T Cell Recognition of Allogeneic Peptide/MHC Ligands

描述(申请人提供)：异体反应性，T细胞识别外来MHC分子的能力，T细胞从未接触过，发生在1-10%的未激发T细胞中，在体内观察为移植物排斥反应，或移植物抗宿主病，体外观察为混合淋巴细胞反应(MLR)。同种异体反应性的现象早已为人所知，然而，其确切的分子基础还没有完全确定。在这些拟议的研究中，我们想要检验这样一个假设，即同种异体T细胞识别多肽是由一系列特异性表示的，这与传统的同基因识别重叠，但包含一个不重叠的退化识别区域。我们实验室以前对两个II类同种异体反应的多肽特异性的研究揭示了一个T细胞，2.102，与同基因反应相比，识别是退化的。相反，对于第二个异体TCR，KRN，同种异体多肽识别与同种异体识别一样特异。对于第三个T细胞3.L2，我们使用酵母展示系统产生了一个高亲和力变体。与3.L2相比，这一高亲和力TCR，M15，表现出多肽简并。重要的是，我们解决了M15与其Hb/I-Ek配体结合的高分辨(2.3A)晶体结构。3.L2和M15对分别代表特定的和简并的多肽识别，因此可以用来模拟特定的同源和简并的同种异体反应。晶体结构为探索多肽的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，我们想要测试同种异体反应是否偏向于简并的识别。我们提出了一系列新颖的、综合的和全面的研究，在这些研究中，我们将以多种方式检查多肽的特异性。在目标I中，我们建议在一系列未启动的同种异体反应性T细胞中鉴定和表征自然呈现的自体多肽。在AIM II中，我们将使用酵母展示系统执行一种新的功能获得方法，在该系统中我们将创建获得I-EP同种异体反应的TCR。从这些研究中，我们可以确定TCR中需要什么变化才能使其产生同种异体反应，以及多肽特异性发生了什么变化。在目标III中，我们建议从结构和生物物理的角度研究3.L2和M15 TCR的多肽特异性/简并性。总体而言，这些研究将为简并与特异性多肽识别的分子基础提供关键的见解，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.