Structure/function studies of CTLD NK immunoreceptors

描述（由申请人提供）：NK细胞介导重要的抗肿瘤和抗病毒先天免疫反应，并有助于同种异体干细胞移植中有效的移植物抗白血病反应。与ctl不同，在ctl中，驱动激活的关键识别事件是由单一受体介导的，即在特定细胞上表达的独特的aB TCR， NK细胞必须通过整合来自多种激活和抑制NK受体的复杂信号来对靶细胞进行审讯。这些研究的最终目的是了解NK细胞信号整合的机制。为了实现这一长期目标，我们从c型凝集素样NK受体NKG2D开始，首先确定受体、配体（MICs、ulbp、RAE-ls）和复杂的晶体结构，通过SPR表征相互作用亲和、动力学和热力学，并计算分析界面——所有这些都有助于理解识别和结构和功能背后的决定因素。在Aim 1中，我们建议通过特定突变来完成这些研究，以确认我们的识别模型、独特的发散配体MIC-A*004和ULBP4的晶体学和SPR分析，并通过突变和基于细胞的激活实验确定NKG2D信号转导通过外畴的结构约束。接下来，我们建议将这些研究扩展到NKG2x-CD94受体家族的其余部分，已经分析了它们的配体（HLA-E）的其他结构及其热稳定性，表达和亲和力之间的关系。在Aim 2中，我们提出对NKG2x-CD94-HLA-E相互作用的结构、识别机制、亲和力和动力学进行晶体学和持续的SPR分析，同时对各种激活和抑制CTLD NK受体在驱动激活过程中传递的信号的相对强度进行基于细胞的研究。完成目标1和目标2将使我们能够充分表征NK细胞信号整合机制的CTLD臂的细胞外成分，并使我们能够通过测量相对信号强度来合理化这些参数。最后，在Aim 3中，我们建议研究与mic相互作用的其他受体：V81 y8 TCRs。在表达了三种不同的mic响应型y8 tcr的可溶性形式后，我们提出了SPR相互作用研究（包括NKG2D-TCR-MIC相互作用是合作的、反合作的还是独立的）和晶体学分析来表征这些配合物。

DESCRIPTION (provided by applicant): NK cells mediate important anti-tumor and anti-viral innate immune responses and can contribute to potent graft-versus-leukemia responses in allogeneic stem cell transplants. Unlike CTLs, where the crucial recognition event driving activation is mediated by a single receptor, the unique aB TCR expressed on that particular cell, NK cells must function by integrating a complex set of signals from the diverse array of activating and inhibitory NK receptors engaged upon interrogation of target cells. The ultimate goal of these studies is to understand the mechanism of NK cell signal integration. To achieve this long-term goal, we have begun with the C-type lectin-like NK receptor NKG2D, first determining receptor, ligand (MICs, ULBPs, RAE-ls) and complex crystal structures, characterizing interaction affinities, kinetics and thermodynamics by SPR and analyzing the interfaces computationally - all leading to an understanding of recognition and the determinants underlying structure and function. In Aim 1, we propose to complete these studies with specific mutations to confirm our recognition model, crystallographic and SPR analyses of the uniquely divergent ligands MIC-A*004 and ULBP4, and by determining the structural constraints on NKG2D signal transduction through the ectodomain, using mutagenesis and cell-based activation assays. We next propose to extend these studies to the rest of the NKG2x-CD94 receptor family, having already analyzed additional structures of their ligand (HLA-E) and the correlation between its thermal stability, expression and affinity. In Aim 2, we propose crystallographic and continuing SPR analyses of the structures, recognition machinery, affinities and kinetics of NKG2x-CD94-HLA-E interactions, concurrent with cell-based studies of the relative strengths of the signals delivered by the various activating and inhibitory CTLD NK receptors in driving activation. Completing Aims 1 and 2 will result in fully characterizing the extracellular components of the CTLD arm of the NK cell signal integration mechanism, and allow us to rationalize those parameters with measurements of relative signal strengths. Finally, in Aim 3, we propose to study other receptors that interact with MICs: V81 y8 TCRs. Having expressed soluble forms of three different MIC-responsive y8 TCRs, we propose SPR interaction studies (including whether NKG2D-TCR-MIC interactions are cooperative, anti-cooperative or independent) and crystallographic analyses to characterize the complexes.