Characterisation of alloreactive helper T-cell epitopes on the platelet membrane GPIIIa (integrin Î²3)
The HPA-1a antigen is the most immunogenic and clinically important antigen of the human platelet antigen (HPA) system. It is a major target in neonatal alloimmune thrombocytopenic purpura (NAITP). As a first step towards peptide immunotherapy for NAITP, the aim was to map immunodominant helper epitopes by challenging peripheral blood T-helper cells in vitro with a panel of linear peptides derived from the GPIIIa protein sequence containing the HPA-1a polymorphic site. A further aim was to determine whether the panel of HPA-1a/1b peptides can stimulate the production of cytokines from helper T-cells of women at risk of FMAIT. 15-mer peptides were synthesised corresponding to the sequences of the HPA-1 polymorphic region, with either Leu33 or Pro33 at each possible position. Peripheral blood mononuclear cells (PBMC) were obtained from the blood of 21 HPA-1b1b women who had recently delivered an HPA-1a positive baby. Fourteen women had developed anti-HPA-1a as a result of an incompatible pregnancy, and 13 of 14 were HLA-DRB3*01 positive. Seven women had not produced anti-HPA-1a and 6 of 7 were HLA-DRB3*01 negative. One unimmunised HPA-1b1b male donor and 11 HPA-1a positive blood donors acted as controls. PBMC from 9 of 14 alloimmunised women responded to particular HPA-1a Leu33 peptides; all 9 responders were HLA-DRB3*01 positive and they were negative for the HLA-DRB1*15 allele. In contrast, only 2 of the 9 also responded to some of the HPA-1b Pro33 peptides. Two of 7 HPA-1b1b women who did not produce anti-HPA-1a also responded to some Leu33 peptides; neither woman was HLA-DRB3*01 positive. Twelve of the 14 alloimmunised women were HLA-DQB1*02 positive and of those, 7 responded to peptides. None of the control donors responded to either Leu33 or Pro33 peptides. Certain peptides induced proliferation more commonly than others. Peptides with Leu33 near the C-terminus were immunodominant, with peptide L1(19-33) eliciting a response in 50% of the alloimmunised women. The immunodominant peptides (L1(19-33), L2(20-34), L3(21-35), L4(22-36) and L5(23-37)) were examined for the presence of motifs that are bound by MHC class II molecules. In addition to Leu33 they were found to also require β3Trp25. The peptide panel was screened for the ability to stimulate PBMC proliferation in an assay designed to map T-cell epitopes, and the responding T-cells were examined by 3-colour flow cytometry for the presence of CD3+ and CD4+ T-cells and the surface expression of CD69 or CD71 activation markers. The majority of the responding cells were CD3+, CD4+ T-helper cells which carry the activation marker CD71 and CD69. HLA class II blocking monoclonal antibodies were utilised to determine whether the HPA-1a response is restricted by MHC class II molecules. The T helper cells responded to a single epitope, presented by peptide L1(19-33), demonstrating that the Th-cell response is focused on one dominant epitope with one major restricting class II molecule. This epitope could enable preparation of a short peptide, suitable for use for the prevention or reversal of the pathogenic alloimmune response to the HPA-1a alloantigen if presented by a tolerogenic route.