Previously it has been shown that sequences containing both Trp<sup>25</sup> and Leu<sup>33</sup> are the most effective at inducing Th cell proliferation in HLA-DRB3*0101 positive women, alloimmunised with anti-HPA-1a. The Leu<sup>33</sup>/Pro<sup>33</sup> polymorphism is embedded in the N-terminal plexin/semaphorin/integrin (PSI) domain of GPIIIa. In the present study, amino acids 1-62 of the GPIIIa (Leu<sup>33</sup> or Pro<sup>33</sup>) PSI domain were cloned into the vector pGEX-6p-1. The recombinant proteins were expressed and tested by ELISA, Luminex and Absorption Assays. The presence of the HPA-1a/-1b epitope was confirmed by the ability of PSI-Leu<sup>33</sup>/-Pro<sup>33</sup> recombinant fragments to specifically capture its corresponding HPA-1 antibody. Cells from a human B cell line (HHKB), homozygous for HLA-DRB3*0101, were pulsed with the recombinant PSI domain fragment of GPIIIa expressing the HPA-1a antigen. MHC class II/peptide complexes were isolated from the pulsed cells using an immunoaffinity column. A nested set of naturally processed and presented HPA-1a derived peptides, each containing the residues Trp<sup>25</sup> – Leu<sup>33</sup> core epitope was identified. For the first time a naturally processed and presented HPA-1a peptide that spans the HPA-1a polymorphism has been identified, bound to the class II molecule encoded by HLA-DRB3*0101. The efficient processing and presentation of this peptide, which includes the putative dominant Th epitope, is likely to be an important contributory factor in the immunogenicity of HPA-1a. Such peptides may also provide the basis for novel treatments to tolerise the corresponding Th response in HPA-1b1b women at risk of NAIT with an HPA-1a-positive fetus.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:531846 |
| Date | January 2010 |
| Creators | Anani Sarab, Gholamreza |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=158493 |
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