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源MHC分子的能力，发生在1-10%的未致敏T细胞中，在体内观察为移植物排斥或移植物抗宿主病，在体外观察为混合淋巴细胞反应（MLR）。同种异体反应性的现象已经知道了很长一段时间;然而，它的精确分子基础尚未完全定义。在这些拟议的研究中，我们要测试的假设，肽识别同种异体T细胞是由一个连续的特异性，它与传统的同源识别重叠，但包含一个非重叠区的简并识别。我们实验室先前对两种II类同种异体反应的肽特异性的研究揭示了一种T细胞，2.102，与同基因反应相比，其识别是简并的。相反，对于第二同种异体TCR，KRN，同种异体肽识别与同基因识别一样特异。对于第三个T细胞，3.L2，我们已经使用酵母展示系统产生了高亲和力变体。与3.L2相比，这种高亲和力TCR M15表现出肽简并性。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。3.L2和M15对分别代表特异性和简并肽识别，因此可用于模拟特异性同基因和简并同种异体应答。晶体结构为探索肽的识别和简并性提供了强有力的结构基础。在这些拟议的研究中，我们想测试同种异体反应性是否偏向于简并识别。我们提出了一系列新的，综合的，全面的研究，我们将在多种方式检查肽的特异性。在目的I中，我们建议识别和表征一系列未引发的同种异体反应性T细胞中天然存在的自身肽。在目标II中，我们将使用酵母展示系统进行一种新的功能获得方法，在该方法中，我们将创建具有获得I-EP同种异体反应性的TCR。从这些研究中，我们可以确定TCR需要什么样的变化才能使其具有同种异体反应性，以及肽特异性发生了什么样的变化。在目的III中，我们提出在结构上和生物药理学上研究3.L2和M15 TCR的肽特异性/简并性。总的来说，这些研究将为简并肽识别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.