Differential effects of donor-specific HLA antibodies in living versus deceased donor transplant.

The presence of donor-specific anti-HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long-term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement-dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10-year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10-year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10-year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence.

The role of gene polymorphism in HLA class I splicing.

Among the large number of human leucocyte antigen (HLA) alleles, only a few have been identified with a nucleotide polymorphism impairing correct splicing. Those alleles show aberrant expression levels, due to either a direct effect of the polymorphism on the normal splice site or to the creation of an alternative splice site. Furthermore, in several studies, the presence of alternatively spliced HLA transcripts co-expressed with the mature spliced transcripts was reported. We evaluated the splice site sequences of all known HLA class I alleles and found that, beside the consensus GT and AG sequences at the intron borders, there were some other highly conserved nucleotides for the different class I genes. In this review, we summarize the splicing mechanism and evaluate what is known today about alternative splicing of HLA class I genes.

Full-length HLA-DRB1 coding sequences generated by a hemizygous RNA-SBT approach.

Currently 1582 HLA-DRB1 alleles have been identified in the IMGT/HLA database (v3.18). Among those alleles, more than 90% have incomplete allele sequences, which complicates the analysis of the functional relevance of polymorphism beyond exon 2. The polymorphic index of each individual exon of the currently known allele sequences, shows that polymorphism is present in all exons, albeit not equally abundant. Full-length HLA-DRB1 RNA sequencing identifies polymorphism of the complete coding region. Here we describe a hemizygous full-length RNA sequence-based typing (SBT) approach based on group-specific HLA-DRB1 amplification and subsequent sequencing. RNA full-length sequences can easily be accessed because of the short amplicon length (801 bp). The RNA-SBT approach was successfully validated on a panel of DRB1 alleles having fully known coding sequences according to the IMGT/HLA database, and cover all serological equivalents. Subsequently, the approach was applied on a panel of 54 alleles with incomplete allele sequences, resulting in full-length coding sequences and the identification of one new and one corrected allele. This study shows the universal applicability of the RNA-based sequencing approach to identify full-length coding sequences and to define the polymorphic content of HLA-DRB1 alleles.

The full length genomic sequence of a novel HLA-C*03 allele: HLA-C*03:219.

HLA-C*03:219 differs from HLA-C*03:04:01:01 by a non-synonymous substitution in the α3 domain (T216I).

Features of a new full length HLA allele: A*02:455.

The full length sequence of HLA-A*02:455 differs from HLA-A*02:01:01:01 by one amino acid substitution: A245E.

Peptide-induced HLA-E expression in human PBMCs is dependent on peptide sequence and the HLA-E genotype.

Human Leukocyte Antigen (HLA)-E is a low-polymorphic non-classical HLA class I molecule which plays a crucial role in immune surveillance by presentation of peptides to T and natural killer (NK) cells. HLA-E polymorphism is related to HLA-E surface expression and is associated with patient outcome after stem cell transplantation. We aim to investigate the regulation of HLA-E expression level in peripheral blood mononuclear cells (PBMCs) of healthy individuals homozygous for HLA-E*01:01 or HLA-E*01:03, by using a panel of HLA-E binding peptides derived from CMV, Hsp60 and HLA class I. Basal and peptide-induced HLA-E surface expression levels were higher in PBMC from HLA-E*01:03 homozygous subjects as compared to PBMC from HLA-E*01:01 homozygous subjects. HLA-E mRNA levels were comparable between the two genotypes and remained constant after peptide stimulation. HLA-E surface expression seemed to be not only dependent on the HLA-E genotype, but also on the sequence of the peptide as evidenced by the profound difference in HLA-E upregulation with the Hsp60 and the B7 peptide. Our results showed that peptide-induced HLA-E expression is regulated at the posttranscriptional level as extracellular peptide stimulation did not influence RNA expression. This study provides new insights in the mechanism by which HLA-E expression is regulated and underlines a new role for extracellular peptides in inducing HLA-E translation, which may represent a defense mechanism against lytic viral infections and necrosis.

Clinical and immunological significance of HLA-E in stem cell transplantation and cancer.

Human leukocyte antigen-E (HLA-E) is a nonclassical HLA class I molecule that canonically binds peptides derived from the leader sequence of classical HLA class I. HLA-E can also bind peptides from stress protein [e.g. heat shock protein 60 (Hsp60)] and pathogens, illustrating the importance of HLA-E for anti-viral and anti-tumor immunity. Like classical HLA class I molecules, HLA-E is ubiquitously expressed, however, it is characterized by only a very limited sequence variability and two dominant protein forms have been described (HLA-E*01:01 and HLA-E*01:03). HLA-E influences both the innate and the adaptive arms of the immune system by the engagement of inhibitory (e.g. NKG2A) and activating receptors [e.g. αß T cell receptor (αßTCR) or NKG2C] on NK cells and CD8 T cells. The effects of HLA-E on the cellular immune response are therefore complex and not completely understood yet. Here, we aim to provide an overview of the immunological and clinical relevance of HLA-E and HLA-E polymorphism in stem cell transplantation and in cancer. We review novel insights in the mechanism via which HLA-E expression levels are controlled and how the cellular immune response in transplantation and cancer is influenced by HLA-E.

An improved and validated RNA HLA class I SBT approach for obtaining full length coding sequences.

The functional relevance of human leukocyte antigen (HLA) class I allele polymorphism beyond exons 2 and 3 is difficult to address because more than 70% of the HLA class I alleles are defined by exons 2 and 3 sequences only. For routine application on clinical samples we improved and validated the HLA sequence-based typing (SBT) approach based on RNA templates, using either a single locus-specific or two overlapping group-specific polymerase chain reaction (PCR) amplifications, with three forward and three reverse sequencing reactions for full length sequencing. Locus-specific HLA typing with RNA SBT of a reference panel, representing the major antigen groups, showed identical results compared to DNA SBT typing. Alleles encountered with unknown exons in the IMGT/HLA database and three samples, two with Null and one with a Low expressed allele, have been addressed by the group-specific RNA SBT approach to obtain full length coding sequences. This RNA SBT approach has proven its value in our routine full length definition of alleles.

Allele and haplotype frequencies of HLA-DPA1 and -DPB1 in the population of Guadeloupe.

Genetic polymorphism of human leukocyte antigen (HLA)-DPA1 and -DPB1 loci was studied in 154 unrelated individuals from Guadeloupe, an archipelago of five islands located in the Carribean Sea. Thirty different DPB1 and eight different DPA1 alleles were observed with a heterozygosity index of 0.87 and 0.78, respectively. This high degree of heterozygosity corresponds with those found in African populations. The DPB1* 01:01:01 allele was most frequent (0.260), followed by 02:01:02 (0.143) and 04:01:01 (0.127). The DPA1 alleles 01:03 (0.380), 02:01 (0.302), 02:02 (0.175) and 03:01 (0.123) were identified in >35 individuals each, whereas 01:04, 01:05 and 04:01 were present only once. Haplotype estimations revealed the presence of 39 different haplotypes, with DPB1*01:01:01-DPA1*02:02 and DPB1*02:01:02-DPA1*01:03 as the most frequent (0.143 and 0.140, respectively). A striking difference was observed in DPB1/DPA1 associations between DPB1*04:02 and *105:01, that have identical exon 2 sequences. DPB1*04:02 was exclusively associated with DPA1*01:03, whereas DPB1*105:01 was present with DPA1*03:01, *03:02 or *04:01. This implies that the DP molecules are actually different, and this difference is relevant to consider in studies on the function of HLA-DP molecules in transplantation. Overall, HLA-DPA1 and DPB1 allele frequencies and haplotypes of the population of Guadeloupe were most similar to African populations, with characteristic alleles and haplotypes that bespeaks the admixture with other ethnicities.

HLA frequencies and associations in cystic fibrosis.

Cystic fibrosis (CF) is classically attributed to the dysfunction of the single CF transmembrane conductance regulator gene. The incidence of human leukocyte antigen (HLA) polymorphisms in different CF-associated diseases raises the question of an unequal distribution of HLA genotypes in CF. This study aimed to evaluate HLA gene frequencies and possible associations in CF patients compared with a control population. Frequencies of HLA-DRB1, HLA-DQA1 and HLA-DQB1, performed by intermediate resolution typing using Luminex sequence-specific oligonucleotide, and epitope counts were similar in 340 CF patients when compared with 400 control subjects. In conclusion, HLA-DRB1, -DQA1 and -DQB1 do not seem to influence susceptibility to CF. Whether HLA plays a role in the severity of CF disease needs to be investigated.

Identification of a new allele polymorphism (HLA-B*40:79) and correlation with the HLA-B40 (B60 and B61) antigens.

Full gene sequence of the HLA-B*40:79 allele; gene polymorphism defines HLA-B60 and B61 lineages.

Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

Full-length sequence of a novel HLA-B*15:220 allele identified in an individual from Guadeloupe.

The new HLA-B*15:220 allele shows a single-nucleotide substitution in exon 1 at position 47 (C>T) when compared to its closest allele HLA-B*15:03:01, resulting in an amino acid substitution from Ala to Val in the signal peptide at codon -9.

Full-length sequence of a novel null allele HLA-A*23:38N identified in an individual from Guadeloupe.

The new HLA-A*23:38N allele shows a single-base deletion in exon 2, resulting in a frame shift and a premature stop codon.

Full-length HLA-DPB1 diversity in multiple alleles of individuals from Caucasian, Black, or Oriental origin.

Despite DP antigens have been shown to be stimulators of the mixed lymphocyte reaction, human leukocyte antigen-DPB1 is not considered in the matching criteria for hematopoietic stem cell transplantation (HSCT). The role of DPB1 matching in HSCT remains inconclusive because of contradictory findings in different studies. The concept of permissible and non-permissible mismatches might clarify these contradictory results. Although several groups have attempted to identify immunogenic epitopes in exon 2 to establish permissive and non-permissive allele groups, the direct correlation between individual exon 2 amino acids and epitopes with DPB1 immunogenicity is still not evident. We hypothesize that polymorphism within the entire molecule, including polymorphic variability in different ethnic groups, is crucial to unravel the function of DPB1 polymorphism. Using an RNA-based approach, we sequenced all frequent and available non-frequent DPB1 alleles full length from 148 samples representing 28 different DPB1 alleles from either Black, Caucasian, or Oriental origin. We identified various DPB1 alleles with, in addition to the exon 2 polymorphism, polymorphisms in exons 1, 3, 4, and 5. Based on this polymorphism outside exon 2, we defined one new allele. Two alleles with identical exon 2 polymorphism but differing outside exon 2 were identified in individuals of different ethnic groups. As T cell binding is not restricted to the polymorphic groove and polymorphism in the ß2 domain of the DP molecule affects CD4 interaction, full-length polymorphism should be considered to determine immunogenicity. Eventually, this knowledge will provide new insights in the classification of DPB1 polymorphism and more importantly will add new perspectives to the concept of permissiveness in transplantation.

HLA-DO polymorphism associated with resistance to type I diabetes detected with monoclonal antibodies, isoelectric point differences, and restriction fragment length polymorphism.

A new HLA-DQ-related genetic system with two alleles, 2B3 and TA10, defined serologically by MAbs and alloantisera, showed an almost perfect correlation with charge differences on DQ beta molecules, as well as with two polymorphic DNA fragments hybridizing with a DQ beta probe and various restriction enzymes on a panel of 14 DR4+ homozygous typing cells. It was therefore concluded that the serologically defined alleles 2B3 and TA10 are coded by the DQ beta gene and situated on the HLA-DQ beta chain. This 2B3/TA10 polymorphism is independent of HLA-D and segregates with HLA in families. The TA10 allele appears to be a new marker for resistance to type I diabetes, which is independent from the known resistance marker DR2, whereas no association was observed between this DQ beta polymorphism and rheumatoid arthritis.

A high-throughput Taqman approach for the discrimination of HLA-E alleles.

An association between human leukocyte antigen (HLA) molecules and the incidence of various diseases has been recognized for years. Molecules of the HLA system also exert an important role on the clinical outcome after transplantation such as stem cell transplantation. The conventional HLA typing method is sequence based typing (SBT) which is reliable but laborious. The goal of this study was to develop a high-throughput Taqman assay to screen large panels for HLA-E alleles. Two functional HLA-E alleles, *0101 and *0103, have been identified. We set up the Taqman assay with genomic DNA as template to discriminate the three HLA-E genotypes: homozygous HLA-E*0101, heterozygous *0101, *0103 and homozygous *0103. This Taqman approach was validated by the comparison of results obtained with the typing results acquired by sequenced base typing (SBT). Additional screening of a large panel showed the pronounced discriminative capacity of the Taqman assay for HLA-E allele typing. The Taqman assay is a fast, reliable and consistent HLA-E allele typing method, especially useful to screen large panels.

A new HLA-C*05 allele, HLA-C*05:156, characterized by full-length hemizygous sequencing.

HLA-C*05:156 allele differs from C*05:01:01:02 by a nucleotide change in exon 2 at codon 9.

Saddlebags: A software interface for submitting full-length HLA allele sequences to the EMBL-ENA nucleotide database.

Submission of full-length HLA allele sequences presents a unique challenge, both for high-throughput sequencing laboratories and smaller diagnostic laboratories. HLA's extensive polymorphism means that accurate representation and annotation of allele sequence is of critical importance, and curators of nucleotide databases must establish submission formats to ensure high-quality data and prevent ambiguities. The IPD-IMGT/HLA database is established as the standard repository for HLA sequences, and it is a major goal of the 17th International HLA and Immunogenetics Workshop to fill the IPD-IMGT/HLA database with full-length HLA sequences. The process of preparing sequence annotation and metadata is cumbersome and error prone, and it is desirable to create a straightforward and concise method of preparing sequence submissions. We introduce Saddlebags, a software tool for rapid generation of HLA (novel) full-length allele sequence submissions. HLA allele sequences are submitted first to EMBL European Nucleotide Archive (EMBL-ENA), and metadata is gathered for subsequent preparation of an IPD-IMGT/HLA formatted submission. Combining these steps into a pipeline reduces effort and minimizes errors for submitting laboratories. This software has been used by Maastricht University Medical Center Transplantation Immunology Laboratory to submit 79 novel alleles to EMBL-ENA, and the tool is freely available for the HLA community.

Chimerism analysis within 6 months of allogeneic stem cell transplantation predicts relapse in acute myeloid leukemia.

The role of chimerism analysis as a prognostic indicator of relapse after hematopoietic stem cell transplantation (SCT) is controversial. We monitored chimerism status by short tandem repeat-based polymerase chain reaction (PCR) in T- and non-T-cell subsets and retrospectively evaluated clinical outcome in 96 patients with acute myeloid leukemia after myeloablative (MA) or reduced-intensity conditioning SCT. Fifty-six percent of 80 patients in the MA group demonstrated complete donor chimerism (CC) at all time points, whereas 6% had decreasing mixed chimerism (MC), 8% stable MC, 25% increasing MC and 3% increasing and decreasing MC. In 16 RIC patients, these percentages were 12, 50, 6, 6 and 19, respectively, together with 6% nonengraftment. Forty-three out of 96 patients experienced relapse. The last chimerism evaluation before relapse revealed increasing MC in only eight patients. In samples taken between 1 and 6 months post SCT, CC/decreasing MC was significantly related with a lower risk of relapse (31 versus 83%, P<0.000) and mortality (38 versus 83%, P<0.000) than with MC/increasing MC. However, the development of relapse was very rapid. Only very frequent monitoring of chimerism status by highly sensitive methods might identify impending relapse and allow early immunological intervention.