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Human chromosome 19q13.4 contains genes encoding killer-cell immunoglobulin-like receptors (KIR). Reported haplotype lengths range from 67 to 269 kilobases and contain 4 to 18 genes. The region has certain properties such as single nucleotide variation, structural variation, homology, and repetitive elements that make it hard to align accurately beyond single gene alleles. To the best of our knowledge, a multiple sequence alignment of KIR haplotypes has never been published or presented. Such an alignment would be useful to precisely define KIR haplotypes and loci, provide context for assigning alleles (especially fusion alleles) to genes, infer evolutionary history, impute alleles, interpret and predict co-expression, and generate markers. In order to extend the framework of KIR haplotype sequences in the human genome reference, 27 new sequences were generated including 24 haplotypes from 12 individuals of African American ancestry that were selected for genotypic diversity and novelty to the reference, to bring the total to 68 full length genomic KIR haplotype sequences. We leveraged these data and tools from our long-read KIR haplotype assembly algorithm to define and align KIR haplotypes at

PURPOSE Ultrahigh resolution (UHR) HLA matching is reported to result in better outcomes following unrelated donor hematopoietic cell transplantation, improving survival and reducing post-transplant complications. However, most studies included relatively small numbers of patients. Here we report the findings from a large, multicenter validation study. METHODS UHR HLA typing was available on 5,140 conventionally 10 out of 10 HLA-matched patients with malignant disease transplanted between 2008 and 2017. RESULTS After UHR HLA typing, 82% of pairs remained 10 out of 10 UHR-matched; 12.3% of patients were 12 out of 12 UHR HLA-matched. Compared with 12 out of 12 UHR-matched patients, probabilities of grade 2-4 acute graft-versus-host disease (aGVHD) were significantly increased with UHR mismatches (overall P = .0019) and in those patients who were HLA-DPB1 T-cell epitope permissively mismatched or nonpermissively mismatched (overall P = .0011). In the T-cell–depleted subset, the degree of UHR HLA mismatch was only associated with increased transplant-related mortality (TRM) (overall P = .0068). In the T-cell–replete subset, UHR HLA matching was associated with a lower probability of aGVHD (overall P = .0020); 12 out of 12 UHR matching was associated with reduced TRM risk when compared with HLA-DPB1 T-cell epitope permissively mismatched patients, whereas nonpermissive mismatching resulted in a greater risk (overall P = .0003). CONCLUSION This study did not confirm that UHR 12 out of 12 HLA matching increases the probability of overall survival but does demonstrate that aGVHD risk, and in certain settings TRM, is lowest in UHR HLA-matched pairs and thus warrants consideration when multiple 10 out of 10 HLA-matched donors of equivalent age are available.

Purpose Disease relapse remains a major challenge to successful outcomes in patients who undergo allogeneic hematopoietic cell transplantation (HCT). Donor natural killer (NK) cell alloreactivity in HCT can control leukemic relapse, but capturing alloreactivity in HLA-matched HCT has been elusive. HLA expression on leukemia cells—upregulated in the post-HCT environment—signals for NK cell inhibition via inhibitory killer immunoglobulin-like (KIR) receptors and interrupts their antitumor activity. We hypothesized that varied strengths of inhibition among subtypes of the ubiquitous KIR3DL1 and its cognate ligand, HLA-B, would titrate NK reactivity against acute myelogenous leukemia (AML). Patients and Methods By using an algorithm that was based on polymorphism-driven expression levels and specificities, we predicted and tested inhibitory and cytotoxic NK potential on the basis of KIR3DL1/HLA-B subtype combinations in vitro and evaluated their impact in 1,328 patients with AML who underwent HCT from 9/10 or 10/10 HLA-matched unrelated donors. Results Segregated by KIR3DL1 subtype, NK cells demonstrated reproducible patterns of strong, weak, or noninhibition by target cells with defined HLA-B subtypes, which translated into discrete cytotoxic hierarchies against AML. In patients, KIR3DL1 and HLA-B subtype combinations that were predictive of weak inhibition or noninhibition were associated with significantly lower relapse (hazard ratio [HR], 0.72; P = .004) and overall mortality (HR, 0.84; P = .030) compared with strong inhibition combinations. The greatest effects were evident in the high-risk group of patients with all KIR ligands (relapse: HR, 0.54; P < .001; and mortality: HR, 0.74; P < .008). Beneficial effects of weak and noninhibiting KIR3DL1 and HLA-B subtype combinations were separate from and additive to the benefit of donor activating KIR2DS1. Conclusion Consideration of KIR3DL1-mediated inhibition in donor selection for HLA-matched HCT may achieve superior graft versus leukemia effects, lower risk for relapse, and an increase in survival among patients with AML.

The killer cell immunoglobulin-like receptor (KIR) region of human chromosome 19 contains up to 16 genes for natural killer (NK) cell receptors that recognize human leukocyte antigen (HLA)/peptide complexes and other ligands. The KIR proteins fulfill functional roles in infections, pregnancy, autoimmune diseases and transplantation. However, their characterization remains a constant challenge. Not only are the genes highly homologous due to their recent evolution by tandem duplications, but the region is structurally dynamic due to frequent transposon-mediated recombination. A sequencing approach that precisely captures the complexity of KIR haplotypes for functional annotation is desirable. We present a unique approach to haplotype the KIR loci using single-molecule, real-time (SMRT) sequencing. Using this method, we have-for the first time-comprehensively sequenced and phased sixteen KIR haplotypes from eight individuals without imputation. The information revealed four novel haplotype structures, a novel gene-fusion allele, novel and confirmed insertion/deletion events, a homozygous individual, and overall diversity for the structural haplotypes and their alleles. These KIR haplotypes augment our existing knowledge by providing high-quality references, evolutionary informers, and source material for imputation. The haplotype sequences and gene annotations provide alternative loci for the KIR region in the human genome reference GrCh38.p8.

Natural killer (NK) cell recognition and killing of target cells are enhanced when inhibitory killer immunoglobulin-like receptors (KIR) are unable to engage their cognate HLA class I ligands. The genes of the KIR locus are organized into either KIR B haplotypes, containing 1 or more activating KIR genes or KIR A haplotypes, which lack those genes. Analysis of unrelated donor (URD) hematopoietic cell transplants (HCT), given to acute myeloid leukemia (AML) patients between 1988 and 2009, showed that KIR B haplotype donors were associated with better outcomes, primarily from relapse protection. Most of these transplants involved marrow grafts, fully myeloablative (MAC) preparative regimens, and significant HLA mismatch. Because the practice of HCT continues to evolve, with increasing use of reduced intensity conditioning (RIC), peripheral blood stem cell grafts, and better HLA match, we evaluated the impact of URD KIR genotype on HCT outcome for AML in the modern era (2010-2016). This analysis combined data from a prospective trial testing URD selection based on KIR genotypes (n = 243) with that from a larger contemporaneous cohort of transplants (n = 2419). We found that KIR B haplotype donors conferred a significantly reduced risk of leukemia relapse and improved disease-free survival after RIC, but not MAC HCT. All genes defining KIR B haplotypes were associated with relapse protection, which was significant only in transplant recipients expressing the C1 epitope of HLA-C. In the context of current HCT practice using RIC, selection of KIR B donors could reduce relapse and improve overall outcome for AML patients receiving an allogeneic HCT.

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is a curative option for blood cancers, but the coupled effects of graft-versus-tumor and graft-versus-host disease (GVHD) limit its broader application. Outcomes improve with matching at HLAs, but other factors are required to explain residual risk of GVHD. In an effort to identify genetic associations outside the major histocompatibility complex, we conducted a genome-wide clinical outcomes study on 205 acute myeloid leukemia patients and their fully HLA-A–, HLA-B–, HLA-C–, HLA-DRB1–, and HLA-DQB1–matched (10/10) unrelated donors. HLA-DPB1 T-cell epitope permissibility mismatches were observed in less than half (45%) of acute GVHD cases, motivating a broader search for genetic factors affecting clinical outcomes. A novel bioinformatics workflow adapted from neoantigen discovery found no associations between acute GVHD and known, HLA-restricted minor histocompatibility antigens (MiHAs). These results were confirmed with microarray data from an additional 988 samples. On the other hand, Y-chromosome–encoded single-nucleotide polymorphisms in 4 genes (PCDH11Y, USP9Y, UTY, and NLGN4Y) did associate with acute GVHD in male patients with female donors. Males in this category with acute GVHD had more Y-encoded variant peptides per patient with higher predicted HLA-binding affinity than males without GVHD who matched X-paralogous alleles in their female donors. Methods and results described here have an immediate impact for allo-HCT, warranting further development and larger genomic studies where MiHAs are clinically relevant, including cancer immunotherapy, solid organ transplant, and pregnancy.

For hematopoietic stem cell transplantation (HCT) HLA 10/10 (HLA-A, B, C, DRB1, DQB1) matched donors are optimal, but are not available for all patients. The identification of permissive/non-immunogenic mismatches may improve the outcome of HLA mismatched transplants. We hypothesize that HLA alleles identical within the antigen recognition domain (ARD), but mismatched outside the peptide binding groove or α-helices are often permissive mismatches. We evaluated the functional impact of non-ARD mismatches by performing in vitro functional T cell assays. Cytotoxic T Lymphocyte precursor assays were performed for 23 HLA class I mismatches and 96% (22 out of 23) were negative. Mixed lymphocyte reaction assays were conducted on 10 HLA class II mismatches and all were negative. However, 4 out of 10 combinations were positive in the Elispot and all involved one direction: a DRB1*14:01/DRB3*02:01 responder against a DRB1*14:54/DRB3*02:02 stimulator. These positive responses were confirmed by Primed Lymphocyte Testing and the DRB1* mismatch seemed to be responsible for the response. In conclusion, HLA mismatches with amino-acid differences outside the ARD are not very immunogenic. However, in some cases weak T cell reactivity in vitro can be observed. The impact of these responses on clinical outcome of HCT remains to be established.

The goals of the KIR component of the 17th International HLA and Immunogenetics Workshop (IHIW) were to encourage and educate researchers to begin analyzing KIR at allelic resolution, and to survey the nature and extent of KIR allelic diversity across human populations. To represent worldwide diversity, we analyzed 1269 individuals from ten populations, focusing on the most polymorphic KIR genes, which express receptors having three immunoglobulin (Ig)-like domains (KIR3DL1/S1, KIR3DL2 and KIR3DL3). We identified 13 novel alleles of KIR3DL1/31, 13 of KIR3DL2 and 18 of KIR3DL3. Previously identified alleles, corresponding to 33 alleles of KIR3DL1/31, 38 of KIR3DL2, and 43 of KIR3DL3, represented over 90% of the observed allele frequencies for these genes. In total we observed 37 KIR3DL1/S1 allotypes, 40 for KIR3DL2 and 44 for KIR3DL3. As KIR allotype diversity can affect NK cell function, this demonstrates potential for high functional diversity worldwide. Allelic variation further diversifies KIR haplotypes. We determined KIR3DL3 similar to KIR3DL1/S1 similar to KIR3DL2 haplotypes from five of the studied populations, and observed multiple population-specific haplotypes in each. This included 234 distinct haplotypes in European Americans, 191 in Ugandans, 35 in Papuans, 95 in Egyptians and 86 in Spanish populations. For another 35 populations, encompassing 642,105 individuals we focused on KIR3DL2 and identified another 375 novel alleles, with approximately half of them observed in more than one individual. The KIR allelic level data gathered from this project represents the most comprehensive summary of global KIR allelic diversity to date, and continued analysis will improve understanding of KIR allelic polymorphism in global populations. Further, the wealth of new data gathered in the course of this workshop component highlights the value of collaborative, community-based efforts in immunogenetics research, exemplified by the IHIW.

Traditional DNA-based typing focuses primarily on interrogating the exons of human leukocyte antigen (HLA) genes that form the antigen recognition domain (ARD). The relevance of mismatching donor and recipient for HLA variation outside the ARD on hematopoietic stem cell transplantation (HSCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) matched unrelated donor transplant pairs (n = 360). Next-generation DNA sequencing was used to characterize both HLA exons and introns for HLA-A, -B, -C alleles; exons 2, 3 and the intervening intron for HLA-DRB1 and exons only for HLA-DQA1 and -DQB1. Over 97% of alleles at each locus were matched for their nucleotide sequence outside of the ARD exons. Of the 4320 allele comparisons overall, only 17 allele pairs were mismatched for non-ARD exons, 41 for noncoding regions and 9 for ARD exons. The observed variation between donor and recipient usually involved a single nucleotide difference (88% of mismatches); 88% of the non-ARD exon variants impacted the amino acid sequence. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D-R pairs will be difficult to assess in HSCT outcome studies because these mismatches do not occur very frequently.

BACKGROUND: Allogeneic hematopoietic stem cell transplants from unrelated donors are routinely used in the treatment of patients with hematologic malignancies. These cellular products are often collected off-site and require transport from the collection site to transplantation centers. However, the effects of transport conditions and media on stem cell graft composition during short-term storage have not been well described. STUDY DESIGN AND METHODS: Five bone marrow (BM), four filgrastim-mobilized peripheral blood stem cell (PBSC), and four nonmobilized peripheral blood mononuclear cell (PBMNC) products were collected from healthy volunteer donors and stored at 4 or 20°C for up to 72 hours in 10% PlasmaLyte A plus anticoagulants such as 10% acid citrate dextran-A (ACD-A) and/or 10 IU/mL heparin. Products were evaluated at 0, 24, 48, and 72 hours for cellular content, viability, and metabolic activities. RESULTS: BM products maintained equivalent cell viability when stored at either 4 or 20°C over 72 hours, but cell viability was better maintained for PBSC products stored at 4°C. The mean viable CD34+ cell recovery for PBSC and BM products stored over 72 hours at 4°C was higher than 75%. Significantly lower CD34+ cell and colony-forming unit recoveries were seen in PBSC products but not BM products stored at room temperature. Faster lactic acid accumulation was observed in PBMNC and PBSC products stored without ACD-A. CONCLUSIONS: Seventy-two-hour storage of BM, PBSC, and PBMNC products at refrigerated temperature maintains optimal cell viability and recovery. Anticoagulation with ACD-A is preferred over heparin to reduce lactic acid accumulation in the product media.

Aim Donors homozygous for the CCR5 delta 32 deletion are of interest in hematopoietic stem cell transplantation (HCT) for patients infected by both HIV-1 and a hematologic malignancy. The mutation has been published to confer natural HIV resistance to individuals carrying two copies, while heterozygous individuals show increased resistance and lower viral loads compared to wild type. The aim of this study was to characterize the CCR5 delta 32 deletion frequency in the diverse unrelated donor populations listed on the National Marrow Donor Program’s Be The Match Registry. Methods Next Generation Sequencing by the Illumina MiSeq was used to target the 32 base pair deletion in the CCR5 gene for 495,530 URDs between 2016-2018. The frequency of donors identified as homozygous, heterozygous and wild type for the mutation was calculated for each of broad race/ethnic groups; Black, Asian/Pacific Islander, White, Hawaiian, Hispanic and Native American Indian. Results Overall, .73% URDs were identified as CCR5 delta 32 homozygous in our cohort. The frequency of homozygotes found in unrelated registry donors self-identified as race group of White was similar (.95%) to previously published data on European individuals (1%). The frequency of homozygotes in the remaining populations was low with .04% observed in Black, .01% in Asian/Pacific Islander, .21% in Hispanic and .24% in Native American. Within donors carrying the homozygous genotype, the population was highly homogeneous, with 98.9% self-identifying as White, given predominate donor recruitment numbers and higher frequency. Conclusions The frequency of self-identified White U.S. registry donors homozygous for the CCR5 delta 32 deletion is consistent with previous studies on individuals of European descent. Given the low frequency of homozygous CCR5 delta32 genotypes in the unrelated donor population, additional treatment methods using multiply mismatched donors can extend the feasibility of this use case for concurrent HCT and infection therapy in HIV patients. Download : Download high-res image (283KB) Download : Download full-size image

Aim In hematopoietic stem cell transplantation (HCT), HLA allele matching between donor and recipient has traditionally focused on the polymorphic antigen recognition domain (ARD). Mismatching at the ARD is known to influence outcomes. This study investigated the genetic differences in the non-ARD regions among ARD-matched donor-recipient pairs to determine possible association with the transplant outcomes. Methods We compared the full-length HLA Class I allele sequences (HLA-A, -B, -C) and partial-length (partial intron 1 through partial intron 3) Class II allele sequences (HLA-DRB1, DQB1) for 4646 high-resolution 10/10 HLA-matched HCT donor-recipient pairs. We developed a comprehensive HLA allele sequence comparison pipeline, which identifies and annotates the mismatched positions between two alleles by their functional region and their protein sequence differences using IMGT/HLA Database (v3.31.0). Results For HLA Class I alleles, 95.4% of the ARD matched alleles have identical sequences outside the ARD, including introns and non-ARD exons. 0.3% of the mismatches were synonymous variants from the ARD region while 0.2% and 0.1% of mismatches found from non-ARD exons were synonymous and nonsynonymous variants, respectively. The intronic variation accounted for 4.2% of the mismatches. Similarly, for HLA Class II alleles, 0.3% of mismatches were synonymous ARD variants, and the mismatches in the non-ARD exons were also very rare (synonymous: 0.3%; nonsynonymous: 0.2%). However, a high degree of polymorphism was observed in the intronic regions of the Class II genes with only 77.3% of the allele pairs having identical sequences. 0.2% and 4.6% of Class I and Class II allele pairs, respectively, showed both exonic and intronic mismatches. Conclusions HCT donor/recipient pairs matched at high resolution for HLA-A, B, C, DRB1 and DQB1 have limited coding variation outside of the ARD. Intronic variation was observed at a higher rate for HLA Class II in our study compared to prior publications (17.3% vs.

Human leukocyte antigen typing of 2578 donor-recipient pairs whose transplantation was facilitated by the National Marrow Donor Program allowed for an in-depth analysis of the accuracy of high-volume allele level testing data. The methods employed provided allele level typing at DRB1/3/5, DQA1, DQB1, DPA1, and DPB1 using sequence-specific oligonucleotide probe hybridization (SSOPH), polymerase chain reaction (PCR) restriction fragment length polymorphism analysis, sequence specific PCR, and direct sequence-based typing (SBT). Each typing was independently tested by two laboratories in Phase 1, and in subsequent phases targeted samples were typed in duplicate by SBT to monitor typing quality. Comparison with prior transplant center typing was also evaluated. SSOPH detected discrepancies ranged from 0.6% at DPB1 to 5.1% at DQB1 in Phase 1. The majority of discrepancies, 62%, resulted from human error such as sample handling, result interpretation, or clerical errors. Alleles that are frequently discrepant have been identified in this predominantly white population.

Aim Allogeneic hematopoietic cell transplantation (AlloHCT) is a curative therapy for certain hematological malignancies, immune deficiencies and inborn errors of metabolism. A major complication of alloHCT is graft-versus-host disease (GVHD) mediated by immunogenic minor histocompatibility antigens (MiHAs). To date, a limited number of MiHAs with HLA restrictions have been identified and well characterized. We developed a comprehensive pipeline to predict potential MHC class I-restricted MiHAs. Methods The MiHA identification pipeline (MiHAIP) integrates diverse biological data sources, including whole genome sequences, HLA genotypes, clinical outcomes, tissue-specific expression, known MiHAs, and minor allele frequencies. MiHAIP incorporates third-party bioinformatics toolboxes, such as RTGtools, snpEff, netchop-3.1 and netMHCpan-3.0, to predict the potential MiHAs in parallel by comparing variants between the donor and recipient. Subsequently, the pipeline simulates antigen processing and presentation on recipients’ cell surface via HLA molecule restriction (figure). Results Whole genome sequences and HLA typing of a cohort of unrelated donor-recipient pairs (n = 205) were loaded to MiHAIP to predict potential MiHAs. It takes on average 22 min to process each donor-recipient pair of whole genome sequence data on a cloud computing environment. Known autosomal MiHAs were examined to validate the sensitivity of the pipeline. MiHAIP outputs the predicted HLA-restricted antigens and their potential GVHD effects. Conclusions The proposed MiHAIP is the first MHC class I-restricted MiHA prediction tool for analyzing whole genome sequences of donor-recipient pairs with HLA matches. It accurately and efficiently identifies known well-characterized MiHAs and new potential MiHAs with high sensitivity.

Aim The goal of this study was to update the 2012 common and well documented (CWD) allele list using data from 3940 class I and 4124 class II assignments obtained by next generation sequencing (NGS) of two registry (USA and Argentina) populations and 360 donor-recipient pairs from the CIBMTR repository. Methods DNA was prepared from blood, buccal swabs, or cell lines. HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs. The sequences of exons and introns were determined by next generation sequencing (NGS) with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Class II NGS sequencing focused on exons 2 and 3; however, some alleles had additional exons and introns characterized. Alleles that were on the well-documented list and alleles not considered CWD based on Mack et al. Tissue Antigens 2013 were identified. Results The majority of alleles detected had previously been designated as common. For example, from the Argentine registry samples, 92% of 2944 HLA-A alleles identified were common. In the total study population, 77 class I alleles previously designated as WD and 97 alleles not defined as C or WD were observed from 1 to >5 times. Thirty-seven of the 174 class I alleles occurred greater than 5 times, e.g., previously undefined A*02:17:02, B*44:02:01:03, and C*17:01:01:05. The majority (90%) of the 19 not CWD alleles were intron variants e.g., A*03:01:01:05, B*39:01:01:03, C*06:02:01:03. Forty-five class II alleles fell into the WD or not defined categories; 29% occurred >5 times. Class II alleles occurring >5 times included DRB1*04:06:02, DQB1*03:01:01:03 and DPB1*04:02:01:02. Conclusions The NGS data expand our knowledge of the frequency of specific full length HLA alleles. The majority of the uncommon alleles characterized in this study are synonymous variations of common alleles.

Aim Traditional DNA-based typing focuses primarily on interrogating the exons of HLA genes that form the ARD (peptide-binding region that interacts with T cell and killer immunoglobulin-like receptors). The relevance of mismatching outside the ARD on hematopoietic stem cell transplantation (HCT) outcomes is unknown. This study was designed to evaluate the frequency of variation outside the ARD in well matched transplant pairs. Methods 360 HLA-A, B, C, DRB1, and DQB1 high resolution matched donor (D)-recipient (R) pairs were selected based on availability of retrospective Sanger-based sequencing and DNA from the CIBMTR Repository. The majority of the population was self-identified Caucasian (80%). Next generation DNA sequencing was performed on the Illumina MiSeq platform and interpreted with Connexio Assign MPS. Class I gene sequences covered 5 ′ UTR-3 ′ UTR; DRB1, intron 1-intron 3; DQA1 5 ′ UTR-exon 4; DQB1, intron 1-3 ′ UTR. DQ noncoding regions were not evaluated. Results The majority (98.1%) of the HLA-A, B, C, DRB1 D-R allele pairs are matched for sequences outside the ARD exons: 0.5% differ in non-ARD exons, 1.9% differ in noncoding regions. A small number (0.2%) differ within ARD exons. Mismatches in non-ARD exons varies from 0.7% for HLA-C and DQA1 to 0% DQB1; noncoding variation ranges from 2.8% for HLA-C to 1.3%, HLA-B and DRB1. Within non-ARD exons, both nonsynonymous (16 allele pairs) and silent (2) variation is present. Intron variation is minor, usually a single nucleotide. Conclusions This is the first study to evaluate the genetic variation and characterize mismatching outside of the ARD in a cohort of HLA-matched donor-recipient pairs. The paucity of exonic mismatches outside of the ARD is striking. Intronic variation is more common but would not contribute to an alloreactive mismatch. At present, it does not appear to be necessary to increase the resolution of HLA typing beyond the ARD in selecting a matched donor except in cases of common non-expressed variants within G-group assignments. The impact of amino acid sequence variation caused by substitutions in exons outside ARD regions in D–R pairs will be difficult to assess in HCT outcome studies since it does not occur very frequently. Further study is warranted to confirm these findings in larger and more diverse cohorts.

Aim The ideal graft source for allogeneic hematopoietic stem cell transplantation (HCT) is an HLA-identical matched sibling (MSD). The Blood and Marrow Transplant Clinical Trials Network recommends that family donors be HLA-A and -B typed at intermediate or higher resolution and HLA-DRB1 typed at high resolution by DNA-based methods as a minimum requirement. Where possible, familial haplotypes should be assigned to establish presumptive high-resolution match identity between the donor and recipient (additionally HLA-C and DQB1 may be considered). Extended typing at additional HLA loci including DPB1 is not commonly performed. Results from unrelated donor HCT studies suggest that mismatches at HLA-DPB1 increase the risk of acute graft versus host disease. Due to the genetic distance from the other classical loci crossover events may lead to mismatching at DPB1 in the MSD setting and therefore may ultimately play a role in transplant outcomes. Methods A cohort of 1199 presumed MSD pairs, from 55 centers, collected by the CIBMTR Research Repository from 2007 to 2015 were analyzed. All pairs were reported to the CIBMTR as HLA identical siblings and received a transplant for either AML (44%), ALL (24%), MDS (26%) and 6% other diseases. The median patient age was 52, 59% were male, 86% received PBSC and 14% BM. To determine the crossover frequency the subjects were typed by targeted exon NGS of exons 2 and 3 for both Class I and Class II yielding G group or better results, at HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1. Results The majority (97.25%) of MSD pairs were identical at all loci tested. A small subset (2.75%) of the pairs contained a mismatch impacting either DPA1, DPB1 or both. The majority of the 33 mismatches, 26 (78.79%) were DPB1, 6 (18.18%) included both DPA1 and DPB1 and 1 (3.03%) only DPA1. Analysis of the T-cell-epitope groups found that 28% of the DPB1 mismatches were not permissive. All were presumed crossover events, but family typing was not available to confirm. Six (18%) of the DPB1 mismatches included a homozygous recipient with a heterozygous donor suggesting a potential loss of heterozygosity. Conclusion The mismatch frequency at the DP loci in MSD pairs was relatively low at 2.75% and 28% were not TCE permissive. The impact of DP mismatching on MSD HCT outcomes are unclear and warrant further study.

The immune responses of natural killer cells are regulated, in part, by killer cell immunoglobulin-like receptors (KIR). The 16 closely-related genes in the KIR gene system have been diversified by gene duplication and unequal crossing over, thereby generating haplotypes with variation in gene copy number. Allelic variation also contributes to diversity within the complex. In this study, we estimated allele-level haplotype frequencies and pairwise linkage disequilibrium statistics for 14 KIR loci. The typing utilized multiple methodologies by four laboratories to provide at least 2x coverage for each allele. The computational methods generated maximum-likelihood estimates of allele-level haplotypes. Our results indicate the most extensive allele diversity was observed for the KIR framework genes and for the genes localized to the telomeric region of the KIR A haplotype. Particular alleles of the stimulatory loci appear to be nearly fixed on specific, common haplotypes while many of the less frequent alleles of the inhibitory loci appeared on multiple haplotypes, some with common haplotype structures. Haplotype structures cA01 and/or tA01 predominate in this cohort, as has been observed in most populations worldwide. Linkage disequilibrium is high within the centromeric and telomeric haplotype regions but not between them and is particularly strong between centromeric gene pairs KIR2DL5~KIR2DS3S5 and KIR2DS3S5~KIR2DL1, and telomeric KIR3DL1~KIR2DS4. Although 93% of the individuals have unique pairs of full-length allelic haplotypes, large genomic blocks sharing specific sets of alleles are seen in the most frequent haplotypes. These high-resolution, high-quality haplotypes extend our basic knowledge of the KIR gene system and may be used to support clinical studies beyond single gene analysis. © 2012 Vierra-Green et al.

DNA sequencing of 268 individuals drawn from four US populations carrying two unresolved DRB1*14 alleles differing only outside the antigen recognition site identified DRB1*1454 in the majority. A database of 4222 human leukocyte antigen (HLA)-matched hematopoietic stem cell transplantation donor–recipient pairs was queried to determine the number likely mismatched for DRB1*140101/DRB1*1454 but matched for class I loci. A power calculation suggests that more than 88,000 transplants among European Americans will be needed to identify sufficient 7/8 allele-matched pairs to evaluate the impact of the DRB1*140101/DRB1*1454 mismatch on transplant outcome. Molecular modeling of the HLA-DR interaction with the T-cell receptor and with CD4 suggests that the amino acid substitution distinguishing the two alleles will have minimal impact on allorecognition.

Aim The knowledge of complete haplotype structure and underlying genetic variation is often key in understanding gene function, phenotype, and medically relevant traits such as susceptibility to disease and individual response to drugs. However, genome wide SNP arrays and shotgun sequencing methods are limited in their ability to identify phased genetic variations at high resolution over extended localized regions. Methods To address this, we improved methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generation that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As specific applications, we targeted subregions of the human major histocompatibility complex (MHC) and the killer Ig-like receptor (KIR) locus, two of the most polymorphic genomic regions in humans. Results For the MHC region, an 800 kb segment of the MHC class II region including the highly polymorphic DR, DQ, and DP genes was targeted. Complete phased sequences were obtained encompassing the DR4 common extended haplotype – a haplotype associated with autoimmune disorders - from four class II heterozygous individuals. For the KIR locus, which spans some 200 kb including tandem repeats of highly homologous polymorphic loci, we targeted several KIR haplotype heterozygous individuals. Conclusions Complete phased sequences for all haplotypes were obtained from all individuals with a high level of data accuracy. The results demonstrate the approach is amenable to cost-effective scale-up to generate scores to hundreds of phased chromosomal sequences of extended lengths that encompass targeted genomic regions associated with disease.

Aim Recent data show that DP matching plays an important role in hematopoietic cell transplantation. Our previous work demonstrated that in European Americans haplotypic associations for the DP heterodimer are determined by near complete linkage disequilibrium between amino acid motifs on DPA1 (pos 31) and DPB1 (pos 85–87). We aimed to generate motif and allele-level haplotype frequencies (HF) for four race groups to extend these insights to minorities. Methods We calculated DPA1∼DPB1 HF at allele and motif level from 16,802 Be the Match Registry® members typed by DNA methods, resolving phase, allelic and motif ambiguity using expectation maximization. Motif frequencies were calculated for position 31 on DPA1 and positions 85–87 on DPB1, which play a critical role in the P1 pocket of the peptide binding region. Normalized linkage disequilibrium (LD) was calculated on each haplotype (D′) and globally (Wn) for each population. Results The top three allele-level haplotypes in all populations account for over 66% of the HF, with DPA1∗01:03 ∼ DPB1∗04:01 most common in the Caucasian (CAU), Asian Pacific Islander (API) and Hispanic (HIS) populations and DPA1∗02:02 ∼ DPB1∗01:01 in the African American (AFA) population. Wn for DPA1∼DPB1 allele associations for CAU, HIS, API, and AFA populations was .55, .45, .49, and .52 respectively. At the motif level there were only four possible haplotypes. M ∼ GPM and Q ∼ EAV were the two most common in every population. M ∼ GPM was the most common in CAU, HIS and API with frequencies 69%, 68% and 56% respectively, with D′ = 0.97 for CAU (complete LD). The most common haplotype for AFA was Q ∼ EAV, at 48% and D′ = .99. Conclusions Low global LD was observed at the allele level for all populations. However, at the motif level haplotypes were in near complete LD suggesting that for the DPA1 ∼ DPB1 heterodimer the unit of selection for all populations is the combined amino acid motif rather than the allele, as previously observed in European Americans.

Aim For hematopoietic stem cell transplantation (HSCT) HLA identical sibling donors or HLA matched unrelated donors are the most ideal donors. As 10/10 (HLA-A, -B, -C,-DRB1, -DQB1) matched donors, are not available for all patients, the identification of permissive/non-immunogenic mismatches is important to inmprove the outcome of HLA mismatched transplants. Alleles that share the amino acid sequence of the antigen recognition site (ARS)(exon 2 and 3 for HLA class I or exon 2 for HLA-DRB1) or differences outside the peptide binding groove or α-helices (as the HLA-C*03:03 vs C*03:04 mismatch) are good candidates for permissive mismatches as these differences are not likely to induce T cell alloreactivity. Methods An inventory of the functional impact of such mismatches was performed by in vitro cellular assays including the Cytotoxic T Lymphocyte precursor test (CTLp) for HLA-class I allele differences and the MLC, IFN-γ elispot and Primed Lymphocyte Test (PLT) for HLA-DRB1 differences. Results CTLp assays were performed for: HLA-A*23:17 vs 23:01 (differ at pos 283, exon 5), HLA-C*07:06 vs *07:01 (pos 307 & 324 exon 5 & 6), HLA-C*04:30 vs 04:01 (pos 327, exon 7), HLA-C*03:62 vs 03:03:01 (pos 197, exon 4), HLA-C*03:03 vs 03:04 (pos 91, exon 3) and HLA-B*35:02 vs 35:04 (pos 109, exon 3). The only positive CTLp test was for HLA-C*07:06 vs *07:01. DRB1*14:01 vs DRB1*14:54 differ at pos 112 and a 2nd mismatch of DRB3*02:01 vs DRB3*02:02, respectively, yielded complicated results. Five of 6 combinations tested did not respond in the MLC and 3 of 6 in the Elispot against the combined DRB1/DRB3 mismatch and only in one direction; DRB1*14:01/DRB3*02:01 against DRB1*14:54/DRB3*02:02. PLT results suggested that the DRB1* mismatch was responsible for the response. Conclusions In conclusion, mismatches involving positions outside the ARS are not very immunogenic. However, some mismatches can lead to T cell reactivity in vitro. The impact of these mismatches on clinical outcome of HSCT remains to be established.

Aim ARS identical alleles are considered high resolution matched for unrelated donor stem cell transplant; however, it is unknown if mismatches outside of the ARS region impact transplant outcome. We investigated ARS identical allele pairs, HLA-A∗02:01/09, HLA-B∗44:02/27, HLA-C∗07:01/06 and C∗07:01/18, to determine if these alleles exist on the same haplotype in the Be the Match Registry®. The goal was to identify cases for in vitro cellular assays of alloreactivity to ARS identical alleles. Methods Over nine million donors on the Be the Match Registry were queried. Haplotypes were selected where both ARS alleles have been reported previously. These haplotypes were paired with common haplotypes within each race. High resolution sequence based typing was completed on 40 Caucasian donors for A∗02:01/09, 58 Caucasian donors for B∗44:02/27, 87 Asian Pacific Islander donors for C∗07:01/06, and 64 African American donors for C∗07:01/18. Results A∗02:01/09 was evaluated in two haplotype pairs: 98% carried A∗02:01, 2% were neither A∗02:01 nor A∗02:09 and none carried A∗02:09. B∗44:02/27 was evaluated in two haplotype pairs: 69% were B∗44:02, 22% were B∗44:27 and 9% were neither B∗44:02 nor B∗44:27; however, B∗44:02 and B∗44:27 alleles were never found on the same haplotype. Within two haplotype pairs tested for C∗07:01/06, 98% of donors carried the C∗07:06 allele, 2% carried neither C allele of interest, while none carried C∗07:01. For the C∗07:01/18, eleven haplotype pairs were investigated, 53% of donors carried C∗07:18 and none carried C∗07:01. The remaining 47% of donors carried other C alleles. Conclusions The results obtained from this study for A∗02:01/09, B∗44:02/27, C∗07:01/06 and C∗07:01/18 suggest that the chance of encountering an isolated mismatch between these ARS identical alleles on the haplotypes investigated in an unrelated donor transplant setting would be quite low. Work will continue to identify isolated ARS mismatches for evaluation of alloreactivity in in vitro cellular assays.

Abstract 349 Natural killer (NK) cells are vital in the control of viral infection and malignancy, in particular AML. Affinity strength between NK receptors and their HLA ligands control both NK effector function and degree of NK inhibition. Differences in binding affinity between allotypes of the NK receptor KIR3DL1 and allotypes of its ligand HLA-Bw4 depend on the expression levels of the receptor and the amino acid residue at position 80 in the ligand. Because receptor-ligand affinities are associated with differences in HIV control, we hypothesized that different affinities between donor KIR3DL1 and donor-recipient HLA-Bw4 allotypes would impact the risk for AML relapse following allogeneic hematopoietic stem cell transplantation (HCT). Methods: We evaluated 299 AML patients who underwent allogeneic HCT from an unrelated donor between 1995 and 2002. Clinical data, HLA allotyping, and donor DNA were provided by the CIBMTR. KIR3DL1 allotyping was executed using PCR- and sequence-based methods. Donors were segregated into those with high-, low- and null expressing KIR3DL1 allele groups [3DL1-H (n=130), 3DL1-L (n=69), 3DL1-N (n=82)] and HLA-B allele groups (Bw6/Bw6, Bw4-I80, Bw4-T80). 3DL1-N genotypes are predictive of poor surface expression and were analyzed separately. Patients and donors were matched at 9 or 10 HLA loci in all cases, with only 3 donor-patient pairs mismatched for HLA-Bw4 ligands. Affinity cohorts were compared using Cox regression for the time-to-event outcomes of relapse and overall mortality (OM). Kaplan-Meier estimates of overall survival and cumulative incidence estimates of relapse were obtained. Results: Recipients of 3DL1-H and 3DL1-L donors were analyzed for high and low-affinity associations with post-HCT AML relapse. Among patients with a 3DL1-H donor, those transplanted from donors with the low-affinity KIR/HLA allotype combination 3DL1-H/Bw4-T80 had lower risk of relapse when compared to those with the high-affinity 3DL1-H/Bw4-I80 combination (HR 0.22; p=.003, Table) and even moreso when compared to the extra-high affinity combinations of 3DL1-H with Bw4-I80-B*2702 or B*57 (HR 0.10; p Conclusions: Protection from relapse of AML after unrelated HCT is associated with the affinity between donor KIR3DL1 and HLA-Bw4, ranging from the highly protective low-affinity donor KIR3DL1/HLA-Bw4 allotype combinations to the susceptible and “super-susceptible” effects of high- and extra-high affinity KIR/HLA combinations. These data support the consideration of KIR and HLA allotype combinations in stem cell donor selection algorithms to minimize the risk of AML relapse following HCT. Because >95% of donors are positive for KIR3DL1 and 69% of patients are positive for HLA-Bw4, incorporation of KIR3DL1/HLA-Bw4 allotypes in donor selection algorithms is a highly relevant and feasible goal. Disclosures: No relevant conflicts of interest to declare.

Animal studies point to a strong role for MHC-specific antibody as a cause for failed donor hematopoietic cell engraftment, but the role of donor-directed HLA-specific allo-antibodies in human transplants has been controversial. To investigate such a role, we used a retrospective case-control design and studied unrelated donor:recipient pairs whose transplants were facilitated through the NMDP. A total of 37 cases with graft failure and 78 matched control pairs were evaluated. The 37 graft failure cases were selected based on survival beyond 28 days of transplant with no sustained engraftment, and available cryopreserved recipient serum to test for HLA-specific alloantibodies. Up to 3 controls were selected for each case, and matched for disease, disease status, graft type, patient age and year of transplant. Patients had AML, ALL, CML or MDS, 98% received myeloablative conditioning regimens, 100% received T replete grafts, 97% received marrow, and 97% received calcineurin-based GVHD prophylaxis. Patients and donors were retrospectively typed for HLA-A,B,Cw,DRB1,DQB1,DQA1,DPB1,and DPA1 by sequencing or other high resolution typing methods. Stored pre-transplant serum samples (patients and controls) were retrieved from the NMDP Research Repository, and assayed for HLA antibodies by solid-phase FlowPRA (One Lambda, Inc). All positive samples were evaluated for HLA specificity by single-antigen microparticles (LabScreen, One Lambda, Inc). Among the 37 failed transplants, 11 (30%) recipients possessed alloantibodies specific for donor HLA Class I or Class II, compared to only 3 (4%) of 78 controls. HLA-DR or DQ specific antibodies were not detected; hence, all HLA Class II-specific antibodies were directed to DP. Recipients with anti-DP antibodies against the donor mismatched DP were at increased risk of graft failure, indicating the importance of recipient antibodies directed against mismatched donor DP antigens. Exact conditional logistic regression analysis for the presence of either Class I HLA-A,B,Cw or Class II HLA-DP antibodies showed similar findings (Class I alone: OR. 6.31: 95% CI 1.17–62.9; p=0.03, Class II alone: OR 12.00; 95% CI 1.46–551.97; p=0.01, Class I and II combined: OR 19.08; 95% CI 2.72–828.49; p=0.0003). Further analyses were conducted to evaluate a limited set of covariates not accounted for in the case:control study, i.e. patient CMV status, cell dose and HLA-C match. Cell dose and CMV status were independently predictive of engraftment, p=0.01 and 0.03, respectively. No effect was observed for HLA-Cw match (p=0.84). The presence of anti-donor HLA Class I or II antibodies was predictive of engraftment when adjustment was made for either cell dose (OR 15.49; 95% CI 2.06–697.83; p=0.002) or CMV status (OR 7.94; 95% CI 0.97–367.84; p=0.05). In summary, these results indicate that donor-specific HLA Class I or Class II antibodies in recipients of unrelated donor hematopoietic cell transplants are associated with failed engraftment. We recommend that, as a “Standard-of-Practice”, all potential recipients be screened for the presence of HLA class I and class II antibodies including HLA DP. Donors should be excluded if they carry mismatched HLA types against which the patient has specific antibodies.

Here, we present results for DPA1 and DPB1 four-digit allele-level typing in a large (n = 5,944) sample of unrelated European American stem cell donors previously characterized for other class I and class II loci. Examination of genetic data for both chains of the DP heterodimer in the largest cohort to date, at the amino acid epitope, allele, genotype, and haplotype level, allows new insights into the functional units of selection and association for the DP heterodimer. The data in this study suggest that for the DPA1-DPB1 heterodimer, the unit of selection is the combined amino acid epitope contributed by both the DPA1 and DPB1 genes, rather than the allele, and that patterns of LD are driven primarily by dimer stability and conformation of the P1 pocket. This may help explain the differential pattern of allele frequency distribution observed for this locus relative to the other class II loci. These findings further support the notion that allele-level associations in disease and transplantation may not be the most important unit of analysis, and that they should be considered instead in the molecular context. Electronic supplementary material The online version of this article (doi:10.1007/s00251-012-0615-3) contains supplementary material, which is available to authorized users.