Molecular Genetic Analysis Of Lymphocyte Function

如上所述，我们的研究导致了对MHC-I与其伴侣TAPBPR相互作用以及伴随着MHC-I分子与多肽相互作用时伴侣释放的三维结构和结构变化的详细了解。我们最近发表的实验将这一分析扩展到MHC-I与Tapasin的相互作用，Tapasin是多肽负载复合体(PLC)的关键伴侣。为此，我们首次表征了人类MHC-I分子与人类伴侣蛋白Tapasin的分子复合体--人类白细胞抗原-B*44：05。其他研究包括一种泛抗MHC-I单抗在体内刺激抗病毒和抗肿瘤免疫反应的特性。对抗体/MHC复合体的结构研究已经确定了单抗的结合部位，并对其在NK细胞激活中的生物学作用机制提供了深入的了解。在MHC-I/肽相互作用研究的第二部分中，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MHC-I易感等位基因结合的自肽谱中所起的作用。特别是，我们在我们开发的模型动物系统中检测了阿巴卡韦与人类白细胞抗原-B*57：01结合的生物学效应。因此，在转基因动物中，当阿巴卡韦单独与人类白细胞抗原B*57：01结合时，可以诱导新的抗原T细胞反应，依赖于CD4T细胞的调节。该动物模型系统为在接受药物治疗的高比例的人类白细胞抗原-B*57：01个体中观察到的严重过敏反应提供了解释。最初的研究是在正常小鼠MHC背景下用人类白细胞抗原-B*57：01转基因动物进行的。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表明，这种特殊的过敏反应有一个相关但不同的机制。该项目的第三部分集中于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，以及这如何导致自身免疫性疾病。在这里，为了为了解T细胞受体(TCR)的抗原特异性结构变化提供基线，我们确定了病毒特异性的MHC-I限制性TCR及其与MHC-I/病毒抗原配体的复合体的X射线结构。值得注意的是，尽管MHC/肽复合体具有相对刚性的结构，但TCR显示其CDR3α和β环有很大的移动，这表明配体结合的飞投机制。核磁共振实验表征了TCR对多肽/MHC相互作用的变构效应。目前的努力是将我们最近在低温EM结构确定方面获得的专业知识应用于MHC/TCR复合体。

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.