Structure/function studies of CTLD NK immunoreceptors

描述（由申请方提供）：NK细胞介导重要的抗肿瘤和抗病毒先天性免疫应答，并可在同种异体干细胞移植中促进有效的移植物抗白血病应答。与CTL不同，其中驱动活化的关键识别事件由单个受体介导，在该特定细胞上表达的独特aB TCR，NK细胞必须通过整合来自在询问靶细胞时参与的各种活化和抑制性NK受体的一组复杂信号来起作用。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NKG 2D开始，首先确定受体、配体（MIC、ULBP、RAE-1）和复合物晶体结构，通过SPR表征相互作用亲和力、动力学和热力学，并通过计算分析界面-所有这些都导致对识别和结构和功能的决定因素的理解。在目标1中，我们建议使用特定突变完成这些研究，以确认我们对独特分歧配体MIC-A*004和ULBP 4的识别模型、晶体学和SPR分析，并通过使用诱变和基于细胞的活化试验确定NKG 2D通过胞外域信号转导的结构限制。我们接下来建议将这些研究扩展到NKG 2x-CD 94受体家族的其余部分，已经分析了其配体（HLA-E）的其他结构及其热稳定性，表达和亲和力之间的相关性。在目标2中，我们提出了对NKG 2x-CD 94-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连续SPR分析，同时对各种激活和抑制性CTLD NK受体在驱动激活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2将导致充分表征NK细胞信号整合机制的CTLD臂的细胞外组分，并允许我们通过测量相对信号强度来合理化这些参数。最后，在目的3中，我们提出研究与MIC相互作用的其他受体：V81 γ 8 TCR。在表达了三种不同MIC响应性γ 8 TCR的可溶形式后，我们提出了SPR相互作用研究（包括NKG 2D-TCR-MIC相互作用是协同的、反协同的还是独立的）和晶体学分析来表征复合物。

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.