Structural correlates of T cell receptor signaling

描述（由申请方提供）：T细胞在整个发育和外周监测过程中与功能不同的配体结合的能力归因于TCR交叉反应性。然而，目前还不清楚TCR在多大程度上真正具有交叉反应性，或者交叉反应性如何影响信号传导。这些问题对于理解正常和致病性免疫中TCR配体歧视的机制和后果是重要的。在这里，我们希望应用一种强大的方法，肽-MHC酵母展示结合深度测序，通过询问以下问题来探索TCR交叉反应性，结构和功能之间的相互作用：1-TCR的交叉反应性如何？我们的肽-MHC库首次实现了TCR/pMHC交叉反应性的实验测定，可以衡量不同TCR与常见pMHC、自身免疫性TCR和从不同类型的T细胞分离的TCR之间的交叉反应性程度。2-TCR/pMHC交叉反应性和结合拓扑结构在TCR信号传导中起指导作用吗？在文献中表征的大多数“替代”激动剂肽仅仅是具有保守取代的同源配体，其以类似的结构模式被TCR识别，导致TCR信号传导在结构上是无差别的。相比之下，我们发现pMHC酵母展示发现的非同源肽被TCR以不寻常的对接几何形状以高亲和力识别，该几何形状未能激活细胞内信号。这是首次证明TCR/pMHC复合物的对接模式是TCR信号传导中的重要变量。我们希望确定的肽，识别由几个TCR在不同的结构模式，并询问如何TCR交叉反应性的生物物理参数调节下游信号在小鼠和人类系统中的最先进的工具存在的询问机制。3-TCR/pMHC交叉反应性和对接几何形状影响人类自身免疫性疾病吗？TCR交叉反应性、分子模拟和自身抗原识别的几种异常结构特征 已经被提出在自身免疫性疾病的发展中起作用。事实上，来自多发性硬化患者的几种MBP反应性TCR呈现与由HLA-DR 15呈递的MBP的非常不寻常的结合模式。我们希望通过探测MBP反应性人TCR的交叉反应特性，并鉴定以与MBP自身抗原不同的结合模式识别的肽来测试这些假设。4-在自然的人类免疫中，非信号TCR/pMHC相互作用的分子基础是什么？出乎意料的是，我们最近发现，使用HIV-DR 4四聚体从人血液中分离的高百分比的T细胞克隆不响应于这些肽抗原而发出信号，显然参与了体内“非生产性”但特异性的TCR/pMHC相互作用。我们希望确定TCR结合如何通过这些肽与信号传导解耦的结构基础，并分离同源内源性激动剂肽。总的来说，我们建议询问和操纵TCR交叉反应的基本方面的方式，是直接密切相关的人类翻译免疫学。

DESCRIPTION (provided by applicant): The ability of T cells to engage functionally distinct ligands throughout development and peripheral surveillance has been attributed to TCR cross-reactivity. Yet, it is still unclear to what extent TCRs really are cross-reactive, or how cross-reactivity impacts signaling. These questions are important to understanding the mechanisms for, and consequences of, TCR ligand discrimination in normal and pathogenic immunity. Here we wish to apply a powerful methodology, peptide-MHC yeast display coupled with deep sequencing, to explore the interplay between TCR cross-reactivity, structure, and function by asking: 1-How cross-reactive are TCRs? Our peptide-MHC libraries enable, for the first time, an experimental determination of TCR/pMHC cross-reactivity that can gauge the extent of cross-reactivity between different TCRs to a common pMHC, autoimmune TCRs, and TCRs isolated from different types of T cells. 2-Do TCR/pMHC cross-reactivity and binding topology play instructive roles in TCR signaling? Most 'alternative' agonist peptides characterized in the literature are simply cognate ligands with conservative substitutions that are recognized in similar structural modes by a TCR, leading to the common assumption that TCR signaling is structurally indiscriminate. In contrast, we found that a non-homologous peptide discovered by pMHC yeast display was recognized with high affinity by a TCR in an unusual docking geometry that failed to activate intracellular signaling. This was the first demonstration that the docking mode of the TCR/pMHC complex is an important variable in TCR signaling. We wish to identify peptides that are recognized by several TCRs in diverse structural modes, and ask how the biophysical parameters of TCR cross-reactivity modulate downstream signaling in mouse and human systems where state-of-the-art tools exist to interrogate mechanism. 3-Do TCR/pMHC cross-reactivity and docking geometry influence human autoimmune disease? TCR cross-reactivity, molecular mimicry, and several aberrant structural features of self-antigen recognition have been proposed to play roles in development of autoimmune disease. Indeed, several MBP-reactive TCRs derived from patients with Multiple Sclerosis assume a very unusual binding mode with MBP presented by HLA-DR15. We wish to test these hypotheses by probing the cross-reactive properties of MBP-reactive human TCRs, and identifying peptides that are recognized in different binding modes than the MBP autoantigen. 4-What is the molecular basis of non- signaling TCR/pMHC interactions in natural human immunity? Unexpectedly, we recently found that a high percentage of T cell clones isolated from human blood using HIV-DR4 tetramers do not signal in response to these peptide antigens, apparently engaged in 'non-productive', but specific, TCR/pMHC interactions in vivo. We wish to determine the structural basis for how TCR binding is decoupled from signaling by these peptides, and isolate cognate endogenous agonist peptides. Collectively, we propose to both interrogate and manipulate basic aspects of TCR cross-reactivity in a manner that is directly germane to human translational immunology.