Investigation on the molecular mechanisms of antigen presentation and recognition.

通过抗原提呈细胞递呈抗原肽，进而刺激CD4^+T细胞，在免疫应答的调节中起着至关重要的作用。在本研究中，我们研究了T细胞受体(TCR)对抗原肽的识别及其在修饰的多肽配体(APL)刺激下的信号转导途径。此外，我们还建立了基于ES细胞的树突状细胞(DC)基因工程方法。1)利用基于片段替换不变链的表位呈递载体，建立了一个从T细胞表位表达文库中识别不同T细胞表位的实验系统。利用将随机氨基酸残基缩小为三个连续氨基酸的文库，我们表征了来自IDDM患者的GAD65反应性Th细胞克隆的表位的简并性，并鉴定了Th细胞克隆与之交叉反应的几种微生物来源的抗原。2)我们发现了…进一步证明，人CD4^+T细胞克隆对高表达的部分激动肽/人类白细胞抗原-DR4复合体有较强的反应能力。然而，蛋白质酪氨酸激酶ZAP-70被认为是TCR介导的T细胞激活所必需的，它没有被酪氨酸磷酸化和激活。相反，我们发现其他激酶B-Raf和PKCm在T细胞中被激活，这表明存在新的信号通路，导致T细胞完全增殖，这不依赖于ZAP-70的激活。3)我们建立了一种产生小鼠ES细胞来源的DC(ES-DC)的方法。ES-DC的基因修饰可以通过ES细胞的转染和随后向DC的分化来完成。通过注射携带OVA表达载体的ES-DC，可在体内有效地激发OVA抗原特异性细胞毒性T淋巴细胞。表达趋化因子的双转基因ES-DC与OVA一起提供了对OVA表达的肿瘤细胞的有效保护。此外，我们还展示了转基因ES-DC治疗对实验性自身免疫性脑脊髓炎的预防作用。较少

Presentation of antigenic peptides by antigen presenting cells and consequent stimulation of CD4^+ T cells is crucial in the regulation of immune response. In this research project, we studied on the recognition of antigenic peptide by T cell receptor (TCR) and signal transduction pathway in CD4^+ T cells stimulated with altered peptide ligands (APL). In addition, we established a method for ES cell-based genetic engineering of dendritic cells (DC). The following results were obtained.1)We developed an experimental system to dentify diverse T-cell epitopes from T-cell epitope-expression library, using an epitope presenting vector based on CLIP-substituted invariant chain. Using the libraries in which randomized amino acid residues were narrowed down into three successive ones, we characterized the degeneracy in the epitopes of the GAD65-reactive Th-cell clones derived from IDDM patients, and identified several microbe-derived antigens to which the Th-cell clones cross-reacted.2)We foun … More d that a human CD4^+ T cell clone showed full proliferation in response to over-expressed partially agonistic peptide/HLA-DR4 complex. However, a protein tyrosine kinase ZAP-70, which is believed to be essential for TCR-mediated T cell activation, was not tyrosine-phosphorylated and activated. Instead, we found that other kinases B-Raf and PKCm were activated in the T cells, suggesting the presence of new signaling pathways leading to full T cell proliferation that is independent of ZAP-70 activation.3)We established a method to generate mouse ES cell-derived DC (ES-DC). Genetic modification of ES-DC can be done by transfection of ES cells and subsequent differentiation to DC. OVA antigen-specific cytotoxic T lymphocytes were efficiently primed in vivo by injecting mice with ES-DCs introduced with an OVA-expression vector. Double-transfectant ES-DC expressing a chemokine along with OVA provided potent protection from OVA-expressing tumor cells. In addition, we demonstrated prevention of experimental autoimmune encephalomyelitis by treatment with genetically modified ES-DC. Less