Revolutionising High Resolution HLA Genotyping for Transplant Assessment: Validation, Implementation and Challenges of Oxford Nanopore Technologies' Q20+ Sequencing.

The advent of third-generation sequencing (TGS) represents a significant shift in the field of genetic sequencing, enabling single-molecule sequencing to overcome limitations of short-read NGS platforms. Several studies have assessed the utilisation of TGS in HLA genotyping, though many of these studies have described the high error rate as an obstacle to achieving a robust and highly accurate HLA typing assay. In 2021, Oxford Nanopore Technologies (ONT) released the high-accuracy sequencing Kit 14 and the MinION flow cell model R10.4.1, which were reported to achieve sequencing accuracies up to 99%. The aim of this study was to validate this novel high-accuracy sequencing kit for HLA genotyping coupled with a full-gene HLA PCR assay. Comparison with historical data obtained using legacy flow cell models such as R9.4, R10.3 and R10.4 was also done to assess progressive improvement in sequencing performance with each sequential release. The workflow was validated based on data throughput, sequence quality and accuracy, and HLA genotyping resolution. An initial validation was performed using an internal reference panel of 42 samples representing common HLA allele groups, followed by an analysis of data obtained from 111 sequencing batch runs since the implementation, to assess assay performance and define quality control metrics to assess inter-run variability and monitor quality. Furthermore, challenges arising from MinION flow cell stability and use, and assessment of barcode contamination are discussed. The findings of this study highlight advantages of ONT sequencing kit 14/R10.4.1 for HLA genotyping and the implementation considerations for the routine diagnostic HLA laboratory.

Report from the extended HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype of the 18th international HLA and immunogenetics workshop.

The primary goal of the HLA-DPA1 ~ promoter ~ HLA-DPB1 haplotype component of the 18th IHIWS was to characterise the extended haplotypes within the HLA-DP region and survey the extent of genetic diversity in this region across human populations. In this report, we analysed single-nucleotide polymorphisms (SNPs) in 255 subjects from 6 different cohorts. The results from the HLA-DP haplotype component have validated findings from the initial pilot study. SNPs in this region were inherited in strong linkage, particularly HLA-DPA1, SNP-linked promoter haplotypes and motifs in exon 2 of HLA-DPB1. We reported 17 SNP-linked haplotypes in the promoter region. Together with HLA-DPA1 and HLA-DPB1 alleles, they formed 74 distinct extended HLA-DP haplotypes in 438 sequences. We also observed the presence of region-specific alleles and promoter haplotypes. Our approach involved phasing extended SNPs including promoter SNPs, HLA-DPA1 and HLA-DPB1 alleles, in a 22 kb region, GRCh38/hg38 (chr6:33,064,111-33,086,679), followed by clustering of these SNPs as one extended haplotype. This hierarchical clustering revealed four major clades, suggesting that haplotypes within each clade may have diverged from a common ancestral haplotype and undergone similar evolutionary processes. The correlation between HLA-DPA1 and the promoter region raises questions about the role of HLA-DPA1 antigen in the heterodimer. This finding requires validation on a larger sample size specifically designed for anthropological analysis. Nevertheless, the results from this study highlight the clinical potential of selecting better-matched donors for patients awaiting haematopoietic stem cell transplants from genetically overlapping groups that share common ancestral haplotypes.

GRAMM: A new method for analysis of HLA in families.

Recently, haplo-identical transplantation with multiple HLA mismatches has become a viable option for stem cell transplants. Haplotype sharing detection requires the imputation of donor and recipient. We show that even in high-resolution typing when all alleles are known, there is a 15% error rate in haplotype phasing, and even more in low-resolution typings. Similarly, in related donors, the parents' haplotypes should be imputed to determine what haplotype each child inherited. We propose graph-based family imputation (GRAMM) to phase alleles in family pedigree HLA typing data, and in mother-cord blood unit pairs. We show that GRAMM has practically no phasing errors when pedigree data are available. We apply GRAMM to simulations with different typing resolutions as well as paired cord-mother typings, and show very high phasing accuracy, and improved allele imputation accuracy. We use GRAMM to detect recombination events and show that the rate of falsely detected recombination events (false-positive rate) in simulations is very low. We then apply recombination detection to typed families to estimate the recombination rate in Israeli and Australian population datasets. The estimated recombination rate has an upper bound of 10%-20% per family (1%-4% per individual).

C3d-binding assay for the detection of complement activating HLA antibodies: A useful tool for allocation to highly sensitised recipients in the post-CDC era?

The CDC crossmatch test is being phased out in solid organ donor allocation, and standard luminex single antigen bead assays do not differentiate complement activating function of HLA antibodies. The current study investigated the LIFECODES C3d-binding assay to determine if it could accurately predict actual T and B cell CDC results in a cohort of highly sensitised patients. Nineteen serum samples from different highly sensitised solid organ patients were crossmatched against cells from 62 unique donors, with 174 total T and B cell crossmatches performed. The sera also underwent SAB assay using OLI and LC platforms, and C3d-binding assay. Complement activating ability of each unique HLA antibody specificity detected using SAB was assigned based on the actual CDC results, which was then used to determine the accuracy of the C3d-binding assay. The C3d-binding assay was found to be highly accurate, with sensitivity of 95%, specificity 89% and negative predictive value 97% for class I DSA and the T cell CDC crossmatch results. Furthermore, we found 100% accuracy for prediction of the complement activating function of HLA-C antibodies. Negative predictive value of above 90% was also found for HLA class II DSA. C3d-binding proved more accurate than virtual crossmatch alone to predict CDC results. This study confirms that the C3d-binding assay predicts actual CDC crossmatch results accurately. In particular, the high negative predictive value of the C3d-binding assay may be extremely useful to define HLA antibodies that do not activate complement in highly sensitised recipients.

Nanopore sequencing data analysis using Microsoft Azure cloud computing service.

Genetic information provides insights into the exome, genome, epigenetics and structural organisation of the organism. Given the enormous amount of genetic information, scientists are able to perform mammoth tasks to improve the standard of health care such as determining genetic influences on outcome of allogeneic transplantation. Cloud based computing has increasingly become a key choice for many scientists, engineers and institutions as it offers on-demand network access and users can conveniently rent rather than buy all required computing resources. With the positive advancements of cloud computing and nanopore sequencing data output, we were motivated to develop an automated and scalable analysis pipeline utilizing cloud infrastructure in Microsoft Azure to accelerate HLA genotyping service and improve the efficiency of the workflow at lower cost. In this study, we describe (i) the selection process for suitable virtual machine sizes for computing resources to balance between the best performance versus cost effectiveness; (ii) the building of Docker containers to include all tools in the cloud computational environment; (iii) the comparison of HLA genotype concordance between the in-house manual method and the automated cloud-based pipeline to assess data accuracy. In conclusion, the Microsoft Azure cloud based data analysis pipeline was shown to meet all the key imperatives for performance, cost, usability, simplicity and accuracy. Importantly, the pipeline allows for the on-going maintenance and testing of version changes before implementation. This pipeline is suitable for the data analysis from MinION sequencing platform and could be adopted for other data analysis application processes.

A Novel Targeted Amplicon Next-Generation Sequencing Gene Panel for the Diagnosis of Common Variable Immunodeficiency Has a High Diagnostic Yield: Results from the Perth CVID Cohort Study.

With the advent of next-generation sequencing (NGS), monogenic forms of common variable immunodeficiency (CVID) have been increasingly described. Our study aimed to identify disease-causing variants in a Western Australian CVID cohort using a novel targeted NGS panel. Targeted amplicon NGS was performed on 22 unrelated subjects who met the formal European Society for Immunodeficiencies-Pan-American Group for Immunodeficiency diagnostic criteria for CVID and had at least one of the following additional criteria: disease onset at age <18 years, autoimmunity, low memory B lymphocytes, family history, and/or history of lymphoproliferation. Candidate variants were assessed by in silico predictions of deleteriousness, comparison to the literature, and classified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology criteria. All detected genetic variants were verified independently by an external laboratory, and additional functional studies were performed if required. Pathogenic or likely pathogenic variants were detected in 6 of 22 (27%) patients. Monoallelic variants of uncertain significance were also identified in a further 4 of 22 patients (18%). Pathogenic variants, likely pathogenic variants, or variants of uncertain significance were found in TNFRSF13B, TNFRSF13C, ICOS, AICDA, IL21R, NFKB2, and CD40LG, including novel variants and variants with unexpected inheritance pattern. Targeted amplicon NGS is an effective tool to identify monogenic disease-causing variants in CVID, and is comparable or superior to other NGS methods. Moreover, targeted amplicon NGS identified patients who may benefit from targeted therapeutic strategies and had important implications for family members.

High-resolution HLA allele and haplotype frequencies in several unrelated populations determined by next generation sequencing: 17th International HLA and Immunogenetics Workshop joint report.

The primary goal of the unrelated population HLA diversity (UPHD) component of the 17th International HLA and Immunogenetics Workshop was to characterize HLA alleles at maximum allelic-resolution in worldwide populations and re-evaluate patterns of HLA diversity across populations. The UPHD project included HLA genotype and sequence data, generated by various next-generation sequencing methods, from 4,240 individuals collated from 12 different countries. Population data included well-defined large datasets from the USA and smaller samples from Europe, Australia, and Western Asia. Allele and haplotype frequencies varied across populations from distant geographical regions. HLA genetic diversity estimated at 2- and 4-field allelic resolution revealed that diversity at the majority of loci, particularly for European-descent populations, was lower at the 2-field resolution. Several common alleles with identical protein sequences differing only by intronic substitutions were found in distinct haplotypes, revealing a more detailed characterization of linkage between variants within the HLA region. The examination of coding and non-coding nucleotide variation revealed many examples in which almost complete biunivocal relations between common alleles at different loci were observed resulting in higher linkage disequilibrium. Our reference data of HLA profiles characterized at maximum resolution from many populations is useful for anthropological studies, unrelated donor searches, transplantation, and disease association studies.

Polymorphism clustering of the 21.5 kb DPA-promoter-DPB region reveals novel extended full-length haplotypes.

DPB1 and DPA1 genes share the same promoter region. Single-nucleotide polymorphisms (SNPs) within the regulatory regions of DP have been reported. This study hypothesizes that by including the SNPs in the promoter region of DP, extended haplotypes are defined, and promoter polymorphism is more extensive than what is currently reported. To identify the SNPs in the region of interest, the DP region spanning 21.5 kb was amplified in three separate long-ranged polymerase chain reactions. A DNA panel consisting of 100 samples was selected to represent a broad range of DPB1 alleles. The panel was amplified and sequenced using a dual sequencing strategy. Binary alignment map (BAM) alignments were generated and the mapped sequence alignments were analyzed using Integrative Genomics Viewer. A total of 76 SNPs were identified, and SNPs were clustered into 12 SNP-linked haplotypes. Multiple sequence alignments of promoter sequences indicated four distinct lineages within the connective region (CR) between two genes. The relationship between DPA1, CR, DPB1, and amino acid motifs was found to be correlated with HV1 and HV6. Of the 12 promoter haplotypes, DPB1 alleles observed with ProDP-4 were in complete linkage with HV1/2/5/6, the rs9277534G SNP, and the highly immunogenic T-cell epitope group. Multiple extended haplotypes of different intronic subtypes of the same DPB1 alleles were also identified. This new view of the full DP haplotype shows the relation of polymorphism, genes, and alleles, and provides a basis for future functionality related nomenclature. The novel clustering of the DP-extended haplotype warrants future investigations of DP haplotype matching in the outcome of haematopoietic stem cell transplantation (HSCT).

Role of HLA-DP Expression in Graft-Versus-Host Disease After Unrelated Donor Transplantation.

PURPOSE: The main aim of this study was to evaluate the significance of HLA-DPB1 expression in acute graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) from HLA-A, -B, -C, -DRB1, -DQB1-matched and -mismatched unrelated donors. PATIENTS AND METHODS: Between January 1, 2017, and January 10, 2019, we assessed 19,136 patients who received HCT from an HLA-A, -B, -C, -DRB1, -DQB1-matched or -mismatched unrelated donor performed in Australia, the European Union, Japan, North America, and the United Kingdom between 1988 and 2016. Among transplant recipients with one HLA-DPB1 mismatch, the patient's mismatched HLA-DPB1 allotype was defined as low or high expression. Multivariable regression models were used to assess risks of GVHD associated with high expression relative to low expression HLA-DPB1 mismatches. The effect of increasing numbers of HLA-DPB1 mismatches on clinical outcome was assessed in HLA-mismatched transplant recipients. RESULTS: In HLA-A, -B, -C, -DRB1,-DQB1-matched transplant recipients, donor mismatching against one high-expression patient HLA-DPB1 increased moderate (odds ratio [OR], 1.36; P = .001) and severe acute GVHD (OR, 1.32; P = .0016) relative to low-expression patient mismatches, regardless of the expression level of the donor's mismatched HLA-DPB1. Among transplant recipients with one HLA-A, -B, -C, -DRB1, or -DQB1 mismatch, the odds of acute GVHD increased with increasing numbers of HLA-DPB1 mismatches (OR, 1.23 for one; OR, 1.40 for two mismatches relative to zero mismatches for moderate GVHD; OR, 1.19 for one; OR, 1.40 for two mismatches relative to zero for severe GVHD), but not with the level of expression of the patient's mismatched HLA-DPB1 allotype. CONCLUSION: The level of expression of patient HLA-DPB1 mismatches informs the risk of GVHD after HLA-A, -B, -C, -DRB1, -DQB1-matched unrelated HCT, and the total number of HLA-DPB1 mismatches informs the risk of GVHD after HLA-mismatched unrelated HCT. Prospective consideration of HLA-DPB1 may help to lower GVHD risks after transplantation.

HLA-B leader and survivorship after HLA-mismatched unrelated donor transplantation.

Hematopoietic cell transplantation (HCT) from HLA-mismatched unrelated donors can cure life-threatening blood disorders, but its success is limited by graft-versus-host disease (GVHD). HLA-B leaders encode methionine (M) or threonine (T) at position 2 and give rise to TT, MT, or MM genotypes. The dimorphic HLA-B leader informs GVHD risk in HLA-B-mismatched HCT. If the leader influences outcome in other HLA-mismatched transplant settings, the success of HCT could be improved for future patients. We determined leader genotypes for 10 415 patients receiving a transplant between 1988 and 2016 from unrelated donors with one HLA-A, HLA-B, HLA-C, HLA-DRB1, or HLA-DQB1 mismatch. Multivariate regression methods were used to evaluate risks associated with patient leader genotype according to the mismatched HLA locus and with HLA-A, HLA-B, HLA-C, HLA-DRB1, or HLA-DQB1 mismatching according to patient leader genotype. The impact of the patient leader genotype on acute GVHD and mortality varied across different mismatched HLA loci. Nonrelapse mortality was higher among HLA-DQB1-mismatched MM patients compared with HLA-DQB1-mismatched TT patients (hazard ratio, 1.35; P = .01). Grades III to IV GVHD risk was higher among HLA-DRB1-mismatched MM or MT patients compared with HLA-DRB1-mismatched TT patients (odds ratio, 2.52 and 1.51, respectively). Patients tolerated a single HLA-DQB1 mismatch better than mismatches at other loci. Outcome after HLA-mismatched transplantation depends on the HLA-B leader dimorphism and the mismatched HLA locus. The patient's leader variant provides new information on the limits of HLA mismatching. The success of HLA-mismatched unrelated transplantation might be enhanced through the judicious selection of mismatched donors for a patient's leader genotype.

Different eplet software programs give discordant and incorrect results: An analysis of HLAMatchmaker vs Fusion Matchmaker Eplet calling software.

HLA eplet matching is a novel approach to define acceptable HLA mismatches for transplant recipients. We performed an eplet analysis of three different transplant case-series to determine if the available software programs gave accurate results. Eplet analysis was performed for three different transplant case-series typed by NGS for all HLA class I and II loci. The three different HLA datasets were entered into both the HLAMatchmaker program (v2.1) and OLI Fusion MatchMaker (v4.2) software tools. Eplet results which were discordant were cross referenced against eplet registry and published HLA allele sequence data to determine the correct assignments. The comparison reveals that there was poor concordance between the two eplet programs. Analysis of the same donor/recipient pair often gave rise to different total eplet scores, incorrect eplet mismatches and antibody verification status, and both programs have eplets assigned to incorrect HLA alleles. Overall, the OLI Fusion MatchMaker eplet tool gave more accurate and useful eplet results. Eplet matching is still primarily a research tool. Before eplet matching can enter routine clinical practice further work is required to validate the accuracy of available eplet software programs. Incorrect eplet assignment could have serious adverse consequences in the clinical transplant setting.

Rapid high-resolution HLA genotyping by MinION Oxford nanopore sequencing for deceased donor organ allocation.

Recently, HLA epitopes on donor HLA molecules have been shown to be important in the success of solid organ transplantation. However, these epitopes can only be defined using high-resolution typing results of which are often not available prior to deceased donor allocation. The ability to perform high-resolution typing at all HLA loci for deceased organ donor allocation prior to transplantation would have major clinical benefits, in particular for highly sensitised recipients. We, therefore, developed a rapid high-resolution next generation sequencing (NGS) HLA typing (ONT-Rapid HR HLA) method for on-call deceased donor allocation using the AllType 11 loci single-tube assay (OneLambda Inc), modified in-house to reduce polymerase chain reaction amplification time, and the Oxford Nanopore single-molecule sequencing platform on the Flongle flow cell. The ONT-Rapid HR HLA method was validated on 42 samples previously typed by current on-call sequence-specific oligonucleotide (HistoSpot) and NGS methods (AllType/Ion Torrent). High-resolution typing obtained using the ONT-Rapid HR HLA typing method was 100% concordant with both the current SSO and NGS methods, and in some cases, obtained higher resolution than either of the current methods. The rapid ONT-Rapid HR HLA typing method was able to obtain these typing results at all loci in 4 to 4.5 hours. The novel ONT-Rapid HR HLA typing method is the first reported NGS HLA typing method utilised for deceased donor allocation. The ability to provide high-resolution HLA typing on deceased donors before implantation will in the future allow improvements in matching, which will ultimately provide clinical benefits to patients.

Allograft and Patient Outcomes Between Indigenous and Nonindigenous Kidney Transplant Recipients.

BACKGROUND: Kidney transplant outcomes of indigenous Australians are poorer compared with nonindigenous Australians, but it is unknown whether the type of acute rejection differs between these patient groups or whether rejection mediates the effect between ethnicity, death-censored graft failure (DCGF), and death with a functioning graft (DWFG). METHODS: Biopsy-proven acute rejection (BPAR) rates and types were compared between indigenous and nonindigenous recipients. The associations between ethnicity, BPAR, DCGF, and DWFG were examined using adjusted competing risk analyses, and mediation analysis was conducted to determine whether BPAR mediated the adverse effects between ethnicity and outcomes. RESULTS: Fifty-seven (9.3%) of 616 patients who have received kidney-only transplants between 2000 and 2010 in Western Australia were indigenous. Compared with nonindigenous recipients, BPAR rates were higher in indigenous recipients (42 versus 74 episodes/100 recipients, P < 0.01), with an excess of antibody-mediated rejections. During a median follow-up of 8 years, indigenous recipients were more likely to experience BPAR, DCGF, and DWFG compared with nonindigenous recipients, with adjusted subdistribution hazard ratio of 1.94 (1.39-2.70), 1.53 (0.85-2.76; P = 0.159), and 2.14 (1.13-4.06; P = 0.020), respectively. Although 70% of the effect between ethnicity and DCGF was mediated by BPAR, no similar association was found for DWFG. CONCLUSIONS: Indigenous recipients experienced poorer allograft and patient outcomes compared with nonindigenous recipients, with BPAR an important determinant for DCGF. Future research identifying other risk factors and mediators associated with patient survival in indigenous recipients should be considered a priority.

A novel multiplexed 11 locus HLA full gene amplification assay using next generation sequencing.

The rapid progress of HLA typing techniques has contributed to improving the outcome of haematopoietic stem cell transplantation (HSCT). However, unambiguous HLA typing remains challenging. Next generation sequencing (NGS) has been shown to resolve the HLA typing ambiguity and simplify HLA typing workflows. The aim of this study is to develop a multiplexed full-gene PCR assay for 11 HLA loci that can be used on any NGS platform to provide additional information to the traditionally sequenced regions. The entire gene of HLA-A, HLA-B, HLA-C, DRB1, DRB3/4/5, DQB1, DQA1, DPB1, and DPA1 were amplified in four multiplexed reactions. A DNA reference panel of 47 samples representing the most common allele groups was selected to evaluate this novel assay using the Ion Torrent sequencing platform. The specificity and sensitivity of this assay was confirmed on additional 158 samples from a local Caucasian control cohort. Full gene sequences from start to stop codons including some UTR regions were obtained for all 11 HLA loci with complete gene coverage and sufficient read-depth for 3619 alleles. The whole amplicon was analysed for HLA class I genes, while only exons were analysed for class II genes. All alleles were amplified as expected with 100% concordance at full gene resolution for HLA class I and exon resolution for HLA class II loci when compared with previously used NGS or Sanger sequencing methods. In summary, the novel multiplexed PCR approach for full-gene HLA typing enabled for a large amount of genetic information to be generated in a simple and fast workflow.

Role of HLA-B exon 1 in graft-versus-host disease after unrelated haemopoietic cell transplantation: a retrospective cohort study.

BACKGROUND: The success of unrelated haemopoietic cell transplantation (HCT) is limited by graft-versus-host disease (GVHD), which is the main post-transplantation challenge when HLA-matched donors are unavailable. A sequence dimorphism in exon 1 of HLA-B gives rise to leader peptides containing methionine (Met; M) or threonine (Thr; T), which differentially influence natural killer and T-cell alloresponses. The main aim of the study was to evaluate the role of the leader dimorphism in GVHD after HLA-B-mismatched unrelated HCT. METHODS: We did a retrospective cohort study of 33 982 patients who received an unrelated HCT done in Australia, Europe, Japan, North America, and the UK between Jan 1, 1988, and Dec 31, 2016. Data were contributed by participants of the International Histocompatibility Working Group in Hematopoietic Cell Transplantation. All cases were included and there were no exclusion criteria. Multivariate regression models were used to assess risks associated with HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 mismatching. Among the 33 982 transplantations, the risks of GVHD associated with HLA-B M and T leaders were established in 17 100 (50·3%) HLA-matched and 1457 (4·3%) single HLA-B-mismatched transplantations using multivariate regression models. Leader frequencies were defined in 2 004 742 BeTheMatch US registry donors. FINDINGS: Between Jan 20, 2017, and March 11, 2019, we assessed 33 982 HCTs using multivariate regression models for the role of HLA mismatching on outcome. Median follow-up was 1841 days (IQR 909-2963). Mortality and GVHD increased with increasing numbers of HLA mismatches. A single HLA-B mismatch increased grade 3-4 acute GVHD (odds ratio [OR] 1·89, 95% CI 1·53-2·33; p<0·0001). Among the single HLA-B-mismatched transplantations, acute GVHD risk was higher with leader mismatching than with leader matching (OR 1·73, 1·02-2·94; p=0·042 for grade 2-4) and with an M leader shared allotype compared with a T leader shared allotype (OR 1·98, 1·39-2·81; p=0·0001 for grade 3-4). The preferred HLA-B-mismatched donor is leader-matched and shares a T leader allotype. The majority (1 836 939 [91·6%]) of the 2 004 742 US registry donors have the TT or MT genotype. INTERPRETATION: The HLA-B leader informs GVHD risk after HLA-B-mismatched unrelated HCT and differentiates high-risk HLA-B mismatches from those with lower risk. The leader of the matched allotype could be considered to be as important as the leader of the mismatched allotype for GVHD. Prospective identification of leader-matched donors is feasible for most patients in need of a HCT, and could lower GVHD and increase availability of HCT therapy. These findings are being independently validated and warrant further research in prospective trials. FUNDING: The National Institutes of Health, USA.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT).

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component "Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing" we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.

Stimulation of HIV-specific T cell clonotypes using allogeneic HLA.

We hypothesized that HIV-specific CD8 T cell clonotypes can be stimulated by allogeneic HLA molecules. Multiple HIV-specific CD8 T cell clones were derived from 12 individuals with chronic HIV infection, specific for 13 different HIV Gag antigens and restricted to 7 different HLA molecules. The generated T cell clones were assayed for alloreactivity against a panel of single HLA class I expressing cell lines (SALs). HIV-specific T cells recognising at least one allogeneic HLA molecule could be identified from 7 of 12 patients tested. Allorecognition was associated with IFNγ cytokine production, CD137 upregulation and cytotoxicity, suggesting high avidity allo-stimulation. Allo-HLA recognition by HIV-specific T cells was specific to the HIV target peptide/HLA restriction and TCR TRBV usage of the T cells. HIV-specific T cells do crossreact against allogeneic HLA molecules in an epitope and TRBV specific manner. Therefore allo-HLA stimulation could be exploited to induce or augment HIV-specific T cell responses.

The detection of NK cell alloreactivity by flow cytometric CD107a assay.

Natural killer (NK) cell alloreactivity can be exploited in haploidentical (one haplotype mismatched) haematopoietic stem cell transplantation (HSCT) to prevent leukaemia relapse, rejection, and graft-vs-host disease (GVHD) (Blood 94:333-339; Science 295:2097-2100). If NK cell alloreactivity is to be exploited in HSCT, it is important to be able to reliably select donors who have NK alloreactivity towards the patient. The detection of donor NK alloreactivity towards patient target cells has traditionally been evaluated by NK cell cloning and (51)Cr-release cytotoxicity assay. This approach is complex and time consuming with results taking up to 6 weeks. Here, we detail a novel flow cytometric CD107a-based assay capable of detecting NK cell alloreactivity in 14 days.