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.

Selection of unrelated donors and cord blood units for hematopoietic cell transplantation: guidelines from the NMDP/CIBMTR.

Allogeneic hematopoietic cell transplantation involves consideration of both donor and recipient characteristics to guide the selection of a suitable graft. Sufficient high-resolution donor-recipient HLA match is of primary importance in transplantation with adult unrelated donors, using conventional graft-versus-host disease prophylaxis. In cord blood transplantation, optimal unit selection requires consideration of unit quality, cell dose and HLA-match. In this summary, the National Marrow Donor Program (NMDP) and the Center for International Blood and Marrow Transplant Research, jointly with the NMDP Histocompatibility Advisory Group, provide evidence-based guidelines for optimal selection of unrelated donors and cord blood units.

HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11). High-resolution HLA genotypes were generated for each subject using next-generation sequencing (NGS) HLA typing systems. We identified the HLA haplotypes in each family using HaplObserve, software that builds haplotypes in families by reviewing HLA allele segregation from parents to children. We calculated haplotype frequencies within each broad group, by treating the parents in each family as unrelated individuals. We also calculated standard measures of global linkage disequilibrium (LD) and conditional asymmetric LD for each ethnic group, and used untruncated and two-field allele names to investigate LD patterns. Finally we demonstrated the utility of consensus DNA sequences in identifying novel variants, confirming them using HLA allele segregation at the DNA sequence level.

Effective desensitization for a strong donor-specific HLA antibody in a case of HLA-mismatched allogeneic hematopoietic cell transplantation.

We describe a unique ABO compatible and 9/10 HLA-matched case of successful allogeneic hematopoietic cell transplantation (HCT) after effective desensitization of a strong anti-HLA-A24 donor-specific antibody (DSA) with mean fluorescence intensity of approximately 18 000. Due to absence of a suitable matched unrelated donor the patient sibling was considered the best available donor, and it was decided to desensitize patient prior to transplant. The strength of HLA-A24 DSA slowly decreased over the course of treatment, necessitating a total of 23 sessions of therapeutic plasma exchange in order to bring the DSA strength to undetectable levels, followed by a successful transplant. In summary, the outcome of this case shows effective application of desensitization treatment to remove strong DSA in HCT patients.

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.

Reanalysis of the role of pronase treatment of B cells in the flow cytometric crossmatch assay: Fc receptor is not the primary target.

Pronase, a mixture of nonspecific bacterial proteases, is used to pretreat human lymphocytes to prevent false-positive B cell results in the flow cytometric crossmatch (FCXM) assay. The target of pronase has been reported to be B cell-expressed Fc receptors, which nonspecifically bind IgG. As pronase use in FCXM can induce other complications, including degradation of HLA leading to inappropriate FCXM results, and false-positive T cell results when testing serum from HIV-positive patients, we tested whether specifically blocking Fc receptor CD32 could replace pronase. Anti-CD32 mAb 6C4 was superior to pronase for blocking binding of aggregated IgG to B cells. However, 6C4 was unable to replace pronase in clinical FCXM, as it did not prevent false-positive B cell FCXM results, or enhance sensitivity of the assay. We conclude that the functional targets of pronase in the FCXM assay are poorly understood, and that B cell-expressed Fc receptor plays an insignificant role.

Characterization of T-cell Receptor Repertoire in Inflamed Tissues of Patients with Crohn's Disease Through Deep Sequencing.

BACKGROUND: Intestinal tissues of patients with Crohn's disease (CD) contain expanded populations of T cells which are believed to mediate inflammation. We performed a detailed characterization of these T-cell repertoires. METHODS: We obtained biopsies from the neoterminal ileum of 12 patients undergoing evaluation for postoperative recurrent CD and 4 individuals with normal terminal ileum and no history of inflammatory bowel disease (controls). Samples of diseased terminal ileum were obtained from 5 patients undergoing surgery for stricturing or penetrating CD. Total RNA was extracted from tissues and peripheral blood mononuclear cells, and cDNAs were generated. We used next-generation sequencing to characterize T-cell receptor (TCR)-α and TCR-ß cDNAs in ileal mucosal tissue and matched peripheral blood mononuclear cells of 17 patients with CD to identify oligoclonal expansions of T-cell populations associated with CD. RESULTS: TCR diversity in mucosal tissue was significantly lower than that of matched peripheral blood mononuclear cells, indicating expansion of certain T-cell populations in inflamed intestinal tissue. A single TCR-ß clonotype, CASSWTNGEQYF (TRBV10-1-TRBJ2-7), was enriched at a frequency of 7.0% to 28.9% in the neoterminal ileum of 4 of 12 patients with recurrent CD. The abundance of this clonotype significantly correlated with the severity of disease recurrence, based on Rutgeerts score (P = 0.015). CONCLUSIONS: Specific populations of T cells are expanded in the inflamed intestinal mucosa of patients with CD; their abundance correlates with severity of disease recurrence. Studies of these T cells could provide information about mechanisms of CD pathogenesis. Deep TCR sequencing is a powerful tool that rapidly provides in-depth, real-time assessment of the T-cell repertoire.

Identification and characterization of novel HLA alleles: Utility of next-generation sequencing methods.

The HLA genes are the most polymorphic of the human genome, and novel HLA alleles are continuously identified, often by clinical Sanger sequencing-based typing (SBT) assays. Introduction of next-generation sequencing (NGS) technologies for clinical HLA typing may significantly improve this process. Here we compare four cases of novel HLA alleles identified and characterized by both SBT and NGS. The tested NGS system sequenced broader regions of the HLA loci, and identified novel polymorphisms undetected by SBT. Subsequent characterization of the novel alleles in isolation of coencoded alleles by SBT required custom-designed primers, while the NGS system was able to sequence both alleles in phase. However, the tested assay was unable to amplify buccal cell DNA for subsequent NGS sequencing, presumably due to the lower quality of these samples. While NGS assays will undoubtedly increase novel allele identification, more stringent DNA sample requirements may be necessary for this new technology.

Amino acid substitution at peptide-binding pockets of HLA class I molecules increases risk of severe acute GVHD and mortality.

HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death.

Predicting HLA class I non-permissive amino acid residues substitutions.

Prediction of peptide binding to human leukocyte antigen (HLA) molecules is essential to a wide range of clinical entities from vaccine design to stem cell transplant compatibility. Here we present a new structure-based methodology that applies robust computational tools to model peptide-HLA (p-HLA) binding interactions. The method leverages the structural conservation observed in p-HLA complexes to significantly reduce the search space and calculate the system's binding free energy. This approach is benchmarked against existing p-HLA complexes and the prediction performance is measured against a library of experimentally validated peptides. The effect on binding activity across a large set of high-affinity peptides is used to investigate amino acid mismatches reported as high-risk factors in hematopoietic stem cell transplantation.

Plasma cell densities and glomerular filtration rates predict renal allograft outcomes following acute rejection.

The contribution of T cells and graft-reactive antibodies to acute allograft rejection is widely accepted, but the role of graft-infiltrating B and plasma cells is controversial. We examined 56 consecutive human renal transplant biopsies classified by Banff schema into T-cell-mediated (N = 21), antibody-mediated (N = 18), and mixed (N = 17) acute rejection, using standard immunohistochemistry for CD3, CD20, CD138, and CD45. In a predominantly African-American population (75%), neither Banff classification nor C4d deposition predicted the return to dialysis. Immunohistochemical analysis revealed CD3(+) T cells as the dominant cell type, followed by CD20(+) B cells and CD138(+) plasma cells in all acute rejection types. Using univariate Cox Proportional Hazard analysis, plasma cell density significantly predicted graft failure while B-cell density trended toward significance. Surprisingly T-cell density did not predict graft failure. The estimated glomerular filtration rate (eGFR) at diagnosis of acute rejection also predicted graft failure, while baseline eGFR ≥6 months prior to biopsy did not. Using multivariate analysis, a model including eGFR at biopsy and plasma cell density was most predictive of graft loss. These observations suggest that plasma cells may be a critical mediator and/or an independently sensitive marker of steroid-resistant acute rejection.

HLA-C antigen mismatch is associated with worse outcome in unrelated donor peripheral blood stem cell transplantation.

The association between HLA matching and outcome in unrelated-donor peripheral blood stem cell (PBSC) transplantation has not yet been established. In the present study, a total of 1933 unrelated donor-recipient pairs who underwent PBSC transplantation between 1999 and 2006 for acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, or chronic myelogenous leukemia and received high-resolution HLA typing for HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1 were included in the analysis. Outcomes were compared between HLA-matched and HLA-mismatched pairs, adjusting for patient and transplant characteristics. Matching for HLA-A, -B, -C, and -DRB1 alleles (8/8 match) was associated with better survival at 1 year compared with 7/8 HLA-matched pairs (56% vs 47%). Using 8/8 HLA-matched patients as the baseline (n = 1243), HLA-C antigen mismatches (n = 189) were statistically significantly associated with lower leukemia-free survival (relative risk [RR], 1.36; 95% confidence interval [CI], 1.13-1.64; P = .0010), and increased risk for mortality (RR, 1.41; 95% CI, 1.16-1.70; P = .0005), treatment-related mortality (RR, 1.61; 95% CI, 1.25-2.08; P = .0002), and grade III-IV graft-versus-host disease (RR, 1.98; 95% CI, 1.50-2.62; P < .0001). HLA-B antigen or allele mismatching was associated with an increased risk for acute GVHD grade III-IV. No statistically significant differences in outcome were observed for HLA-C allele (n = 61), HLA-A antigen/allele (n = 136), HLA-DRB1 allele (n = 39), or HLA-DQ antigen/allele (n = 114) mismatches compared with 8/8 HLA-matched pairs. HLA mismatch was not associated with relapse or chronic GVHD. HLA-C antigen-mismatched unrelated PBSC donors were associated with worse outcomes compared with 8/8 HLA-matched donors. The study's limited power due to small sample size precludes conclusions about other mismatches.

One-antigen mismatched related versus HLA-matched unrelated donor hematopoietic stem cell transplantation in adults with acute leukemia: Center for International Blood and Marrow Transplant Research results in the era of molecular HLA typing.

Approximately 13% of patients lacking an HLA-identical sibling have a one-antigen-mismatched related donor (MMRD). Historically, outcomes from the use of a one-antigen MMRD were considered equivalent to those from the use of a matched unrelated donor (UD). Recent improvements in UD stem cell transplantation (SCT) resulting from better molecular HLA matching justifies investigating whether UD should be preferred over MMRD in adult patients with acute leukemia. Here, we compared the outcomes of MMRD (n = 89) and HLA-A, -B, -C, and -DRB1 allele-matched UD (n = 700) SCT reported to the Center for International Blood and Marrow Transplant Research between 1995 and 2005. The patients underwent transplantation for acute myelogenous leukemia or acute lymphoblastic leukemia in first or second complete remission. Donor type was not associated with hematologic recovery. Univariate and multivariate comparisons of MMRD versus HLA-matched UD transplants showed no statistically significant differences in overall survival, disease-free survival, treatment-related mortality, relapse, or 100-day grade III-IV acute graft-versus-host disease (GVHD). MMRD SCT was associated with a lower rate of chronic GVHD at 1 year (35% vs 47%; P = .03), which was confirmed by multivariate analysis (relative risk, 0.58; 95% confidence interval, 0.39-0.85; P < .01). According to our data, HLA-matched UD and MMRD SCT are associated with comparable survival. Given that less chronic GVHD was observed in the MMRD transplantations, this option, when available, remains the first choice in patients with acute leukemia without an HLA-identical sibling in need of allogeneic SCT.