Structural correlates of T cell receptor signaling

描述（由申请人提供）：T细胞在整个发育和外周监测过程中参与功能不同的配体的能力归因于TCR交叉反应性。然而，目前尚不清楚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免疫中TCR配体歧视的机制和后果非常重要。在这里，我们希望应用一种强大的方法，肽- mhc酵母展示与深度测序相结合，通过以下问题来探索TCR交叉反应性、结构和功能之间的相互作用：1- TCR交叉反应性如何？我们的肽- mhc文库首次实现了TCR/pMHC交叉反应性的实验测定，可以测量不同TCR与普通pMHC、自身免疫TCR和从不同类型T细胞分离的TCR之间的交叉反应程度。TCR/pMHC的交叉反应性和结合拓扑在TCR信号传导中起指导作用吗？文献中描述的大多数“替代”激动剂肽都是具有保守取代的同源配体，它们被TCR识别为类似的结构模式，这导致了TCR信号在结构上是不分青红皂白的普遍假设。相比之下，我们发现pMHC酵母显示发现的非同源肽被TCR以高亲和力识别，其不寻常的对接几何形状无法激活细胞内信号传导。这是首次证明TCR/pMHC复合物的对接模式是TCR信号传导的重要变量。我们希望确定在不同结构模式下被几种TCR识别的肽，并询问TCR交叉反应性的生物物理参数如何调节小鼠和人类系统中的下游信号，其中存在最先进的工具来询问机制。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的交叉反应性、分子拟态和自身抗原识别的一些异常结构特征

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.