Analysis of Naturally Processed HPA-1a Peptides Bound to Human MHC Class II Molecules

Background: Human platelet HPA-1a and HPA-1b alloantigens are determined respectively by the polymorphism Leu or Pro at position 33 of the β₃ chain (GPIIIa) of the platelet integrin αIIbβ₃. In neonatal alloimmune thrombocytopenia (NAITP) the HPA-1b1b mothers who are at risk of producing anti-HPA-1a antibody through carrying an HPA-1a fetus are almost always HLA-DRB3*0101 (DR52a) positive. The T-helper response that drives anti-HPA-1a production remains to be fully characterized. The Th epitope(s) must include the Leu³³ polymorphism, which is also predicted to be an anchor residue for binding to the class II molecule encoded by HLA-DRB3*0101. We have previously used a panel of 15-mer peptides to map the immunodominant Th epitope on GPIIIa (

Methods: Cells from a Human B cell line (HHKB), homozygous for HLA-DRB3*0101, were pulsed with 1mg/ml of recombinant PSI domain fragment of GPIIIa expressing the HPA-1a antigen. MHC classII/peptide complexes were isolated from the pulsed cells using an immunoaffinity column. The peptides were acid eluted and fractionated by HPLC. Fractions were introduced into the electron spray ionization source of a tandem mass spectrometer (Bruker Daltonics) and the mass data analyzed using the Mascot search engine.

Results: A naturally processed and presented peptide, derived from the HPA-1a antigen, and spanning the Leu³³ polymorphism, has been identified. The peptide is 16 amino acids in length and the sequence spans from Met at position 22 to Arg at position 37 of GPIIIa (MCAWCSDEALPLGSPR). The sequence thus includes the residues Trp²⁵ and Leu³³ that are predicted to form the dominant Th cell epitope. Despite the presence of other proteins that could be processed, the GPIIIa derived peptide Met²² - Arg³⁷ is amongst those most abundantly presented by HLA-DRB3*0101 positive antigen presenting cells in culture. The sequences of further GPIIIa derived peptides forming a “nested set” surrounding the epitope, including Ala²⁴ - Arg²⁷ and Trp²⁵ - Arg³⁷, may also have been generated, but at levels too low for confidence in their identification.

Conclusion: 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 HLADRB3* 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 tolerize the corresponding Th response in HPA-1b1b women at risk of NAIT with an HPA-1a positive fetus.