Molecular Genetic Analysis Of Lymphocyte Function

如上所述，我们的研究已经详细了解了MHC-I与其分子伴侣TAPBPR相互作用以及分子伴侣在与肽相互作用时从MHC-I分子释放的三维结构和结构变化。我们最近发表的实验将这种分析扩展到MHC-I与tapasin（肽加载复合物（PLC）的关键伴侣）的相互作用。为此，我们首次表征了人MHC-I分子HLA-B*44：05与人伴侣Tapasin的分子复合物。其他研究包括泛抗MHC-I单克隆抗体在刺激抗病毒和抗肿瘤免疫应答中的体内表征。抗体/MHC复合物的结构研究已经确定了单克隆抗体的结合位点，并提供了其在NK细胞活化中的生物学功能机制的见解。在MHC-I/肽相互作用研究的第2部分中，我们探索了抗逆转录病毒药物阿巴卡韦在与MHC-I结合和扭曲易感MHC-I等位基因结合的自身肽库中所起的作用。特别是，我们研究了阿巴卡韦结合HLA-B*57：01在我们开发的模型动物系统中的生物学效应。因此，当在转基因动物中被HLA-B*57：01结合时，单独的阿巴卡韦可以引发新抗原T细胞应答，这依赖于CD 4 T细胞的调节。该动物模型系统为在接受药物的高比例HLA-B*57：01个体中观察到的重度超敏反应提供了解释。在正常小鼠MHC背景下，用HLA-B*57：01转基因动物进行初始研究。最近对涉及抗生素氟氯西林的类似药物/MHC-I相互作用的合作研究证明了这种特定超敏反应的相关但不同的机制。该项目的第三部分重点是T细胞受体识别抗原的结构和功能研究，这如何导致T细胞信号传导，以及这如何导致自身免疫性疾病。在这里，提供一个基线，了解抗原特异性的结构变化的T细胞受体（TCR），我们已经确定了X射线结构的病毒特异性，MHC-I限制性TCR，以及它的复合物与它的MHC-I/病毒抗原配体。值得注意的是，尽管MHC/肽复合物具有相对刚性的结构，但TCR显示出其CDR 3 α和β环的巨大运动，表明配体接合的飞抛机制。NMR实验已经表征了TCR对肽/MHC相互作用的变构效应。目前的努力是为了将我们最近获得的cryo-EM结构测定的专业知识应用于MHC/TCR复合物。

As indicated above, our studies have resulted in a detailed understanding of the three-dimensional structure and structural changes that accompany MHC-I interaction with its chaperone, TAPBPR, and with the release of the chaperone from the MHC-I molecule on interaction with peptide. Our recently published experiments extend this analysis to the interaction of the MHC-I interaction with tapasin, the crucial chaperone of the peptide loading complex (PLC). To this end, we have for the first time characterized a molecular complex of a human MHC-I molecule, HLA-B*44:05 with the human chaperone, tapasin. Additional studies include the in vivo characterization of a pan-anti-MHC-I monoclonal antibody in stimulating anti-viral and anti-tumor immune responses. Structural studies of the antibody/MHC complex have defined the binding site of the monoclonal antibody, and provide insight into the mechanism of its biological function in activation of NK cells. In Part 2 of the studies of MHC-I/peptide interactions, we have explored the role that the anti-retroviral drug, abacavir, plays in binding to MHC-I and distorting the self-peptide repertoire bound by susceptible MHC-I alleles. In particular, we examined the biological effects of abacavir binding to HLA-B*57:01 in a model animal system that we have developed. Thus, abacavir alone, when bound by HLA-B*57:01 in a transgenic animal, can elicit a neoantigen T cell response, dependent on regulation of CD4 T cells. This animal model system provides an explanation for the severe hypersensitivity reactions that are observed in a high proportion of HLA-B*57:01 individuals who receive the drug. The initial studies were performed with HLA-B*57:01 transgenic animals on a normal mouse MHC background. Recent collaborative studies of a similar drug/MHC-I interaction involving the antibiotic, flucloxacillin, demonstrate a related but distinct mechanism for this particular hypersensitivity reaction. The third part of this project is focused on structural and functional studies of T cell receptor recognition of antigens, how this leads to T cell signaling, and how this leads to autoimmune disease. Here, to provide a baseline for understanding antigen-specific structural changes in the T cell receptor (TCR), we have determined the X-ray structure of a virus specific, MHC-I-restricted TCR, as well as its complex with its MHC-I/viral antigen ligand. Remarkably, although the MHC/peptide complex has a relatively rigid structure, the TCR shows great movement of its CDR3 alpha and beta loops, indicative of a fly-casting mechanism for ligand engagement. NMR experiments have characterized allosteric effects of the TCR on peptide/MHC interaction. Current efforts are geared to apply our recently acquired expertise in cryo-EM structure determination to the MHC/TCR complex.