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.
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.
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.
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-β 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.
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-β 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).
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.
Abstract 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.
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.
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.
Key Points
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.
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.
P079 Weidner, Jerome; Ramahi, Sana; Marino, Susana R
Human immunology,
10/2014, Letnik:
75
Journal Article
Recenzirano
Aim DNA isolation from frozen plasma may be necessary to determine inherited vs. non-inherited umbilical cord blood (UCB) maternal HLA haplotypes. Our aim was to determine if DNA isolated from frozen ...plasma can yield high quality DNA that could consistently be used in HLA class I and II rSSOP typing for hematopoietic UCB transplant donors. Methods Plasma was collected from 26 recipients and healthy donors then frozen to mimic future storage conditions. 20 samples were previously typed. Samples were thawed and isolated using the DNA Purification from Blood and Body Fluids Spin Protocol as described in the QIAamp DNA Mini and Blood Mini Handbook . Four samples were eluted in 50 μL and 200 μL of AE buffer. The remaining 22 samples were eluted in 50 μL of AE buffer. The concentration and purity (260/280 ratio) of all samples were determined using a NanoDrop™ 1000 spectrophotometer. All 26 samples eluted in 50 μL were typed using the LABType® SSO methodology at HLA-A, B, C, DRB1, DQB1, and DQA1 loci. For samples with less than optimal purity, additional adjustments were made in an attempt to improve purity, using higher concentrations of protease during lysis, as well as alcohol precipitations. Results DNA was obtained from all samples. Purity differences between samples eluted in 50 μL versus 200 μL AE buffer were negligible; however the concentrations were considerably higher in 50 μL eluted samples. The concentrations ranged from 2.83 to 39.95 ng/μL (Average: 10.65 ng/μL). The 260/280 ratios ranged from 1.32 to 2.71 (Average: 1.72). The increased protease isolations and alcohol precipitations did not improve purity. 9/26 (34.6%) samples yielded complete typing results at all 6 loci. 10/26 (38.5%) samples yielded a complete typing for class I loci. 22/26 (84.6%) samples yielded a complete typing for class II loci. 160 assignments across all loci could be compared to previous typing results. 2/160 (1.3%) were mistyped due to allele dropout, both were HLA-C alleles. Conclusion DNA isolated from plasma using the QIAamp DNA Blood kit can be used in rSSOP HLA typing methods. However the isolated DNA does not yield quality template consistently enough to replace traditional sources of DNA in HLA labs for typing at HLA-A, B, C, DRB1, DQB1, and DQA1 loci by rSSOP methods. Additional methods should be tested for reliable DNA isolation from this source.
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