T Cell Recognition of Allogeneic Peptide/MHC Ligands

描述（由申请人提供）：同种异体反应性，即T细胞识别从未接触过的外来MHC分子的能力，发生在1-10%未启动的T细胞中，在体内以移植物排斥反应或移植物抗宿主病的形式观察，在体外以混合淋巴细胞反应（MLR）的形式观察。自体反应性现象早已为人所知；然而，其确切的分子基础尚未完全确定。在这些拟议的研究中，我们想要验证一个假设，即异体T细胞的肽识别是由一个连续的特异性代表的，它与传统的同基因识别重叠，但包含一个非重叠的退化识别区域。我们实验室之前对两种II类异体反应的肽特异性的研究表明，与同基因反应相比，一种T细胞2.102的识别能力下降。相反，对于第二个等位基因TCR， KRN，同种异体肽识别与同基因识别一样特异性。第三个T细胞是3。L2，我们使用酵母展示系统生成了一个高亲和力的变体。与3.L2相比，这种高亲和力的TCR M15表现出肽退化。重要的是，我们已经解决了M15结合其Hb/I-Ek配体的高分辨率（2.3A）晶体结构。的3。L2和Ml5对分别代表特异性和退化肽识别，因此可以用来模拟特异性的同基因和退化同种异体反应。该晶体结构为探索多肽识别和简并提供了强有力的结构基础。在这些拟议的研究中，我们想测试自体反应性是否偏向于退化的识别。我们正在提出一系列新颖、综合和全面的研究，在这些研究中，我们将以多种方式检查肽的特异性。在Aim I中，我们建议在一系列未启动的同种异体反应性T细胞中识别和表征自然呈现的自肽。在Aim II中，我们将使用酵母展示系统执行一种新的功能增益方法，在该系统中，我们将创建具有I-EP异反应性的tcr。从这些研究中，我们可以确定TCR需要哪些变化才能使其具有同种异体反应性，以及肽特异性发生了哪些变化。在Aim III中，我们建议研究3.的肽特异性/退化性。L2和M15 tcr在结构和生物物理上。总的来说，这些研究将为退化与特异性肽识别的分子基础提供关键见解，并将促进我们对同种异体反应性的理解。

DESCRIPTION (provided by applicant): Alloreactivity, the ability of a T cell to recognize foreign MHC molecules, to which the T cell had never been exposed, occurs in 1-10% of unprimed T cells, and is observed in vivo as graft rejection, or graft versus host disease, and in vitro as a mixed lymphocyte reaction (MLR). The phenomenon of alloreactivity has been known for a long time; however, the precise molecular basis for it is yet to be fully defined. In these proposed studies, we want to test the hypothesis that peptide recognition by allogeneic T cells is represented by a continuum of specificity, which overlaps with conventional syngeneic recognition, but contains a nonoverlapping area of degenerate recognition. Previous studies from our laboratory on the peptide specificity of two class II alloresponses have revealed one T cell, 2.102, where the recognition is degenerate compared to the syngeneic response. Conversely, for a second allo TCR, KRN, the allogeneic peptide recognition is as specific as the syngeneic recognition. For a third T cell, 3.L2, we have generated a high affinity variant using a yeast display system. This high affinity TCR, M15, exhibits peptide degeneracy compared to 3.L2. Importantly, we have solved a high resolution (2.3A) crystal structure of M15 bound its Hb/I-Ek ligand. The 3.L2 and Ml5 pair represent specific and degenerate peptide recognition respectively, and can therefore be used to model a specific syngeneic and degenerate alloresponse. The crystal structure provides a strong structural basis to explore the peptide recognition and degeneracy. In these proposed studies we want to test whether alloreactivity is biased towards degenerate recognition. We are proposing a series of novel, integrated, and comprehensive studies in which we will examine peptide specificity in multiple ways. In Aim I, we propose to identify and characterize the naturally presented self-peptides in a series of unprimed alloreactive T cells. In Aim II, we will perform a novel gain of function approach using a yeast display system in which we will create TCRs which have acquired I-EP alloreactivity. From these studies, we can ascertain what changes in a TCR are required to make it alloreactive and what changes in peptide specificity have occurred. In Aim III, we propose to study the peptide specificity/degeneracy of the 3.L2 and M15 TCRs structurally and biophysically. Overall, these studies will provide key insights into the molecular basis for the degenerate vs. specific peptide recognition and will advance our understanding of alloreactivity.