Expression of a single-chain human leukocyte antigen-DRA/DRB3*01:01 molecule and differential binding of a monoclonal antibody in the presence of specifically bound human platelet antigen-1a peptide.

BACKGROUND: Studies show that 1 in 1200 neonates have a low platelet (PLT) count due to alloimmunization against human PLT antigen (HPA)-1a (ß3 -L33). This mainly occurs in HPA-1a-negative mothers who are positive for the human leukocyte antigen (HLA)-DRB3*01:01 allele, but only about one-third of cases will mount an effective alloimmune response. The development of specific treatment modalities requires that the mechanisms driving the maternal alloimmune response against the fetal PLTs be further explored. An antibody reagent that has a different binding affinity to HLA-DRA/DRB3*01:01 with and without the ß3 -L33 peptide would be a valuable reagent to study peptide presentation on maternal antigen-presenting cells. STUDY DESIGN AND METHODS: To identify such antibodies, HLA-DRA/DRB3*01:01 was recombinantly expressed in Drosophila S2 cells. To delineate the epitope of interesting antibodies, seven mutant HLA-DRA/DRB3*01:01 molecules were generated by site-directed mutagenesis introducing naturally occurring amino acid changes encoded by DRB3*02 and DRB3*03 alleles. RESULTS: The murine monoclonal antibody (MoAb) DA2 showed robust binding by enzyme-linked immunosorbent assay to recombinant HLA-DRA/DRB3*01:01, but binding was reduced in the presence of ß3 -L33 peptide. The binding affinity of DA2 to the mutant HLA-DRA/DRB3*0101 in which serine at Position 60 of the ß1-chain was replaced by tyrosine was greatly enhanced. Interestingly the binding of DA2 to the mutant was not reduced by the presence of ß3 -L33 peptide. CONCLUSION: The results of this study generate a molecular model of the interaction of the HLA-DRA/DRB3*01:01 molecule with MoAb DA2. This will inform functional studies with the recombinant Class II molecules.

Identification of a novel DRB1 allele through intergenic recombination between HLA-DRB1 and HLA-DRB3∗02 in a Chinese family.

In this study, a novel DRB1 allele was revealed by routine HLA-SBT typing noted for its extensive mismatches to any known DRB1 alleles within the exon 2. Sequences containing the exons 2, 3 of HLA-DRB1, their surrounding introns, and the full-length cDNA of DRB1 were analyzed to determine a possible recombination event. Interestingly, the sequences of entire exon 2 were characterized as DRB3(∗)02:02:01:01/02; while exon 3 were characterized as DRB1(∗)14 like alleles. Further analysis of the sequences using Simplot software suggested that an intergenic recombinant event (i.e. exchange of sequence between non-allelic genes) may have occurred between DRB3(∗)02 allele and DRB1(∗)14 like allele, and the recombination sites are located at intron 1 and the boundary of exon 2 and intron 2 of DRB1. There are 5 CGGGG sequences flanking each side of exon 2 could serve as potential recombination site. Moreover, the full-length cDNA of the novel allele has been identified. The exon 1 and exon 3 to exon 6 share the same sequence as DRB1(∗)14 like alleles. At the mRNA level, the new allele has no significant difference when compared with the other DRB1 allele. This novel recombinant allele is also found to be paternally inherited. In conclusion, this is the first report of a DRB1 and DRB3 intergenic recombination event involving whole exon 2, which generate a new DRB1(∗)14:141.

Compound heterozygosity of HLA-DRB3*01:01 and HLA-DRB4*01:01 as a potential predictor of fetal neonatal alloimmune thrombocytopenia.

BACKGROUND: Fetal neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder in the fetus or neonate caused by maternal alloantibodies directed against fetal platelet (PLT) antigens inherited from the father. The immune-dominant antigen leading to severe FNAIT is the human PLT antigen (HPA)-1, whose polymorphism constitutes an epitope for human leukocyte antigens (HLAs), usually DRB3*0101 leading to an immune response. STUDY DESIGN AND METHODS: In this study our aims were to find whether other allele variants of the ß subunit of the HLA-DR family specifically focused on the HLA residues that bind Position 33 of the HPA-1 integrin contribute to FNAIT development and affect response to treatment and whether coexistence of both anti-HPA-1a and anti-HLA class I specific against the father's antigens leads to a more severe thrombocytopenia in the newborn. We examine the genotype of 23 mothers to newborns with FNAIT compared to a control group. RESULTS: Our results suggested that, when HPA-1 incompatibility with the husband is found, the presence of two HLA alleles (DRB3*01:01 and DRB4*01:01) in the mother increases the risk and severity of FNAIT and reduces the success of a preventive immunoglobulin G treatment. We provide a structural model for the molecular basis of the rational effects of the different HLA alleles. In addition, we found that the presence of both anti-HPA-1 and anti-HLAs did not aggravate FNAIT in comparison to mothers harboring only anti-HPA-1. CONCLUSION: Overall, we suggest that a specific genotyping of the mother in relation to HLA-DRB as well as HPA-1 can serve as an antenatal diagnostic tool, particularly in siblings of women who gave birth to neonates with FNAIT.