis in a Chinese Population

Natural killer (NK) cells serve as primary immune surveillance and are partially regulated by combinations of killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen-C (HLA-C) ligands. Alterations in NK cell activity have been associated with Hashimoto thyroiditis (HT). The aim of this study was to determine whether certain KIR/HLA-C genotype combinations play a role in HT pathogenesis. The present study enrolled 107 unrelated HT patients and 108 random healthy individuals in a case-control study. Blood was collected for DNA extraction; typing of KIR genes and HLA-C alleles was performed by polymerase chain reaction with sequence specific primers (PCR-SSP), followed by electrophoresis on agarose gels. Among a panel of KIR2D/HLA-C genotype combinations, the frequency of KIR2DS2/HLA-C1 was significantly increased in HT patients compared to controls (33.64% vs. 12.96%, P<.001). To further analyze the precise genotype, we investigated inhibitory or activating KIR/HLA-C gene pairs when their corresponding activating or inhibitory KIR genes were absent in the 2 groups. Only the frequency of KIR2DS2(-)2DL2/3(+)HLA-C1(+) was significantly decreased in HT patients compared to controls (48.60% vs. 70.37%, P = .001). Our data suggest that KIR2DS2/HLA-C1 may correlate with HT pathogenesis. On the contrary, the predominance of KIR2DL2/3/HLA-C1 in the absence of KIR2DS2 suggests a potential inhibitory role in HT pathogenesis. In conclusion, our findings may further elucidate the mechanisms underlying the pathogenesis of HT and other autoimmune diseases. HLA-C = human leukocyte antigen-C HT = Hashimoto thyroiditis KIR = killer immunoglobulin-like receptor NK = natural killer PCR = polymerase chain reaction.

KIR Gene Frequencies in Women with Infertility Problems

Introduction: Natural killer (NK) cells are the predominant lymphocyte population in the decidua. Being the most abundant leucocytes, the activity of NK cells is important in different immuno-pathological conditions, such as recurrent spontaneous abortions, infertility and problems in implantation. The NK cells recognize HLA class I molecules on trophoblasts trough killer immunoglobulin-like receptors (KIRs) found on their surface. The KIRs are classified as either activating or inhibitory, regarding the effect they produce on NK cells upon interaction with corresponding ligand. Since KIR genes exhibit extensive polymorphism and individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes, it is hypothesized that the KIR gene content might influence the pregnancy outcome. Aim: The aim of this pilot study is to analyze the frequency of different KIR genes in women with infertility problems, and compare them to healthy women. Material and Methods: Total of 122 healthy women (Control) and 25 women with reproductive problems (MISSC) participated in this study. After signing of written consent DNA was isolated from peripheral blood using phenol/chloroform method. The genotyping of 16 KIR genes was performed using commercially available kit from Dynal Biotech, (Pel-Freez Clinical Systems, Brown Deer, WI, USA), based on SSP method. Results: We found that inhibitory KIR are present in similar observed frequency in both control and patients with MISSC, except KIR2DL5 which was found in lower frequency in patients with MISSC. There are no significant differences of all noninhibitory KIR between control and patients with MISSC. The number of inhibitory KIR genes in patients with MISSC was lover, except for seven inhibitory KIR genes which was almost doubled. The number of noninhibiotry (stimulatory) KIR genes was lower in patients with MISSC, except for those with three KIR genes which were almost four times more frequent. We found significantly bigger percentage of 0,34 – 0,60 activating/inhibitory KIR gene number ratio in the patients with MISSC. Conclusion: In conclusion, there are differences in the KIR gene distribution, gene number, and activating/inhibitory KIR gene number ratio between control and Macedonian patients with MISSC. Further analysis of frequencies of corresponding KIR genotypes or in the ratio of activating/inhibiting genes content in two groups are needed.

Specific KIR and HLA Genotypes Affect Outcomes Of Single-Agent Anti-CD20 Immunotherapy Of Follicular Lymphoma

Lymphoma immunotherapy with anti-CD20 antibody (rituximab) acts primarily through Antibody Dependent Cellular Cytotoxicity (ADCC), implying a role for NK cells. The activation and inhibition of NK cells are influenced by Killer Immunoglobulin-like Receptors (KIRs) and their HLA ligands. To evaluate the involvement of NK cells in response to immunotherapy, we investigated the role of both KIR and HLA polymorphisms in response to single agent rituximab. We have analyzed the relationship between patient genotype and clinical parameters to determine if there are beneficial KIR/HLA interactions that can lead to improved clinical outcome. Methods204 follicular lymphoma (FL) patients participating in the Eastern Cooperative Oncology Group (ECOG) RESORT trial (E4402) were treated with rituximab. The patients who showed an initial response were randomized into two different rituximab treatment regimens. Approximately 70% of FL patients showed initial response to 4 weekly doses of rituximab, and were randomized between arm A: therapy “as needed” (4 weekly doses, only upon progression) or arm B: scheduled maintenance (1 dose every 13 weeks). Clinical outcomes analyzed included duration of response, time to rituximab failure (TTRF), time to cytotoxic therapy (TCT) and decrease in tumor size. The data presented here represent both treatment arms combined, i.e. arms A+B, for all clinical data parameters mentioned. Genomic DNA samples were used for genotyping patients for the presence of 15 KIR loci using a Real Time PCR-based method as previously published (Alves, Tissue Antigens, 2008). HLA specificities corresponding to KIR ligands (HLA-C1, C2, Bw4 and ABw4) were typed using a combination of PCR-SSP methods (Olerup and Invitrogen). ResultsThose patients homozygous for HLA-C1 (C1C1) showed a trend towards a longer duration of response (p=0.06) as compared to those that were heterozygous (C1C2) or homozygous for HLA-C2 (C2C2). A similar difference was observed where patients that were HLA-C1 positive [C1+ (C1C1 or C1C2)] showed a trend towards longer TTRF (p=0.071), as well as a significantly longer TCT (p=0.003), compared to C2C2 patients. Further analyses revealed that the influence of these HLA C specificities on clinical outcome could be related to the presence or absence of inhibitory and/or activating KIR genes. Specifically, HLA-C1 is a ligand for the inhibitory KIR genes 2DL2 and 2DL3. Among the patients who had the KIR2DL2 inhibitory gene (KIR2DL2+), those that were C1+ showed a prolonged duration of response (p=0.049), TCT [Figure 1A, (p=0.003)] and TTRF (p<0.001)] compared to KIR2DL2+ patients who were C2C2 and thus lacked the C1 cognate ligand. Similarly, KIR2DL3+ patients that were C1+ had a significantly longer TCT (p=0.007) than patients that were C2C2, but significant effects were not observed for these KIR2DL3+/C1+ patients with respect to the three other analyzed clinical parameters. Therefore, further studies are required to confirm that KIR2DL3 has an effect independent of other KIRs. [Display omitted] [Display omitted] HLA-C2 alleles are ligands for an activating KIR gene, KIR2DS1. The observed HLA-C genotype effect on clinical outcome could potentially be conveyed by the interaction of HLA-C2 with KIR2DS1. Among the C2C2 patients, KIR2DS1+ patients had a significantly shorter TTRF than those without KIR2DS1 (p=0.023), as well as a significantly smaller decrease in tumor size (p=0.039), implying that the interaction of the activating KIR receptor (KIR2DS1) with its cognate ligand (HLA-C2) conveys a disadvantage to patient outcome. ConclusionSingle-agent rituximab immunotherapy of FL reveals differences in outcomes based on KIR and HLA polymorphisms. The HLA-C2 homozygous individuals seem to be at a disadvantage. This may relate to 2 significant observations highlighting diverse effects of inhibitory and activating KIR-HLA interactions leading to NK cell “licensing” and “hyporesponsiveness”, respectively. Specifically, the data from this study are consistent with: A) a beneficial effect from inhibitory KIR2DL2 and KIR2DL3 interaction with HLA-C1, and B) a detrimental effect due to the contribution of activating KIR2DS1 interacting with its ligand HLA-C2.BG, AKE and WW are co-first authors. Disclosures:Horning:Genentech: Employment, Equity Ownership. Kahl:Genentech: Consultancy, Research Funding.

Allelic Polymorphisms of Inhibitory Killer Immunoglobulin–Like Receptor Natural Killer Cell Function Can Also Influence the Graft-Versus-Leukemia Response

Allogeneic hematopoietic stem-cell transplantations (HSCT) are recognized as a potentially curative treatment of choice for patients with acute leukemia and certain types of lymphoma who fail to sustain an initial complete remission, and are a favored option for patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in first remission who are at high risk of relapse. Although allogeneic HSCTs entail significant risks, they also confer on the transplantation recipient an enhanced resistance to disease recurrence that is not observed after autologous or syngeneic HSCT. The basis for this graft-versus-leukemia effect is complex and still poorly understood. However, both innate and adaptive immune responses that are directed against leukemia cells have been implicated, particularly the contributions of donor-derived natural killer (NK) cells and those of T cells that are specific for either alloantigens that are broadly expressed or differentially expressed by hematopoietic cells of the host, or oncofetal and disease-specific antigens that are aberrantly expressed by leukemia cells. NK cells can be activated through a broad array of activating receptors, such as NKG2D, the natural cytotoxicity receptors NKp30, NKp44, NKp46, and NKp80, and activating receptors of the killer immunoglobulin–like receptor (KIR) complex. These receptors endow NK cells with the capacity to distinguish and respond to autologous cells that differentially express their respective ligands in response to viral infection, malignant transformation, or other types of stress. The KIRs are encoded by a complex of genes on chromosome 19. The KIRs inherited on each haplotype differ in content and function and include KIRs that inhibit or activate the NK cell on binding to their cognate ligands. The ligands for inhibitory KIRs are HLA class I alleles. KIR2DL3 binds HLA-C alleles that contain asparagine at position 80 (C1 alleles), whereas KIR2DL1 binds HLA-C alleles bearing lysine at that position (C2 alleles). KIR3DL1 binds HLA-BW4, whereas KIR3DL2 binds HLA-A3 and -A11 alleles. The activating KIR2DS4 is also known to bind HLA alleles (HLA-A3, -A11, or certain HLA-C alleles). KIR2DS1, like 2DL1, can also bind to HLA-C2 alleles. However, if the 2DS1 donor is also homozygous for an HLA-C2 allele (C2/C2), the NK cells are rendered hyporesponsive or tolerant. The ligands for the other activating KIRs that induce NK cytotoxic activity are unknown. The KIR genes exhibit significant diversity at two levels: allelic polymorphisms within the KIR genes themselves, and differences in the number and type of activating and inhibitory KIR genes inherited on a given haplotype. Analysis of the KIR gene complex has led to the recognition of a series of KIR gene haplotypes that can be divided into two major groups, each containing distinctive sets of KIR genes in their centromeric and telomeric regions. Group A KIR haplotypes differentially contain genes for four inhibitory receptors, KIR2DL3 and KIR2DL1 in the centromeric region and KIR3DL1 and KIR3DL2 in the telomeric region, but only one activating receptor, KIR2DS4. In contrast, KIR B haplotypes contain the inhibitory receptor KIR2DL2 and the activating receptor 2DS2 in the centromeric region and variably contain three activating receptors, 2DS1, 2DS3, and 2DS5, and one inhibitory receptor, 2DL5, in the telomeric region (Fig 1). Because NK cells can be activated through many receptors that encounter their ligands on stressed cells, control of NK cell activity is both essential and necessarily nuanced. Active NK cell tolerance is provided by inhibitory KIR receptors that continuously encounter their cognate class I HLA ligand on normal cells in their environment. These so-called licensed NK cells are differentially armed to distinguish cells that do or do not express their cognate HLA ligand. Detection and binding of this self-ligand on a potential target by inhibitory KIRs induces an inhibitory response. However, if the NK cell’s inhibitory KIR does not encounter its HLA ligand, and expression of ligands for activating KIRs is sufficient, the NK cell’s cytolytic complex is activated and the target is lysed. Additional mechanisms are also operative in the control of NKcell–mediated autoreactivity. Because inheritance of HLA haplotypes on chromosome 6 and KIR haplotypes on chromosome 19 are independent, most individuals inherit inhibitory KIRs for which the cognate class I ligand is not present in the host’s HLA genotype. Unlike T cells, NK cells that express KIRs that develop in the absence of cognate HLA ligands are not deleted. Rather, these so-called unlicensed NK cells continue to be generated but are functionally hyporesponsive JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 31 NUMBER 30 OCTOBER 2

Homozygosity for killer immunoglobin-like receptor haplotype A predicts complete molecular response to treatment with tyrosine kinase inhibitors in chronic myeloid leukemia patients

Several recent reports suggest a possible role for killer immunoglobulin-like receptors (KIR) in the onset of chronic myeloid leukemia (CML) and response to therapy with tyrosine kinase inhibitors (TKIs). To explore this hypothesis, we studied KIRs and their human leukocyte antigen class I ligands in 59 consecutive patients with chronic-phase CML (mean age, 53 years; range, 23-81 years) and a group of 121 healthy control participants belonging to the same ethnic group as the patients. The 2-year cumulative incidence of complete molecular response, obtained after a median of 27 months (range, 4-52 months), was 51.2%. An increased frequency of the activating receptor KIR2DS1 (pm = 0.05) and a reduced frequency of the KIR-ligand combination KIR2DS2/2DL2 absent/C1 present (pm = 0.001) were significantly associated with CML. Moreover, KIR repertoires in patients appeared to influence response to TKI therapy. Homozygosity for KIR haplotype A (pm = 0.01), a decreased frequency of the inhibitory KIR gene KIR2DL2 (pm = 0.02), and low numbers of inhibitory KIR genes (pm = 0.05) were all significantly associated with achievement of complete molecular remission. These data suggest that a decrease in properly stimulated and activated NK cells might contribute to the occurrence of CML and indicate homozygosity for KIR haplotype A as a promising immunogenetic marker of complete molecular response that could help clinicians decide whether to withdraw treatment in patients with CML.

Frequencies of killer immunoglobulin-like receptor genotypes influence susceptibility to spontaneous abortion

Natural killer (NK) cells are the most abundant lymphocyte population in the decidua. These cells express killer immunoglobulin-like receptors (KIRs), which upon recognition of HLA class I molecules on trophoblasts may either stimulate NK cells (activating KIRs) or inhibit them (inhibitory KIRs) to produce soluble factors necessary for the maintenance of pregnancy. KIR genes exhibit extensive haplotype polymorphism; individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes. This polymorphism affects NK cell reactivity and susceptibility to diseases, including gynecological disorders. Therefore we KIR-genotyped 149 spontaneously aborting women and 117 control multiparae (at least 2 healthy-born children). Several genotypes (i.e. combinations of various KIR genes) were differently distributed among the patients and control subjects. Differences were observed in the numbers and the ratios of activating to inhibitory KIRs between patients and healthy women: (i) genotypes containing 6 activating KIR genes were less frequent and those containing 6 inhibitory KIR genes were more frequent in patients than in control subjects, and (ii) an excess of inhibitory KIRs (activating-to-inhibitory KIR gene ratios of 0.33 to 0.83) was associated with miscarriage, whereas ratios close to equilibrium (0.86-1.25) seemed to be protective. In addition, the results suggest for the first time that sporadic and recurrent spontaneous abortions as well as miscarriage in the presence or absence of autoantibodies may have different KIR genotypic backgrounds.

Lack of killer immunoglobulin-like receptor 2DS2 (KIR2DS2) and KIR2DL2 is associated with poor responses to therapy of recurrent hepatitis C virus in liver transplant recipients

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer and natural killer T cells are involved in activation of these cells and can influence antiviral immunity in the liver. This study investigated the association between KIR genetic diversity and sustained virologic response (SVR) to Peginterferon and Ribavirin (Peg/RBV) therapy in liver transplant (LT) recipients with hepatitis C virus (HCV) recurrence. We tested KIR genotypes in 44 HCV-infected LT recipients treated with Peg/RBV for 48 weeks. Patients were categorized as having KIR genotypes A/A or B/x and analyzed for association with SVR. Fifteen of 44 (34%) patients had SVR. Only 2 of 18 (11%) who lacked KIR2DS2/KIR2DL2 achieved SVR compared to 13 of 26 (50%) who carried these two genes (odds ratio: 8.0, 95% confidence interval: 1.5-42.0, P = 0.008). The association between lack of KIR2DS2/KIR2DL2 and SVR remained significant after exclusion of 10 patients with non-genotype 1 HCV. No correlation was found with other activating or inhibitory KIR genes. Absence of KIR2DS2 and/or KIR2DL2 is associated with failure of Peg/RBV therapy in patients with recurrent HCV after LT. These findings support the role of natural killer and natural killer T cells in HCV clearance after LT and might be generalizable to treatment of HCV infection outside the setting of LT.

Demethylating treatment suppresses natural killer cell cytolytic activity

NK cells as a component of the innate immune system provide a first line of defense against viral infections and malignancies. Killer immunoglobulin-like receptors (KIRs) are a polymorphic gene family expressed on NK cells. The crucial role of DNA methylation in determining the variegated expression pattern of KIRs has recently been reported. In this study the direct effect of DNA demethylating treatment on human NK cell cytotoxicity was analyzed. In a human NK cell line NK-92MI, inhibitory KIR genes exhibited characteristic epigenetic repression, whose promoter regions are densely methylated. Treatment of NK-92MI cells with methyltransferase inhibitor 5-azacytidine significantly increased the expression levels of inhibitory KIRs and strongly suppressed their cytolytic activity against human K562 leukemic cells. Cytolytic activity of human polyclonal NK cells was also suppressed upon 5-azacytidine-treatment. The suppression of NK cell cytotoxicity was associated with 5-azacytidine-induced overexpression of inhibitory KIRs and impaired granzyme B and perforin release by these cells. The results demonstrate for the first time that demethylating treatment can suppress NK cell cytolytic activity. The aberrant methylation patterns of KIR genes during NK cell differentiation and maturation may have importance for its abnormal function.

Killer immunoglobulin-like receptor repertoire on uterine natural killer cell subsets in women with recurrent spontaneous abortions

Objective The objective of this study is to investigate the distribution of inhibitory and activating killer immunoglobulin-like receptors (KIRs) on uterine natural killer (uNK) cells and the compatibility of KIR/HLA-C at the maternal–fetal interface in women with unexplained recurrent spontaneous abortion (RSA) in the Chinese population. Study design Sixteen patients with unexplained recurrent spontaneous abortion were enrolled in this study. The PCR sequence-specific primers (SSP) method was used to detect the inhibitory/activating KIRs in uterine NK cells and the HLA-C gene polymorphism expressed on the trophoblast. Results The frequencies of inhibitory KIR2DL2 in the RSA group were increased significantly compared with those of the controls. The other inhibitory KIR2DL families did not show significantly different frequencies in the RSA group. No difference in numbers of inhibitory KIR genes with statistical significance was observed between the RSA group and the controls. When analyzing activating KIRs, none of the KIR2DS1–5 family showed statistically different frequencies in the RSA group compared with the controls. Similarly, there was no statistically significant difference between the numbers of activating KIR genes in the RSA group and the controls. Finally, the matching of the inhibitory or activating KIRs/HLA combination at the maternal–fetal interface did not play a dominant role in the pathogenesis of pregnancy loss. Conclusions This study suggests that the imbalance of inhibitory and activating KIRs in uterine NKs might confer susceptibility to the occurrence of pregnancy loss. The maternal inhibitory/activating KIRs-HLA-C polymorphism expressed on trophoblast cells from decidual tissues seems to play a limited role in abortion.

Global diversity and evidence for coevolution of KIR and HLA

The killer immunoglobulin-like receptor (KIR) gene cluster shows extensive genetic diversity, as do the HLA class I loci, which encode ligands for KIR molecules. We genotyped 1,642 individuals from 30 geographically distinct populations to examine population-level evidence for coevolution of these two functionally related but unlinked gene clusters. We observed strong negative correlations between the presence of activating KIR genes and their corresponding HLA ligand groups across populations, especially KIR3DS1 and its putative HLA-B Bw4-80I ligands (r = -0.66, P = 0.038). In contrast, we observed weak positive relationships between the various inhibitory KIR genes and their ligands. We observed a negative correlation between distance from East Africa and frequency of activating KIR genes and their corresponding ligands, suggesting a balance between selection on HLA and KIR loci. Most KIR-HLA genetic association studies indicate a primary influence of activating KIR-HLA genotypes in disease risk; concomitantly, activating receptor-ligand pairs in this study show the strongest signature of coevolution of these two complex genetic systems as compared with inhibitory receptor-ligand pairs.

Low Number of Donor Activating Killer Immunoglobulin-Like Receptors (KIR) Genes but not KIR-Ligand Mismatch Prevents Relapse and Improves Disease-Free Survival in Leukemia Patients After In Vivo T-Cell Depleted Unrelated Stem Cell Transplantation

The effect of natural killer (NK) cell alloreactivity on outcome of unrelated stem cell transplantation (SCT) remains controversial. Killer immunoglobulin-like receptors (KIRs) recognize human leukocyte antigen C and B epitopes on target cells, thereby regulating NK cell activity. The KIR genes are polymorphic and two broad haplotypes exist: KIR-haplotype A mainly encode for inhibitory receptors and only for one activating (KIR2DS4), whereas the group B haplotype encodes more for activating KIRs (KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 und KIR3DS1). The impact of KIR ligand mismatch on the number of activating and inhibitory donor KIR genes and on KIR-haplotype was studied on outcome of 142 patients with leukemia, who received standard myeloablative conditioning followed by in vivo T-cell depleted (ATG) unrelated SCT. In a multivariate analysis KIR ligand mismatch had significantly higher treatment related mortality (RR 2.206, P = 0.03), resulting in reduced overall (RR 2.015, P = 0.02) and disease-free survival (RR 1.924, P = 0.03). In contrast, SCT from donors with group A haplotype (P = 0.003) or with low number of activating KIR genes (P = 0.005) resulted in reduced relapse rate with improved disease-fee survival (P = 0.04). This effect was seen only in acute myeloid leukemia/myelodysplastic syndrome and to a less extent in chronic myeloid leukemia. No effect was seen for acute lymphoblastic leukemia. After in vivo T-cell depleted (ATG) unrelated stem cell transplantation with donors carrying low number of activating KIR genes (group A KIR haplotype), the risk of relapse is reduced and resulted in a significantly better disease-free survival.

KIR Gene Distribution in Hematologic Disorders.

Killer immunoglobulin-like receptors (KIR) shape the response of NK cells and some CTL. At the genetic level, a KIR mismatch can occur in an individual if the ligand for a particular KIR is absent. The role that such mismatches and certain KIR gene constellations may play in regulation of pathologic immune responses has been rarely explored. It is possible that KIR/HLA combinations can influence disease susceptibility by altering surveillance against microbial agents or transformed cells or by predisposing to autoimmunity. In hematology, KIR/HLA donor/recipient mismatch can influence stem cell transplantation outcome, and dysregulated KIR expression is found in NK cell leukemia. Despite progress in understanding the immune pathophysiology of aplastic anemia (AA), the determinants of susceptibility to immune-mediated bone marrow failure remain unknown. We hypothesized that the immunogenetic background of a specific set of KIR and HLA genes could be involved in the pathophysiologic mechanisms (autoimmune CTL and NK response) in these disorders. Since a number of HLA class I alleles serving as ligands for KIR is known, it is possible to examine the frequency of ligand sets among patient populations. When we studied 282 patients with AA, 82 with PNH, 119 with MDS, and 38 with T-LGL leukemia, no difference in the frequency of KIR C1 & C2 and Bw4 & Bw6 ligand contellations was found between these cohorts. Since there is extensive diversity in the number and types of KIR genes carried by individuals in the population, we next compared the KIR profiles in patients typed by sequence-specific primer amplification of 14 KIR genes and 2 pseudogenes (for 3 KIR, allele specific PCR was performed) to control populations (published cohorts and control groups from our laboratory [N=65]). Since KIR can occur in stimulatory and inhibitory forms, the balance between these two KIR types can determine the quality of the effector cell response to specific targets and globally. Therefore, we analyzed the global ratio between activating and inhibitory KIR genes in patients and controls (AA, N=26; MDS, N=36; PNH, N=29 and LGL, N=22), but no significant differences were found. In addition, the frequency of “unopposed” KIR (absent HLA ligand) in patients did not differ. When the prevalence of individual KIR genes was compared, no differences were found between controls and MDS or T-LGL leukemia patients. However, AA and PNH patients had a decrease in KIR-2DS1 and KIR-2DS5 genes: KIR-2DS1 (Control = 35%, AA = 21% (p=.015), PNH = 19% (p=.014)); KIR-2DS5 (Control = 33%, AA = 14% (p=.038), PNH = 12% (p=.027)). To determine whether absence of 2DS1 and 2DS5 was associated with AA and/or PNH, all patients with a PNH clone and those with AA without a detectable PNH clone were compared; no difference in the frequency of these KIR genes was found. The reduced frequency of KIR-2DS1 and -2DS5 in AA and PNH patients suggests that KIR-positive effector cells may be involved in the pathophysiology of these diseases. Several possible mechanisms could be operative. These activating KIR may be involved in the clearing of a hypothetical agent that triggers a pathologic immune response in AA. Alternately, the absence of these genes may simply identify haplotypes that carry the actual disease predisposing genes. The nature of the KIR gene association aside, these results further support the close pathophysiologic relationship of AA and PNH.