Killer Immunoglobulin-like Receptors Receptors Research Articles

Conformational Alterations of the Cell Surface of Monomeric and Dimeric β2m-Free HLA-I (Proto-HLA) May Enable Novel Immune Functions in Health and Disease.

Human leukocyte antigens (HLAs) are polymorphic glycoproteins expressed on the cell surface of nucleated cells and consist of two classes, HLA class I and HLA class II. In contrast, in mice, these molecules, known as H-2, are expressed on both nucleated cells and erythrocytes. HLA-I molecules (Face-1) are heterodimers consisting of a polypeptide heavy chain (HC) and a light chain, B2-microglobulin (B2m). The heterodimers bind to antigenic peptides and present them to the T-cell receptors of CD8+ cytotoxic T lymphocytes. The HCs can also independently emerge on the cell surface as B2m-free HC monomers without peptides (Face-2). Early investigators suggested that the occurrence of B2m-free HCs on the cell surface resulted from the dissociation of B2m from Face-1. However, others documented the independent emergence of B2m-free HCs (Face-2) from the endoplasmic reticulum (ER) to the cell surface. The clustering of such HC molecules on either the cell surface or on exosomes resulted in the dimerization of B2m-free HCs to form homodimers (if the same allele, designated as Face-3) or heterodimers (if different alleles, designated as Face-4). Face-2 occurs at low levels on the cell surface of several normal cells but is upregulated on immune cells upon activation by proinflammatory cytokines and other agents such as anti-CD3 antibodies, phytohemagglutinin, and phorbol myristate acetate. Their density on the cell surface remains high as long as the cells remain activated. After activation-induced upregulation, Face-2 molecules undergo homo- and heterodimerization (Face-3 and Face-4). Observations made on the structural patterns of HCs and their dimerization in sharks, fishes, and tetrapod species suggest that the formation of B2m-free HC monomers and dimers is a recapitalization of a phylogenetically conserved event, befitting the term Proto-HLA for the B2m-free HCs. Spontaneous arthritis occurs in HLA-B27+ mice lacking B2m (HLA-B27+ B2m-/-) but not in HLA-B27+ B2m+/+ mice. Anti-HC-specific monoclonal antibodies (mAbs) delay disease development. Some HLA-I polyreactive mAbs (MEM series) used for immunostaining confirm the existence of B2m-free variants in several cancer cells. The conformational alterations that occur in the B2m-free HCs enable them to interact with several inhibitory and activating receptors of cellular components of the innate (natural killer (NK) cells) and adaptive (T and B cells) immune systems. The NK cells express killer immunoglobulin-like receptors (KIRs), whereas leukocytes (T and B lymphocytes, monocytes/macrophages, and dendritic cells) express leukocyte immunoglobulin-like receptors (LILRs). The KIRs and LILRs include activating and inhibitory members within their respective groups. This review focuses on the interaction of KIRs and LILRs with B2m-free HC monomers and dimers in patients with spondylarthritis. Several investigations reveal that the conformational alterations occurring in the alpha-1 and alpha-2 domains of B2m-free HCs may facilitate immunomodulation by their interaction with KIR and LILR receptors. This opens new avenues to immunotherapy of autoimmune diseases and even human cancers that express B2m-free HCs.

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

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

KIR- Ligand Interactions in Hypertensive Disorders in Pregnancy.

HypothesisThe activity of natural killer (NK) cells is considered an important factor for the tolerance of the fetus during pregnancy. The complications of pregnancy, such as hypertensive disorders (HDP), may be therefore associated with this immune compartment.MethodsThe current study included 41 pregnant women diagnosed with HDPs (Gestational Hypertension; GH or Preeclampsia; PE) and 21 healthy women. All the patients were under continuous obstetric care during the pregnancy and labour. The number of mother-child mismatches within killer immunoglobulin-like receptors (KIRs), their ligands [MM], and missing KIR ligands [MSLs] was assessed. KIRs and their ligands were assessed with Next Generation Sequencing (NGS) and Polymerase Chain Reaction Sequence-Specific Oligonucleotide (PCR-SSO) typing. The subsets of NK cells were assessed with multicolor flow cytometry and correlated to the number of MSLs.ResultsThe number of MSLs was significantly higher in HDP patients when compared to healthy non-complicated pregnancy patients. Some MSLs, such as those with 2DS2 activating KIR, were present only in HDP patients. The percentage of CD56+CD16-CD94+ NK cells and CD56+CD16-CD279+ NK cells correlated with the number of MSLs with inhibiting KIRs only in healthy patients. In HDP patients, there was a correlation between the percentage of CD56-CD16+CD69+ NK cells and the number of MSLs with inhibiting and activating KIRs. As compared to the healthy group, the percentage of CD56+CD16-CD279+ NK cells and CD56-CD16+CD279+ NK cells were lower in HDP patients. HDP patients were also characterized by a higher percentage of CD56+CD16+perforin+ NK cells than their healthy counterparts.ConclusionsPatients with HDP were characterized by a higher number of MSLs within the KIRs receptors. It seemed that the number of MSLs in the healthy group was balanced by various receptors, such as CD94 or inhibitory CD279, expressed on NK cells. Conversely, in HDP patients the number of MSLs was associated with the activation detected as the increased level of CD69+ NK cells.

KIR Receptors as Key Regulators of NK Cells Activity in Health and Disease.

Natural killer (NK) cells are part of the cellular immune response. They target mainly cancer and virally infected cells. To a high extent cytotoxic activity of NK cells is regulated inter alia by signals from killer immunoglobulin-like receptors (KIR). The major histocompatibility complex (MHC) class I molecules are important ligands for KIR receptors. Binding of ligands (such as MHC I) to the KIR receptors has the important role in solid organ or hematopoietic cell transplantation. Of note, the understanding of the relationship between KIR and MHC receptors may contribute to the improvement of transplant results. Donor-recipient matching, which also includes the KIR typing, may improve monitoring, individualize the treatment and allow for predicting possible effects after transplantation, such as the graft-versus-leukemia effect (GvL) or viral re-infection. There are also less evident implications of KIR/MHC matching, such as with pregnancy and cancer. In this review, we present the most relevant literature reports on the importance of the KIR/MHC relationship on NK cell activity and hematopoietic stem cell transplantation (HSCT)/solid organ transplantation (SOT) effects, the risk of allograft rejection, protection against post-transplant cytomegalovirus (CMV) infection, pregnancy complications, cancer and adoptive therapy with NK cells.

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.

Impact of killer immunoglobulin-like receptor-human leukocyte antigens ligand incompatibility among renal transplantation

Killer immunoglobulin-like receptor (KIR) gene shows a high degree of polymorphism. Natural killer cell receptor gets activated once they bind to self-human leukocyte antigens (HLAs) with specific ligand. KIR gene and HLA ligand incompatibility due to the presence/absence of KIR in the recipient and the corresponding HLA ligand in the allograft may impact graft survival in solid organ transplantation. This study evaluates the effect of matches between KIR genes and known HLA ligands. KIR genotypes were determined using sequence specific primer polymerase chain reaction. Presence of certain KIR in a recipient, where the donor lacked the corresponding HLA ligand was considered a mismatch. The allograft was considered matched when both KIR receptor and HLA alloantigen reveald compatibility among recipient and donor. The data revealed better survival among individuals with matched inhibitory KIR receptors and their corresponding HLA ligands (KIR2DL2/DL3-HLAC2, KIR3DL1-HLABw4). On the contrary, no adverse effect was seen for matched activating KIR receptors and their corresponding HLA ligands. One of the activating gene KIR2DS4 showed risk (P = 0.0413, odds ratio = 1.91, 95% confidence interval = 1.02-3.57) association with renal allograft rejection. We conclude that the presence of inhibitory KIR gene leads to better survival; whereas activating motifs show no significant role in renal allograft survival.

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

KIR‐HLA‐A and B alleles of the Bw4 epitope against HIV infection in discordant heterosexual couples in Chaco Argentina

Activating and inhibitory killer immunoglobulin-like receptors (KIR) and their ligands HLA-Bw4 (loci A and B) were studied by way of establishing whether they can contribute to protection against HIV-1 infection in highly exposed and persistently seronegative (HESN) patients. Twenty-three HIV-1 serodiscordant heterosexual couples, 100 HIV-1(+) patients and 200 healthy individuals were included in this retrospective case-control study. HLA typing was performed by means of PCR followed by sequence-specific oligonucleotide probe reverse hybridization. KIR3DL1 and KIR3DS1 were studied by PCR sequence-specific primers. The frequency of KIR3DS1(3DS1/3DL1)-Bw4 combination was significantly higher in HESN patients versus the discordant couples (P=0·0003) and HIV-1(+) patients (P=0·0001). Conversely, the KIR3DL1/KIR3DL1 homozygosity was significantly decreased in HESN patients versus the discordant couples (P=0·00003), and HIV-1(+) patients (P=0·00066). The frequency of HLA-A*32 and HLA-B*44 was higher in HESN versus their discordant couples (P=0·009; P=0·049), and HIV-1(+) patients (P=0·00002; P=0·0001). This had greater significance in combination with KIR3DS1 (3DS1/3DL1). KIR3DS1(3DS1/3DL1) could have a greater effect on protection against HIV-1 infection in HESN patients when bound to a specific HLA allele, in this case HLA-A*32 and HLA-B*44, both Bw4 alleles. The differences probably arise both in the HLA alleles and in the subtypes of KIR receptors depending on the ethnic group studied.

Distribution of Killer cell immunoglobulin like receptor genes in end stage renal disease among North Indian population

NK cell function is regulated by cell surface inhibitory and activating receptors including the C-type lectin receptors and Killer Immunoglobulin-like receptors (KIR). The effect of immune modulating cytokines produced by NK cells in the pathogenesis of end stage renal disease (ESRD) remained intriguing. In this regard the present study assesses the combinatorial association of KIR gene content and KIR receptor-HLA ligand in the North Indian ESRD patients. KIR gene polymorphism as a susceptible marker in ESRD among 512 patients and 512 ethnically matched controls was analyzed. PCR-SSP based genotyping for KIR gene content and HLA-A, B, C typing was carried out. Significant difference in frequencies of KIR2DS1-HLA-C2 (p≤0.0001, OR=1.98, CI=1.50-2.61), KIR2DS2-HLAC1 (p≤0.0001, OR=1.87, CI=1.42-2.46), KIR3DS1-HLA-Bw4 (p=0.0038, OR=1.46, CI=1.13-1.88) combinations for ESRD was found. In the combinatorial analysis Bw4(+)/3DL1(-)/3DS1(+) (p≤0.0001, OR=4.90, CI=2.75-8.71) and C1(+)/2DL2(-)/2DL3(-)/2DS2(+)/2DS3(+) (p=0.0037, OR=2.50, CI=1.35-4.63) showed risk association. KIR3DS1 was observed to be susceptible for all four primary kidney disease groups. NK cell de-regulation due to HLA ligand binding KIR receptors may be involved in the patho-physiology of ESRD. Upon analyzing the data in this context it was found that C2/C2 donor might improve the clinical outcome of patients having C2 ligands.

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.

CD158k/KIR3DL2 and NKp46 are frequently expressed in transformed mycosis fungoides

Malignant Sezary cells express the natural killer (NK) receptors KIR3DL2 (CD158k) and Nkp46 and may co-express activating killer immunoglobulin-like receptors (KIR) that may participate to neoplastic T-cell activation through the JNK pathway. Little is known regarding NK receptor expression in other cutaneous T-cell lymphomas. We studied the expression of KIR and natural cytotoxicity receptor (NCR) transcripts, and KIR3DL1/2 at the protein level, in 16 skin biopsies from 10 patients with transformed mycosis fungoides (tMF). Some KIR and NCR transcripts were found in all cases, with various repertoires. Two to nine different KIR receptors were expressed in a single biopsy. Among them, KIR3DL2 was the most frequent, with the highest level of expression in quantitative analyses and correlated with in situ protein expression, while phosphorylated JNK was never detected. Among NCR, NKp46 was expressed in all investigated cases. The role of KIR3DL2 and NKp46 in tMF oncogenesis remains to be studied.

A predictive model of treatment outcome in patients with chronic HCV infection using IL28B and PD-1 genotyping

The advent of new chronic hepatitis C virus (HCV) therapies requires characterization of patients in order to predict adequate treatment. A good candidate marker is Programmed Cell Death-1 (PD-1) which is involved in progression of HCV infection. The aim of this study was to analyse the effect of several single nucleotide polymorphisms of PD-1 gene and several previously associated factors (IL28B and KIR receptors) on treatment responses. 407 HCV chronic infected patients treated with PEG-IFN-α and ribavirin were recruited and classified according to their response to treatment. They were genotyped for PD-1 and IL28B polymorphisms, killer immunoglobulin-like receptors (KIR) and HLA genes. A multivariate logistic regression analysis and a Chi-squared Automatic Interaction Detector (CHAID) prediction model of response included these and other clinical parameters. Our results showed that PD-1.3/A allele was significantly associated with sustained virological response (SVR) in a multivariate logistic regression analysis (p<0.01, OR=2.57). Additionally, IL28B C/C genotype was the most significant predictor of an SVR to treatment in all HCV genotypes (74.5%). In IL28B C/C patients, the presence of PD-1.3/A allele increased the probability of an SVR to 93.3%. Moreover, when this analysis was made only with patients infected by HCV-1, the predictive value of IL28B C/C genotype with PD-1.3/A allele was 90%. PD-1.3/A allele is associated with SVR to treatment and notably increases the predictive value of IL28B C/C genotype. Both markers in conjunction could be a useful tool, more relevant than HCV genotype in some cases, in clinical practice.

Recovery of KIR Expression After Allogeneic Stem Cell Transplantation in Multiple Myeloma Patients

Abstract 4555 Introduction:Activating and inhibitory killer immunoglobulin like receptors (KIR) are predominantly expressed on natural killer (NK) cells. KIR mismatch allogeneic stem cell transplantation (alloSCT) has been reported to provide beneficial effects for Multiple Myeloma (MM). However, their recovery in MM patients remains poorly understood. We, therefore, analysed KIR recovery in 90 MM patients after alloSCT. Methods:KIR expression (CD158a/h, CD158b/b2, CD158e1/e2) on NK cells and T cell subsets was measured by flow cytometry at different time points after alloSCT. Results:During the first 90 days after alloSCT NK cells represent the largest lymphocyte subset. Activating receptors like NKp30 and NKp44 showed a fluctuating expression while members of the KIR family were expressed at a constant rate (20% of NK cells). There was no significant difference in the early post transplantation period (day 0–90) compared to later time points (day 360).In contrast, T cells showed increased KIR expression during the first 30 days after alloSCT, which was highly significant for CD158e (p=0,0001). After 30 days the expression declined to baseline. Furthermore, T cell activation marker HLA-DR reached its highest expression between days 60 and 90 when KIR receptors were expressed at their lowest level (27% vs. 8%, p < 0,0001). Conclusions:We conclude that KIR receptors were differentially expressed on NK and T cells. Because KIR receptors are constantly expressed by NK cells and NK cells are the most frequent lymphocyte populations early after alloSCT, NK cells may be useful for KIR mismatch cellular therapy. Disclosures:No relevant conflicts of interest to declare.

Doenças reumatológicas autoimunes e sua associação com os genes killer immunoglobulin-like receptors

Killer Immunoglobulin-like Receptor (KIR) genes express as receptors that activate or inhibit Natural Killer (NK) cells. The NK cells are part of the innate immune response and, through their KIR receptors, they identify target cells that have modified or different HLA (Human Leukocyte Antigen) molecules, inducing their lysis. The KIR receptors result from the expression of KIR genes (19q13.14) on the cell membrane of NK cells, which are polymorphic, and form haplotypes. The diversity of the frequency of KIR haplotypes in certain populations suggests that some individuals have different levels of protection against some diseases. The balance between cell inhibition and activation enables the NK cell to help the organism in immunological surveillance. In addition, there is evidence of the association of activating KIR genotypes with an increased risk for autoimmune disease.

KIRs and their HLA ligands in Remitting–Relapsing Multiple Sclerosis

Killer Immunoglobulin-like Receptor (KIR) genes may affect both resistance and susceptibility to autoimmune disorders, but their role in the pathogenesis of Multiple Sclerosis (MS) is still unclear. To evaluate the involvement of KIRs and their HLA ligands in the development of MS we performed genotyping of HLA -A, -B, -Cw, -DRB1 and KIRs loci in 121 RRMS patients and 103 healthy controls (HC). Results evidenced a possible protective role of the activating KIR2DS1 gene (p y = 0.001; OR:0.38), enhanced in the presence of its ligand group HLA-C2 (p y = 0.0001; OR:0.23). Our data suggest that the presence of functional compounds of activating KIR receptors together with their HLA ligands, allowing the immunomodulatory function of NK cells, may have a protective role against the disease.

Lack of expression of inhibitory KIR3DL1 receptor in patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes

Natural killer cell-type lymphoproliferative disease of granular lymphocytes is a disorder characterized by chronic proliferation of CD3(-)CD16(+) granular lymphocytes. By flow cytometry analysis, we previously demonstrated a dysregulation in killer immunoglobulin-like receptor (KIR) expression in natural killer cells from patients with this lymphoproliferative disease, the activating KIR receptors being mostly expressed. We also found that patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes usually had KIR genotypes characterized by multiple activating KIR genes. We investigated the mRNA levels of the KIR3DL1 inhibitory and the related KIR3DS1 activating receptors in 15 patients with natural killer cell-type lymphoproliferative disease of granular lymphocytes and in ten controls. These genes are usually expressed when present in the genome of the Caucasian population. We demonstrated the complete lack of KIR3DL1 expression in most of the patients analyzed, with the receptor being expressed in 13% of patients compared to in 90% of controls (P<0.01). Interestingly, studies of the methylation patterns of KIR3DL1 promoter showed a significantly higher methylation status (0.76 ± 0.12 SD) in patients than in healthy subjects (0.49±0.10 SD, P<0.01). The levels of expression of DNA methyl transferases, which are the enzymes responsible for DNA methylation, did not differ between patients and controls. In this study we showed, for the first time, a consistent down-regulation of the inhibitory KIR3DL1 signal due to marked methylation of its promoter, thus suggesting that together with the increased expression of activating receptors, the lack of the inhibitory signal could also play a role in the pathogenesis of natural killer cell-type lymphoproliferative disease of granular lymphocytes.

KIR Genes and Their HLA-C Ligands in B-Cell Chronic Lymphocytic Leukemia in a Polish Population.

Abstract 4402 IntroductionDysfunction in cellular and humoral immunity entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). It has been suggested that innate immunity, especially natural killers cells play key role in antitumor cytotoxicity regulated by interaction between killer immunoglobulin-like receptors (KIRs) on NK cells and some T-cell subpopulations and their HLA-class I ligands on target cells. KIRs receptors have been divided into two functional groups: with long cytoplasmic tails (DL) - inhibitory and with short cytoplasmic tails (DS) - activating. Two broad haplotypes of KIR genes have been defined: the A haplotype characterized by presence of single activating KIR gene (2DS4) and the B haloptype characterized by two or more activating KIR genes (2DS1, 2DS2, 2DS3, 2DS5 and 3DS1).The 2DL2, 2DL3 and 2DS2 KIRs bind to HLA-C1 allotype (Asp80), while 2DL1 and 2DS1 KIRs bind to HLA-C2 allotype (Lys80). Due to lack of representative data in literature the study was undertaken to determine the association between polymorphism of KIR genes and HLA-C allotypes and susceptibility to B-CLL. Patients and methodsThe following KIR genes were typed: 2DL1, 2DL2, 2DL3, 3DL1, 2DS1, 2DS2, 2DS3, 2DS4fl, 2DS4del, 2DS5, 3DS1, in 200 B-CLL patients and 199 healthy individuals using PCR-SSP method. The HLA-C1 and HLA-C2 were determined using Olerup SSP typing kit in 187 B-CLL patients and 104 controls. ResultsIndividual KIR gene content was similar in B-CLL cases and healthy controls although patients with KIR2DS3 gene were more common among cases compared to controls (36% vs. 27%, p=0.06) (Table1). The frequency of AA haplotype did not differ between studied groups (29% vs. 28%). Moreover no differences were found between incidence of HLA-C allotypes (HLA-C1: 74.3% vs. 76.0%, HLA-C2: 67.4% vs.70.2%), genotypes (C1/C1: 32.6% vs 29.8%, C1/C2: 41.7% vs 46,2% and C2/C2: 25.7% vs 24.1) and KIR genes in combination with genes for their HLA-C ligands in B-CLL patients and controls (Table 2).Table 1Frequency of KIR genes in B-CLL patients and controls.2DL12DL22DL32DS12DS22DS32DS4del2DS4fl2DS53DL13DS1B-CLL (n=200)1951061767510854166565318670[%]97.553.088.037.554.027.083.028.026.593.035.0Controls (n=199)1971061808111171162586118178[%]99.053.390.540.755.835.781.429.130.791.039.2Table.2Frequency of the KIR-HLA gene combinations in B-CLL patients and controls.B-CLL (n = 187)Controls (n = 104)N%N%2DL1+/C2+12265.247370.192DL2/3+/C1+13974.337975.962DS1+/C2+4926.202423.082DS2+/C1+7137.974038.462DL1+/C2-6032.083028.802DL2/3+/C1-4825.692524.042DS1+/C2-2211.761817.312DS2+/C1-1910.371110.58 ConclusionsThe data obtain in this study imply that there may be not direct association between KIR and HLA-C gene content in the genome and the presence of B-CLL. Disclosures:No relevant conflicts of interest to declare.

NK Cytotoxicity Is Decreased During the Early Posttransplant Period Following Pediatric Allogeneic Transplantation From Unrelated Donors Using CD3/CD19-Depleted Graft.

Abstract 2434Poster Board II-411 Introduction and objectives:Unrelated donors, match unrelated (MUD) and haploidentical donors (HSCT), have been described as a therapeutic option for high-risk childhood acute leukemia. CD3/CD19 depleted graft has been used in order to decrease the incidence of graft versus host disease (GvHD) and post-transplant lymphoproliferative disease, in the unrelated transplantation setting. Donor-derived NK cell alloreactivity has been reported to mediate early graft-versus-leukemia (GvL) effect after allogeneic hematopoietic stem cell transplantation. NK cells are components of the innate immunity playing an important role in the surveillance of human tumors. NK cell recognition of malignant cells depends on a dynamic balance between activating and inhibitory receptors. NK cell alloreactivity can be predicted by donor Killer Immunoglobulin like Receptors (KIRs), Natural Killer Receptors (NCRs), C-type Lectin receptors (NKG2D), Toll Like Receptors (TLRs) and recipient human leukocyte antigen (HLA) class I alleles as ligands. Reduced risk of relapsed has been described in malignant cancer after haploidentical stem cell transplantation when HLA ligands against the inhibitory KIRs present in the donor were absent in the recipient (KIR–HLA receptor–ligand mismatch). We prospectively investigated NK function and NK reconstitution in 18 CD3/CD19 depleted graft unrelated hematopoietic stem cell transplantation (7 MUD and 11 HSCT) using fludarabine-based reduced intensity conditioning regimen. Results:NK cells peaked around day 30 after transplantation. The median number of NK cells on day +30 was 403±88/μL . On day 100 after transplantation the median number of NK cells/μL was 221±58. While the CD56bright NK cell subset was above normal during the first 100 days post-transplant, the “effector” NK cell subset, CD56dim CD16bright, was significantly reduced early after transplantation. The median percentage of CD56bright cells among NK cells in peripheral blood was 25.8±4.6% at day +30, and it was 24.5±5.7 at day +100. The decreased in CD56dim CD16bright NK cell subset was correlated with the decreased of the inhibitory KIR receptors (KIR2DL1, KIR2DL2, KIR3DL1) expression. We also observed a lower expression than donors of the activating receptors NKG2D, TLR4 at day +30, NKp46, TLR 9 at day 60 and NKp46, NKp30 at day +100. Although absolute NK-cell counts rapidly increased after transplant, their cytotoxicity against K562 was much lower compared to that of their donors. At day 100 after transplantation, patients NK cytotoxicity was lower than donor values. These results suggest that the low NK cell cytotoxicity could be related to an “immature” NK phenotype during the early period after HSCT. As other authors have published, activating receptors can be significantly upregulated in cytokine-stimulated NK cells. In our experience, overnight incubation with IL-15 overcomes this limitation, enhancing three times NK cytotoxicity, in vitro. Conclusion:The phenotype of NK cells and NK cytotoxicity ability are significantly altered early after allogeneic transplantation from unrelated donors using CD3/CD19-depleted graft. NK repertoire observed in patients was associated with the imbalance between CD56bright and CD56dim NK subsets and the expression of KIRs and NCRs. These data suggest a pattern consistent with an ongoing NK maturation after MUD and HSCT transplantation. In our experience, the phenotype and functional pattern of NK cells observed is suggestive of a cytokine-driven process. IL-15 stimulated NK cells could be helpful to optimize adoptive antitumor NK immunotherapy to enhance GvL effect as early as possible after transplantation. Disclosures:No relevant conflicts of interest to declare.

Reply: Influence of activating and inhibiting killer immunoglobulin-like receptors on predisposition to recurrent miscarriages

Understanding of the immune events and mechanisms occurring at the feto-maternal interface is likely to help in understanding the ability of the fetus to survive within the maternal body. Evidence supporting extensive roles of natural killer cells during pregnancy gives rise to a possibility that these NK cells can be mis-regulated and involved in fetal allograft rejection. Killer immunoglobulin-like receptors (KIR) play an important role in regulating the NK cell activity through their activating and inhibiting isoforms. Since there exists a considerable, genetically determined variation in the repertoire of KIR receptors between different individuals, a particular maternal KIR repertoire may predispose to recurrent miscarriages (RMs).Gene-specific PCR amplification (PCR-SSP) was used to determine the individual KIR genotypes in women experiencing RM and controls.A higher prevalence of activating KIR genes was seen in patients than in controls. Among women experiencing RM, the BB genotypes were more prevalent (P < 0.0001, OR = 4.4, 95% CI = 2.89-6.69) compared with controls.Our results indicate that the balance between inhibitory and activating receptor-mediated signals present in natural killer cells is inclined toward a more activating state that may contribute to pregnancy loss.

Influence of activating and inhibitory killer immunoglobulin-like receptors on predisposition to recurrent miscarriages

Understanding of the immune events and mechanisms occurring at the feto-maternal interface is likely to help in understanding the ability of the fetus to survive within the maternal body. Evidence supporting extensive roles of natural killer cells during pregnancy gives rise to a possibility that these NK cells can be mis-regulated and involved in fetal allograft rejection. Killer immunoglobulin-like receptors (KIR) play an important role in regulating the NK cell activity through their activating and inhibiting isoforms. Since there exists a considerable, genetically determined variation in the repertoire of KIR receptors between different individuals, a particular maternal KIR repertoire may predispose to recurrent miscarriages (RMs). Gene-specific PCR amplification (PCR-SSP) was used to determine the individual KIR genotypes in women experiencing RM and controls. A higher prevalence of activating KIR genes was seen in patients than in controls. Among women experiencing RM, the BB genotypes were more prevalent (P < 0.0001, OR = 4.4, 95% CI = 2.89-6.69) compared with controls. Our results indicate that the balance between inhibitory and activating receptor-mediated signals present in natural killer cells is inclined toward a more activating state that may contribute to pregnancy loss.