Mechanism and in vitro reconstitution of tapasin-mediated peptide exchange

抗原呈递到T细胞是由主要组织相容性复合体（MHC）编码的蛋白质提供的。这种高度多态基因位点的等位基因导致了人群中数千种不同MHC同种异体的表达。每个同种异体都能与多种不同的抗原肽结合。当显示在细胞表面时，它们可以被T细胞监视，潜在地调用适应性免疫的细胞臂。在两大类典型的MHC分子中，存在于所有有核细胞中的MHC I类（MHC-I）通常从蛋白酶体中获得抗原。蛋白酶体切割后长度为9-15个氨基酸的肽通过与抗原加工相关的转运体进入内质网，由氨基肽酶ERAP进一步修剪，并在肽装载复合物（PLC）中装载到mhc -1分子上。在PLC的核心，交换催化剂tapasin稳定MHC-I分子，用高仿射肽取代低仿射肽，从而影响MHC-I显示的免疫肽体，最终影响T细胞的反应性。tapasin的结合和肽交换活性在不同的MHC-I同种异型中有很大的不同，这就提出了同种异型区分背后的机制问题。在这里，这个中心问题首先由结构生物学和生物化学方法来解决。将研究几种人和啮齿动物mhc - 1分子对tapasin的结合和交换敏感性。此外，将开发一种体外重组系统，包括MHC-I装载复合物的基本成分。建立这样一个极简的实验系统，可以鉴定来自病毒蛋白或具有新抗原的肿瘤蛋白的MHC-I结合肽段。mhc - i肽显示的tapasin依赖性将被测量，并与机制见解相结合，以获得同种异体特异性特征。这些知识既可以用于对来自新病毒或患者特异性肿瘤新抗原的蛋白质的MHC-I呈递进行个性化预测，也可以用于开发可用于可视化、丰富或扩增抗原特异性T细胞克隆的工具。

Presentation of antigens to T cells is provided by proteins encoded by the Major Histocompatibility Complex (MHC). The alleles of this highly polymorphic gene locus lead to the expression of thousands of different MHC allotypes across the population. Each allotype is capable of binding to a manifold of different antigenic peptides. When displayed on the cell surface, they can be surveilled by T cells, potentially invoking the cellular arm of adaptive immunity. Of the two major classes of canonical MHC molecules, MHC class I (MHC-I), which are present on all nucleated cells, typically derive their antigens from the proteasome. Peptides of 9-15 amino acid in length after proteasomal cleavage enter the endoplasmic reticulum through the transporter associated with antigen processing, are further trimmed by the aminopeptidase ERAP, and are loaded onto the MHC-I molecules in the peptide loading complex (PLC). At the heart of the PLC, the exchange catalyst tapasin stabilizes the MHC-I molecules and acts to replace lower affine by higher affine peptides, thus influencing the MHC-I displayed immunopeptidomes and ultimately T cell reactivity. The binding and peptide exchange activity of tapasin strongly differs for the individual MHC-I allotypes and raises the question of the mechanism behind allotypic distinction. Here, this central question is first addressed by structural biology and biochemical methods. Several human and rodent MHC-I molecules will be investigated for their binding and exchange susceptibility to tapasin. Furthermore, an in vitro reconstituted system will be developed that comprises essential components of the MHC-I loading complex. Establishing such a minimalistic experimental system allows for the identification of MHC-I bound peptidomes derived from viral proteins or from tumor proteins featuring neo-antigens. The tapasin dependency of MHC-I-peptide display will be measured and combined with the mechanistic insights to derive allotype-specific features. This knowledge can either be used to make individualized predictions of MHC-I presentation of proteins from novel viruses or patient-specific tumor neo-antigens or it will allow to develop tools that can be used to visualize, enrich, or expand antigen-specific T cell clones.