KIR Haplotype Research Articles

Host Genetics of Cytomegalovirus Pathogenesis.

Human cytomegalovirus (HCMV) is a ubiquitous herpes virus (human herpes virus 5) with the highest morbidity and mortality rates compared to other herpes viruses. Risk groups include very young, elderly, transplant recipient, and immunocompromised individuals. HCMV may cause retinitis, encephalitis, hepatitis, esophagitis, colitis, pneumonia, neonatal infection sequelae, inflammatory, and age-related diseases. With an arsenal of genes in its large genome dedicated to host immune evasion, HCMV can block intrinsic cellular defenses and interfere with cellular immune responses. HCMV also encodes chemokines, chemokine receptors, and cytokines. Therefore, genes involved in human viral defense mechanisms and those encoding proteins targeted by the CMV proteins are candidates for host control of CMV infection and reactivation. Although still few in number, host genetic studies are producing valuable insights into biological processes involved in HCMV pathogenesis and HCMV-related diseases. For example, genetic variants in the immunoglobulin GM light chain can influence the antibody responsiveness to CMV glycoprotein B and modify risk of HCMV-related diseases. Moreover, CMV infection following organ transplantation has been associated with variants in genes encoding toll-like receptors (TLRs), programmed death-1 (PD-1), and interleukin-12p40 (IL-12B). A KIR haplotype (2DS4+) is proposed to be protective for CMV activation among hematopoietic stem cell transplant patients. Polymorphisms in the interferon lambda 3/4 (IFNL3/4) region are shown to influence susceptibility to CMV replication among solid organ transplant patients. Interestingly, the IFNL3/4 region is also associated with AIDS-related CMV retinitis susceptibility in HIV-infected patients. Likewise, interleukin-10 receptor 1 (IL-10R1) variants are shown to influence CMV retinitis development in patients with AIDS. Results from genome-wide association studies suggest a possible role for microtubule network and retinol metabolism in anti-CMV antibody response. Nevertheless, further genetic epidemiological studies with large cohorts, functional studies on the numerous HCMV genes, and immune response to chronic and latent states of infection that contribute to HCMV persistence are clearly necessary to elucidate the genetic mechanisms of CMV infection, reactivation, and pathogenesis.

The European Society for Blood and Marrow Transplantation (EBMT) consensus recommendations for donor selection in haploidentical hematopoietic cell transplantation.

The number of HLA-haploidentical hematopoietic cell transplants continues to increase worldwide due to recent improvements in outcomes, allowing more patients with hematological malignancies and non-malignant disorders to benefit from this procedure and have a chance to cure their disease. Despite these encouraging results, questions remain as multiple donors are usually available for transplantation, and choosing the best HLA-haploidentical donor for transplantation remains a challenge. Several approaches to haploidentical transplantation havebeen developed over time and, based on the graft received, can be grouped as follows: T-cell depleted haploidentical transplants, either complete or partial, or withT-cell replete grafts, performed with post-transplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis, or G-CSF-primed bone marrow graft and enhanced GVHD prophylaxis. Carefully selecting the donor can help optimize transplant outcomes for recipients of haploidentical donor transplants. Variables usually considered in the donor selection include presence of donor-specific antibodies in the recipient, donor age, donor/recipient gender and ABO combinations, and immunogenic variables, such as natural killer cell alloreactivity or KIR haplotype. Here we provide a comprehensive review of available evidence for selecting haploidentical donors for transplantation, and summarize the recommendations from the European Society for Blood and Marrow Transplantation (EBMT) on donor selection for different transplant platforms.

Class II HLA Epitope Level Mismatch Can Predict Chronic Graft-Versus-Host Disease and Survival Following Haploidentical Transplant Using Post-Transplant Cyclophosphamide

Post-transplant cyclophosphamide (PTCy) has improved the outcomes and expanded the use of haploidentical hematopoietic cell transplantation (haplo-HCT). Unlike many other allogeneic HCT settings, the impact of HLA disparity on graft-versus-host disease (GVHD) and transplant outcome in this setting remains unclear. HLAMatchmaker is a computer algorithm that assesses HLA compatibility at the structural level by determining what and how many functional epitopes (eplets), defined as patches of polymorphic residues within a radius of 3.0-3.5 Ångstroms, are shared between donor and recipient. It has been useful in the identification of acceptable mismatches (mm) for alloimmunized kidney transplant candidates. In order to determine the effects of HLA class I (HLA-A, B, C) and II (HLA-DR, DQ, DP) epitope mm on transplant outcome, we retrospectively analyzed 208 consecutive donor-recipient pairs receiving haplo-HCT with PTCy for hematologic malignancy. The impact of epitope mm (GVH direction) on GVHD and survival endpoints was evaluated by Cox multivariate analysis (MVA), controlling for other significant patient, donor and transplant-related factors. Median (range) recipient and donor age was 52 (19-75) and 38 (15-73) years respectively. Patients were transplanted for AML (34%), MDS/MPS/CML (20%), ALL (17%), NHL/HD/CLL (25%). PBSC was used as the stem cell source in 66% of patients, and conditioning intensity was myeloablative in 41%. The donor was a child, sibling, or parent in 47%, 38%, and 14% respectively. Median (range) follow-up for surviving patients was 33 (7-130) months. HLA class I epitope mm had no effect on GVHD or survival. In contrast, increased HLA class II epitope mm (>16) was significantly correlated to an increased frequency of chronic GVHD (figure 1). In MVA, higher degree of class II epitope mm was associated with chronic GVHD, total (HR 1.91, p=0.012) and moderate-to-severe (HR 2.37, p=0.006). The positive effect of increased class II epitope mm on chronic GVHD was driven mostly by HLA-DQ epitope mm (HR 1.7 for >7 vs. ≤7, p=0.047) with a non-significant contribution from HLA-DP (HR 1.36 for >2 vs. ≤2, p=0.24). In contrast, increased HLA-DR epitope mm had a protective effect on chronic GVHD (HR 0.52 for >7 vs. ≤7, p=0.021). Epitope mm was not significantly associated with acute GVHD, grade 2-4 or 3-4. There was also no effect of allele-level mm at any HLA loci on acute or chronic GVHD. We next tested the impact of class I and II epitope mm on survival, including the individual impact of HLA-DR, -DQ and -DP epitope mm. Although class I epitope mm had no impact in univariate analysis, a higher number of class II epitope mm (>16) was correlated with better overall survival and the effect was primarily driven by HLA-DQ epitope mm (figure 2). To better assess the impact of class II epitope mm on survival, we analyzed this variable in the context of a previously published MVA (Solomon et al. Biol Blood Marrow Transplant. 2018;24:789-798). Controlling for other significant variables (age, race, CMV status, donor relationship, HLA-DR mm, nonpermissive HLA-DP mm, KIR receptor-ligand mm and KIR haplotype), only increased HLA-DQ epitope mm (>7) was independently associated with decreased non-relapse (HR 0.34, p=0.021) and overall mortality (HR 0.60, p=0.039). These results indicate a significant effect of class II epitope mm on chronic GVHD and survival following haplo-HCT with PTCy. Higher level of class II epitope mm and HLA-DQ epitope mm is associated with increased chronic GVHD incidence, whereas HLA-DR epitope mm is protective. Higher HLA-DQ epitope mm is independently associated with better survival, when controlling for the presence of HLA-DR allele-level mm or a nonpermissive HLA-DP mm, which have been shown previously to improve survival. Class II HLA epitope level matching provides important prognostic information in the setting of haplo-HCT and PTCy, which is not reflected by conventional allele-level matching. [Display omitted] DisclosuresSolh:Amgen: Speakers Bureau; Celgene: Speakers Bureau; ADC Therapeutics: Research Funding.

P063Killer-cell immunoglobulin-like receptors genotypes & haplotypes among kuwaiti families

Aim Inhibitory and activating immunoglobulin like receptor (KIR) family is a major determinant of NK cells activity against cancer. Specific KIR genotypes and/or haplotypes have been reported to influence the outcomes of hematopoietic cell transplantation and in association with a number of autoimmune disorders. The repertoire of KIR genes varies within and between populations, creating a diverse pool of KIR genotypes. The aim of this study was to characterize the genotypes and haplotypes of the KIR genes among 25 Kuwaiti Arab families. Methods A total of 75 individuals from 25 Kuwaiti Arab trios families (including father, mother and a child) were enrolled in this study. KIR genotyping was performed using Real-time PCR based KIR genotyping kit (Linkage Biosciences) from peripheral blood. Sequence specific primer amplification combined with a melting curve analysis method was used to identify 14 KIR genes and 2 KIR pseudogenes including both full length and deleted forms of 3DP1 and 2DS4. Interpretation and reporting of genotype with SureTyperTM software. Results The percentage for the presence of the KIR genes was found to be: KIR2DL1 97.2%, KIR2DL2 56.3%, KIR2DL3 88.7%, KIR2DL4 100%, KIR2DL5 62%, KIR2DP1 97.2%, KIR2DS1 35.2%, KIR2DS2 56.3%, KIR2DS3 42.3%, KIR2DS4 95.8%, KIR2DS5 31%, KIR3DL1 95.8%, KIR3DL2 100%, KIR3DL3 100%, KIR3DP1 100%, KIR3DS1 36.7%. The framework genes were present in 100% for all 75 individuals. The highest frequency of 97% is in 2DP1 and 2DL1 gene and lowest frequency of 31% among KIR genes is in 2DS5. Only 32% of the tested subjects were of AA haplotype genotype, making 68% Bx haplotype genotype. Conclusions Findings of this study are crucial to characterize and compare KIR gene and haplotype frequencies with other populations and also to provide a new data on KIR genotypes and haplotypes of the Kuwaiti Arab population based on family study to understand the population’s genetic makeup. Further, majority of Kuwaiti patients have a several healthy haploidentical relatives who could potentially donate, therefore donor activating KIR-based donor selection is an area of current interest.

P006 NK KIR receptor genotypes are associated with relapse in mexican HLA matched sibling patients with acute lymphoblastic leukemia (ALL)

Aim The outcome of Hematopoietic Stem Cell Transplantation (HSCT) is strongly associated with the HLA identity of donor/recipient, and GVHD remains the most frequent associated factor with mortality and impact on the quality of life. A successful HSCT depends also on T-cell mediated graft vs. leukemia (GvL) effect. Relapse is a major important challenge. NK cells, KIR and their HLA ligands are involved with an increase in overall survival, as well as better engraftment and reduced incidence of GVHD in patients with AML. The aim of the study was to determine the KIR gene content, their HLA ligands and their association on the outcome of ALL patients transplanted with HLA-identical sibling donors. Methods Twenty-seven Mexican patients with their HLA-identical siblings were included. Of these, 20 had ALL and 7 AML. KIR gene content was determined with the LinkSēq™ KIR kit and was run on an ABI 7500 Real-Time PCR instrument. Analysis was done with SureTyper 5. HLA intermediate resolution typing was performed by SSOP on a Luminex. HLA alleles were analyzed with the Match it DNA 1.2.0 software. KIR haplotypes and HLA ligands were assessed using https://www.ebi.ac.uk/ipd/kir/ligand.html . Allele Frequency (AF) and multiple parameter comparison analysis was performed using the SPSS17 software. Results We looked if activating or inhibitory KIR genes had any effect on relapse, long term disease free survival (DFS), GVHD or mortality. An association with relapse was observed in ALL patients, when the matched sibling donor carried the 3DL1 -Bw4 ligand (50% of ALL patients were carriers); (Chi2 = 5.93; p = 0.03) with no differences in Bw4 80I and 80T ligand. The AA and Bx haplotypes did not have any association with mortality, GVHD, relapse or DFS. Conclusions The results in ALL regarding relapse show that the presence of 3DL1 -Bw4, conveys higher susceptibility for relapse, as shown for AML. Contrary to AML, no effect was detected with 80I or 80T with intermediate relapse and mortality, implying a non benefit of education and sensitivity to inhibition as reported for AML. More cases of ALL are being analyzed to confirm if HLA and KIR allele typing in donor selection, enables the antileukemic benefits of NK alloreactivity in HSCT, in ALL, as it has been pointed out in AML, since the affected cell population in ALL, is totally different of the latter.

Spectrum of clinical manifestations of SLE patients from India and its correlation with KIR gene polymorphism

BackgroundSLE is an autoimmune disease presenting with diverse symptoms. Complement components and autoantibodies targeted against them play a major role in the disease pathogenesis. In this study, we have tried to identify the association between KIR genes with the clinical-serological aspects of SLE. MethodsTotal of 250 consecutive cases of SLE (ACR/SLICC 2015 Criteria) were included in this study. Demographic data and clinical presentations were recorded. Autoantibody profiling (disease-associated) was performed to assess the presence of various autoantibodies in patients (serum). A duplex PCR was set up for detecting the presence or absence 14 KIR genes and 2 pseudogenes. Statistical analysis was done using Graph-pad software. ResultsA total of 230 female patients and 20 male SLE patients following the ACR criteria were included in the study. Their age ranged from 5 to 53 years (mean ± SD; 28.14 ± 9.99) in females and 7–45 years (mean ± SD; 25.75 ± 13.76) in males with about 78% patients in the age group of 11–30 years. Clinical manifestation of the patients included arthritis (63.6%), alopecia (51%), mucosal ulcers (49.8%), hematological manifestations (44.5%) and Lupus Nephritis (38.5%). Inhibitory KIR - 2DL1, 2DL5 and 3DL2 were significantly reduced in SLE patients except for 2DL2 gene, which was significantly increased in patients than controls. The activating gene 2DS3 was significantly reduced in the patients as compared to controls. The KIR haplotype B, containing many activating genes was identified as the most common haplotype in patients as well as control samples. ConclusionQuite a few of the genes of the KIR, which are not uncommon in Indian population are also significantly associated with SLE in Indian patients. Few KIR genes can be used as biomarkers to understand the severity of the disease. The KIR genes in this cohort lead to decreased inhibitory signal and increased activation of NK cells and therefore causing increased apoptosis and formation of autoantibodies.

What Is the most Important for Elite Control: Genetic Background of Patient, Genetic Background of Partner, both or neither? Description of Complete Natural History within a Couple of MSM

BackgroundWe describe a homosexual man who strongly controlled HIV-1 for ten years despite lack of protective genetic background. MethodsHIV-1 DNA was measured in blood and other tissues. Cell susceptibility was evaluated with various strains. HIV-1-specific (CD4 and CD8 activation markers and immune check points) and NK cells responses were assessed; KIRs haplotypes and HLA alleles were determined. FindingsTwo HIV-1 RNA copies/mL of plasma were detected in 2009, using an ultra-sensitive assay. HIV-DNA was detected at 1.1 and 2 copies/106 PBMCs in 2009 and 2015 respectively, at 1.2 copies/106 cells in rectal cells in 2011. WBs showed weak reactivity with antibodies to gp160, p55 and p25 from 2007 to 2014, remaining incomplete in 2017. CD4 T cells were susceptible to various strains including HIVKON, a primary isolate of his own CRF02_AG variant. CD8 T cells showed a strong poly-functional response against HIV-Gag, producing mainly IFN-γ; a robust capacity of antibody-dependant cell cytotoxicity (ADCC) was observed in NK cells. Case patient was group B KIR haplotype. Neutralizing antibodies were not detected. CD4 and CD8 blood T cells showed normal proportions without increased activation markers. Phylogenetic analyses identified the same CRF02_AG variant in his partner. The patient and his partner were heterozygous for the CCR5ΔD32 deletion and shared HLA-B*07, C*07 non-protective alleles. InterpretationThis thorough description of the natural history of an individual controlling HIV-1 in various compartments for ten years despite lack of protective alleles, and of his partner, may have implications for strategies to cure HIV-1 infection.

Prevalence of KIR haplotypes and association with mortality in a cohort of critical care patients

Prevalence of KIR haplotypes and association with mortality in a cohort of critical care patients

P137 Next generation sequencing reveals HLA and KIR susceptibility alleles for rheumatoid arthritis

Aim Previous associations of KIR with rheumatoid arthritis, RA, have been reported in some but not all populations studied, possibly due to limited genotyping in some studies, and gene content homogeneity in some populations. In this study, HLA and KIR typing was carried out, including KIR haplotype and allele typing, using state of the art sequencing methodologies in a re-examination of the association of these gene families with RA in a Japanese cohort. An additional cohort of pollen allergy patients was examined in an effort to distinguish common genetic elements in a phenotype functionally reciprocal to RA. Methods DNA from 116 RA patients, 167 pollen allergy patients, and 185 healthy controls were examined for KIR haplotype, allele type and HLA class I and II allele types using next generation sequencing. Association analysis was carried out with healthy controls classified into two groups, positive and negative for allergen specific IgE antibodies, including a pollen allergy group for comparison with RA. Results Significant results were observed with allele types KIR2DS4∗007 and KIR3DL1∗00501, which strongly associated with disease, while KIR 3DL1∗001 and 3DL1∗02901 associated with a protective phenotype. These findings were significant when compared with the IgE positive control group while the IgE-negative group did not demonstrate significance. Conclusions Given that KIR3DL1 is an inhibitory receptor and the KIR3DL1∗00501 allele has been reported as a low expression allele, these findings are consistent with a model of weak suppression of NK cytotoxic activity as a contributing factor in RA. Further support for this model was observed from the reciprocity of the genetic associations between RA and pollen allergy.

Resurrecting KIR2DP1: A Key Intermediate in the Evolution of Human Inhibitory NK Cell Receptors That Recognize HLA-C.

KIR2DP1 is an inactive member of the human lineage III KIR family, which includes all HLA-C-specific receptor genes. The lethal, and only, defect in KIR2DP1 is a nucleotide deletion in codon 88. Fixed in modern humans, the deletion is also in archaic human genomes. KIR2DP1 is polymorphic, with dimorphism at specificity-determining position 44. By repairing the deletion, we resurrected 11 alleles of KIR2DP1F , the functional antecedent of KIR2DP1 We demonstrate how K44-KIR2DP1F with lysine 44 recognized C1+HLA-C, whereas T44-KIR2DP1F recognized C2+HLA-C. Dimorphisms at 12 other KIR2DP1F residues modulate receptor avidity or signaling. KIR2DP1 and KIR2DL1 are neighbors in the centromeric KIR region and are in tight linkage disequilibrium. Like KIR2DL1, KIR2DP1 contributed to CenA and CenB KIR haplotype differences. Encoded on CenA, C1-specific K44-KIR2DP1F were stronger receptors than the attenuated C2-specific T44-KIR2DP1F encoded on CenB The last common ancestor of humans and chimpanzees had diverse lineage III KIR that passed on to chimpanzees but not to humans. Early humans inherited activating KIR2DS4 and an inhibitory lineage III KIR, likely encoding a C1-specific receptor. The latter spawned the modern family of HLA-C receptors. KIR2DP1F has properties consistent with KIR2DP1F having been the founder gene. The first KIR2DP1F alleles encoded K44-C1 receptors; subsequently KIR2DP1F alleles encoding T44-C2 receptors evolved. The emergence of dedicated KIR2DL2/3 and KIR2DL1 genes encoding C1 and C2 receptors, respectively, could have led to obsolescence of KIR2DP1F Alternatively, pathogen subversion caused its demise. Preservation of KIR2DP1F functional polymorphism was a side effect of fixation of the deletion in KIR2DP1F by micro gene conversion.

Characterisation of B killer cell immunoglobulin-like receptor genes and telomeric and centromeric motifs in hematopoietic stem cell transplantation donors in Vojvodina, Serbia

The outcome of HSCT is strongly in?uenced by the genetic similarity or identity in the HLA genes that affects the incidence of graft-versus-host disease (GvHD). Successful allogeneic HSCT, however, depends also on T-cell mediated graft-versus-leukemia (GvL) effect, in which donor-derived T cells and natural killer (NK) cells kill these malignant cells in the patient, therefore playing a crucial role in relapse prevention. The aim of this study was to make the predictive analysis of the structure and distribution of B KIR alleles and centromeric and telomeric KIR genotypes in HSCT donors in Vojvodina with regard to their contribution to protection from relapse. A total of 124 first-degree relatives of patients with hematological malignancies were examined for the presence or absence of 15 KIR genes by using of PCR-SSO technique with Luminex xMap technology. The percentage of individuals carrying each KIR gene, centromeric and telomeric KIR haplotypes and genotypes was determined by direct counting. Sixty two percent of the HSCT donors in Vojvodina carry A KIR haplotype, while nearly 38% carry B KIR haplotype. The distribution of B KIR genes showed that among 124 studied HSCT donors, 31(25%) do not carry none of the KIR genes belonging to B group, 71.77% of donors have two or more B KIR genes, 61.29% of them carry KIR 2DL2 and 2DS2 or more B KIR genes. The analysis of centromeric and telomeric KIR genotypes, showed that Cen-A1/Tel-A1 genotype had a highest frequency of 51.47% and Cen-B2/Tel-B1 the lowest frequency of 1.30%. The usage of donor KIR B gene content and centromeric and telomeric KIR gene structure could be used in development of a simple algorithm to identify donors who will provide the most protection against the relapse in related HSC transplants.

KIR gene haplotype: an independent predictor of clinical outcome in MDS patients

AbstractMyelodysplastic syndromes (MDSs) are a group of hematopoietic disorders affecting the myeloid lineage, characterized by cytopenias and clonal evolution to acute myeloid leukemia (AML). We hypothesized that natural killer (NK) cells and their activating killer immunoglobulin-like receptors (aKIRs) influence the immune surveillance and clinical outcome of patients with MDSs. Here, we first examined the distribution of aKIR genes and haplotype in 2 independent cohorts of MDS and AML patients. The median number of aKIR genes was lower in MDS patients than healthy controls (2 vs 3 genes; P = .001), and lower in patients with secondary AML (progressed from MDSs) compared with de novo AML patients (2 vs 3; P = .008) and healthy controls (2 vs 3; P = .006). In a multivariate analysis, the presence of KIR haplotype A (characterized by low aKIR content 0-1) independently predicted a higher risk of conversion to AML (relative risk [RR] with 95% confidence interval [CI], 2.67 [1.13-6.71]; P = .02) and worse adjusted progression-free survival (RR with 95% CI, 2.96 [1.59-5.52]; P = .001) and overall survival (2.25 [1.17-4.31]; P = .02), compared with KIR haplotype B (multiple aKIR genes). These novel findings may help to identify MDS patients with a high risk of disease progression who would likely benefit from adoptive NK-cell therapy.

A Naive NK Cell Repertoire in the Circulation of Haploidentical Stem Cell Donors Pre Mobilization Predicts Rejection of Cord Myeloid Cells in Patients Undergoing Combined Haploidentical and Unrelated Cord Blood HSCT

Background: For patients (pts) with severe aplastic anemia (SAA) lacking an HLA identical donor, outcomes of hematopoietic stem cell transplantation (HSCT) using unrelated cord blood (UCB) units or haplo-identical donors (HDs) have historically been associated with high graft failure rates and poor survival. In an ongoing clinical trial at the NHLBI, we have observed excellent engraftment (100%) and survival (91%) in SAA pts (n=27) receiving a transplant that co-infuses a single UCB unit with CD34-selectedCD3-depletedcells from a haplo-identical relative. Although cord myeloid engraftment(defined as cord ANC >500/μL) occurred at<day 100 in the majority of pts, a significant fraction of pts had delayed (>day 100) or no cord myeloid engraftment. In this analysis, we investigated factors that may have impeded cord myeloid engraftment following UCB/HD transplantation.Methods: Flow-based NK cell phenotyping using a BD Fortessa II instrument was performed on blood obtained pre-transplant from HDs used for the first 18 SAA pts undergoing UCB/HD transplantation. Lineage specific chimerism was measured by PCR of microsatellites (PowerPlex 16 HS Systemkit/Promega) using DNA from flow sorted cells (BD FACSAria) collected multiple time points post-transplant.KIR-ligand incompatibility in the HD vs UCB directionwas defined using high-resolution HLA typing.Results: 13/18 (72%) pts had cord myeloid engraftment before day 100 while 5/18 (28%) had delayed or no cord myeloid engraftment. Remarkably, delayed or no cord myeloid engraftment occurred exclusively in pts transplanted with KIR-ligand incompatibility in the HD vs UCB direction (n=9) (Figure 1A). In contrast, all 9 pts transplanted with KIR-ligand compatibility in the HD vs UCB direction achieved cord myeloid engraftment by ²day 48 (median day 35) post-transplant. Chimerism analysis performed on blood obtained 30+ days post-transplant revealed NK cell chimerism was ³ 90% cord in origin in all 9 pts transplanted with KIR-ligand compatible grafts. In contrast, amongst the 9 pts receiving a KIR-ligand incompatible transplant, NK cell chimerism was predominantly HD in origin with only a minor fraction of cord NK cells detected 30-200 days post-transplant (Figure 1B). Predominant HD NK cell chimerism in pts receiving a KIR-ligand incompatible transplant was associated with lower degrees of cord myeloid chimerism compared to KIR-ligand compatible recipients. Further analysis of the KIR-ligand incompatible cohort revealed distinct heterogeneity in the time to cord myeloid engraftment (Figure 1A). Although delayed or no cord myeloid engraftment was observed in 5/9 recipients of KIR-ligand incompatible transplants, 4/9 pts in this cohort had cord engraftment at a similar time as pts transplanted with KIR-ligand compatible grafts (median 35 vs. 35 days). This variability in time to cord myeloid engraftment was not associated with stem cell dose, degree of HD NK cell chimerism, type of KIR-ligand incompatibility or KIR haplotype. However, we observed a strong correlation between the proportion of naive NK cells in circulation of HDs before stem cell mobilization with delayed or no myeloid cord engraftment (Figure 1C). With the exception of one patient who had failed HD engraftment, only transplants of CD34+ cells from HDs with a predominantly naive NK cell repertoire, expressing high frequencies of the NKG2A receptor concomitant with low frequencies of NKG2C, Lir-1 and CD57 resulted in delayed or no cord myeloid engraftment (p<0.05).Conclusions: Our study provides the first evidence that NK cells from engrafting CD34+ cells from selected HDs can significantly delay or completely inhibit cord myeloid engraftment following UCB/HD transplantation. Suppression of cord hematopoiesis appears to be restricted to NK cells originating from HDs withHD vs UCB KIR-ligand incompatibility who have a large naive NK cell repertoire in their circulation prior to stem cell mobilization. The myelosuppressive effects of these NK cells are consistent with recentlypublished data showing a naive NK cell repertoire in stem cell donors predicts a reduced risk of AML relapse post-allogeneic HSCT.Further studies defining the mechanisms through which naive NK cells suppress cord hematopoiesis followingUCB/HDtransplantation could shed insights into methods to optimize NK cell mediated graft-vs-leukemia followingallogeneicHSCT of myeloid leukemias. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

KIR repertory in patients with hematopoietic diseases and healthy family members.

BackgroundSince the discovery of specific histocompatibility, literature has associated genes involved in the immune response, like the Human Leucocyte Antigen (HLA), with a better prognosis in transplantation. However, other non-HLA genes may also influence the immune process, such as the genes encoding the immunoglobulin-like receptors of natural killer cells (KIRs). The discovery that NK cell KIR receptors interact with conservative epitopes (C1, C2, Bw4) presented in HLA class I molecules that are genetically polymorphic, also observed in KIR genes, led to the investigation of the relevance of the KIR system to hematopoietic stem cell transplant. The cure of patients with leukemias and other hematological malignancies after bone marrow transplantation (BMT) has been attributed in part to the ability of the donor immune cells, present in the graft, to recognize and eliminate neoplastic cells of the patient. The cytotoxic activity of NK cells is mediated by the absence of HLA class I-specific ligands on the target cell surface to inhibitory KIR receptors (hypothesis of “missing-self”).MethodsWe analyzed, by PCR typing-SSOP technique, the presence or absence of 16 KIR genes and haplotypes of 39 patients with hematopoietic disorders and 136 healthy individuals from Paraná State. The comparisons made between the patient and control group were performed using χ2 test or Fisher exact test (bilateral p-value), as appropriated. Significance level was considered when p-value ≤ 0.05.ResultsFramework genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2 were positive in all samples. The comparison between KIR repertoire of patients and healthy individuals revealed significant differences (p < 0.05) in inhibitors genes KIR2DL2 (p = 0.0005) and KIR2DL5 (p = 0.0067) and activating genes KIR2DS1 (p = 0.0013), KIR2DS2 (p = 0.0038), KIR2DS3 (p = 0.0153) that are more frequent in controls than in patients. The KIR2DS3 was significantly more frequent (p = 0.0031) in patients with acute myeloid leukemia (AML) when compared to patients with acute lymphoblastic leukemia (ALL). We observed a higher frequency of haplotype A (59 %) in the patients.ConclusionOur data suggests that susceptibility to leukemia can be influenced, at least, partly byKIR receptors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12878-016-0064-6) contains supplementary material, which is available to authorized users.

P110 Resolution potential of KIR typing based on high-throughput NGS genotyping data

Aim Recently, we implemented an amplicon based next generation sequencing (NGS) workflow for routine KIR typing, which currently yields presence/absence calls for all KIR genes. While presence/absence data is a good starting point, donor selection might further benefit from allele level resolution. In the current setting, high resolution KIR typing results could, however, hardly be achieved as sequencing relies on exons 4, 5 and 7 only. We therefore analyzed whether expanding the sequenced regions by exons 3, 8 and 9 would significantly reduce allele level ambiguities. Methods Taking previously published KIR haplotype frequency data into account, we randomly generated 1000 artificial KIR genotypes, each consisting of two KIR haplotypes. Based on the IPD-KIR database for each genotype the exon specific sequence compositions were calculated and fed back into an algorithm to recalculate the genotype. We then compared the level of allelic resolution that could be achieved when using exons 4, 5, 7 alone or in combination with exons 3, 8, 9. Here the number of alleles that could not be resolved at 3-digit nomenclature determines the remaining level of ambiguity (LA), where an LA of one refers to an unambiguous typing. Results When performing the recalculation based on exon 4, 5 and 7 alone, we are able to achieve an overall low to medium allelic resolution, with an LA ranging from 1 to 16. We found that the majority of cases, with LA >1 are due to sequence coverage limitations rather than missing phase information. With LAs ranging from 1 to 4, the recalculation based on exons 3, 4, 5, 7, 8 and 9 showed a significantly higher overall allelic resolution. Particularly KIR2/3DL alleles were shown to benefit from additional exons. Interestingly, when analyzing the reasons for remaining ambiguities there is a balance between phasing and sequence coverage issues. Conclusion Expanding the sequenced regions by exons 3, 8 and 9 significantly reduces the KIR allele level ambiguity and thus enables high resolution KIR typings. In order to improve donor selection as well as transplantation outcome we will add exons 3, 8, 9 to our standard KIR typing workflow this summer.

OR36 Defining KIR and HLA class I alleles at highest resolution using next generation sequencing

Aim Combinatorial diversity of KIR and HLA class I alleles directly affects NK cell activity and impacts the outcome of human immune responses to infections, and success in reproduction and transplantation. Therefore it is important to identify genetic variation in these highly polymorphic genes at high resolution. Here we have used our high throughput capture/NGS method to target KIR and HLA class I genes. Methods Libraries were prepared using fragmented genomic DNA samples from 1000 European individuals, 97 IHWG cell lines and 31 family trios from West Africa. After capture of fragments with oligonucleotide probes, the entire KIR locus and HLA class I genes were sequenced to high depth (100×) using Illumina paired-end technology. We also developed a bioinformatics pipeline (PING: Pushing Immunogenetics to the Next Generation) specifically to extract and analyze the KIR-specific reads from any NGS data source. PING attributes sequencing reads to specific KIR genes, determines their copy number by read depth and virtual sequence probes, and then calls high-resolution genotypes. PING also returns information on newly-identified SNPs and recombinant alleles. PING is available as an R package at https://github.com/ . Results The method was validated using data from the 97 IHWG cell lines. KIR gene copy numbers were independently verified by complete correlation with real time PCR, and the allele calls using pyrosequencing and Sanger sequencing. We determined the composition of all the KIR haplotypes represented in the 97 IHWG cells, which are available for reference. PING was further validated by analyzing the 1000 genomes data set for 3DL1/S1 and 3DL2, where we identified and validated 100 novel alleles. This analysis of just two KIR genes doubled the number of known 3DL2 alleles, and showed that 22% of the 1000 Genomes subjects have a novel allele or a structural variant. Subsequently in analyzing the European and African we find few novel HLA class I alleles, but in contrast we find one novel KIR allele per ten individuals. Conclusions The KIR locus is likely to be as diverse as HLA. The method we describe is suited to the large-scale analyses that are needed for characterizing human populations, matching transplant donors with recipients, and defining the precise HLA and KIR factors associated with disease.

Next generation sequencing reveals HLA and KIR susceptibility alleles for rheumatoid arthritis

Abstract Previous associations of KIR with rheumatoid arthritis (RA) have been reported in some but not all populations studied, possibly due to limited genotyping in some studies, and gene content homogeneity in some populations. In this study, HLA and KIR typing was carried out, including KIR haplotype and allele typing, using state of the art sequencing methodologies in a re-examination of the association of these gene families with RA in a Japanese cohort. An additional cohort of pollen allergy patients was examined in an effort to distinguish common genetic elements in a phenotype functionally reciprocal to RA. DNAs from 116 RA patients, 167 pollen allergy patients, and 185 healthy controls were examined for KIR haplotype, allele type and HLA class I and II allele types using next generation sequencing (NGS). Association analysis was carried out with healthy controls classified into two groups, positive and negative for allergen specific IgE antibodies, including a pollen allergy group for comparison with RA. Significant results were observed with allele types KIR2DS4*007 and KIR3DL1*00501, which strongly associated with disease, while KIR 3DL1*001 and 3DL1*02901 associated with a protective phenotype. These findings were significant when compared with the IgE positive control group while the IgE-negative group did not demonstrate significance. Given that KIR3DL1 is an inhibitory receptor and the KIR3DL1*00501 allele has been reported as a low expression allele, these findings are consistent with a model of weak suppression of NK cytotoxic activity as a contributing factor in RA. Further support for this model was observed from the reciprocity of the genetic associations between RA and pollen allergy.

Interactions between maternal killer cell immunoglobulin receptor genes and foetal HLA ligand genes contribute to type 1 diabetes susceptibility in Han Chinese.

In this study, we aimed to test the hypothesis that KIR haplotypes (that interact with HLA class I molecules) are associated with susceptibility in patients with T1DM in utero through maternal-foetal interaction of KIR and their HLA class I ligands in Han Chinese population. We determined the KIR genes and KIR/ligand gene combination frequencies in 59 Han Chinese children with T1D and their mothers and compared it with 159 healthy control children and their mothers. The absence of KIR-2DS1 in the mother and the presence of HLA-C2 ligand in the child were negatively associated with type 1 diabetes in the child. Our results indicate that maternal KIR genes and their interaction with foetal HLA-C2 may contribute to the risk of type 1 diabetes among Han Chinese children.

The killer immunoglobulin-like receptor KIR3DL1 in combination with HLA-Bw4 is protective against multiple sclerosis in African Americans.

We investigated the role of the KIR loci and their HLA class I ligands in a large cohort of African American MS patients (N=907) and controls (N=1456). No significant differences in carrier frequencies for any KIR locus or haplotype were observed between cases and controls. However, examination of KIR in the context of their cognate HLA ligands revealed a strong protective effect for KIR3DL1 in combination with HLA-A and –B alleles bearing the Bw4 motif (p=10−8; OR=0.60, CI=0.50–0.71) and the Bw4 ligand alone (Table 3; p<10−6; OR=0.63, CI=0.53–0.75). The observed effect cannot be explained by either a specific HLA-B allele or by linkage disequilibrium with HLA-DRB1 or HLA-A. The protective effect was observed only in individuals who were not positive for the MS risk allele HLA-DRB1*15:01 (p<10−6; OR=0.61, CI=0.51–0.74). Our study, the first investigation of KIR and MS in African Americans, confirms and refines previous findings in a European cohort.

Exploring the Role of Killer Cell Immunoglobulin-Like Receptors and Their HLA Class I Ligands in Autoimmune Hepatitis.

BackgroundNatural killer cells are involved in the complex mechanisms underlying autoimmune diseases but few studies have investigated their role in autoimmune hepatitis. Killer immunoglobulin-like receptors are key regulators of natural killer cell-mediated immune responses.Methods and FindingsKIR gene frequencies, KIR haplotypes, KIR ligands and combinations of KIRs and their HLA Class I ligands were investigated in 114 patients diagnosed with type 1 autoimmune hepatitis and compared with a group of 221 healthy controls. HLA Class I and Class II antigen frequencies were compared to those of 551 healthy unrelated families representative of the Sardinian population. In our cohort, type 1 autoimmune hepatitis was strongly associated with the HLA-B18, Cw5, DR3 haplotype. The KIR2DS1 activating KIR gene and the high affinity HLA-C2 ligands were significantly higher in patients compared to controls. Patients also had a reduced frequency of HLA-Bw4 ligands for KIR3DL1 and HLA-C1 ligands for KIR2DL3. Age at onset was significantly associated with the KIR2DS1 activating gene but not with HLA-C1 or HLA-C2 ligand groups.ConclusionsThe activating KIR gene KIR2DS1 resulted to have an important predictive potential for early onset of type 1 autoimmune hepatitis. Additionally, the low frequency of the KIR-ligand combinations KIR3DL1/HLA-Bw4 and KIR2DL3/HLA-C1 coupled to the high frequency of the HLA-C2 high affinity ligands for KIR2DS1 could contribute to unwanted NK cell autoreactivity in AIH-1.