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

本研究的目的是明确GPIIb反应性T细胞在特发性血小板减少性紫癜(ITP)中的作用和优势表位。我们从一名ITP患者那里建立了一株识别GPIIb#429-443氨基酸序列的CD4^+T细胞系，该细胞系以人类白细胞抗原DR^*0405-限制性方式识别。当细胞与负载了血小板裂解物的自体未成熟树突状细胞共同培养时，检测到干扰素-γ的产生，这表明在HLA-DRB1^*0405的背景下，GPIIb429-443周围的区域可以呈现给CD4^+T细胞。我们从几个ITP患者身上分离出了更多的T细胞克隆。这些克隆主要是CD4~+辅助T细胞，但也可以分离CD8~+细胞毒性T细胞。为了检测T细胞的辅助活性，我们用本实验室制备的抗GPIIb-IIIa单抗建立了检测GPIIb抗体的夹心ELISA法。然后我们报道了WT1特异性和人类白细胞抗原II类限制性的CD4^+T细胞对白血病细胞具有直接的细胞毒活性，表明建立了特异性的CD4^+T细胞细胞毒活性检测方法。我们还报道了穿孔素在记忆性CD8^+细胞毒性T细胞中的组成性表达，但在记忆性CD4^+细胞毒性T细胞中的表达依赖于细胞的激活，提示在CD8^+和CD8^+T细胞中存在细胞毒性的不同调节。然后，我们试图通过丙氨酸扫描突变来确定对GPIIb-IIIa激活至关重要的氨基酸区域。我们确定了三个与GPIIb-IIIa配体结合至关重要的氨基酸，以及GPIIb胞内区域中一个抑制GPIIb-IIIa激活的氨基酸基序。

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.