Identification of T cell epitopes and development of a new immunotherapy for idiopathic thrombocytopenic purpura

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The purpose of this study is to define the role and dominant epitopes of GPIIb-reactive T cells in idiopathic thrombocytopenic purpura (ITP). We established a CD4^+ T cell line recognizing the GPIIb #429-443 amino acid sequence in a HLA-DR^*0405-restricted manner from an ITP patient. Interferon-gamma production was detected when the cells were co-cultured autologous immature dendritic cells which had been loaded with platelet lysate, suggesting that the region around GPIIb 429-443 can be presented to CD4^+ T cells in the context of HLA-DRB1^*0405. We isolated further T cell clones from several ITP patients. These clones were mainly CD4^+ helper T cells, but CD8^+ cytotoxic T cells could also be isolated. To measure helper activity of T cells, we established a sandwich ELISA assay for GPIIb antibody using anti-GPIIb-IIIa monoclonal antibodies produced in our laboratory. Then we reported WT1-specific and HLA class II-restricted CD4^+ T cells possessing direct cytotoxic activity against leukemia cells, indicating establishment of specific assay for cytotoxic activity of CD4^+ T cells. We also reported that perforin is expressed constitutively in memory CD8^+ cytotoxic T cells, but is dependent on cell activation in memory CD4^+ cytotoxic T cells, suggesting differential regulation of cytotoxicity in CD4^+ and CD8^+ T cells. Then we attempted to identify the amino acid regions critical for GPIIb-IIIa activation using alanine-scaninng mutagenesis. We identified three amino acids critical for ligand binding to GPIIb-IIIa and an amino acid motif in the intracellular domain of GPIIb that suppresses GPIIb-IIIa activation.