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的先天免疫反应，并可在同种异基因干细胞移植中促进强大的移植物抗白血病反应。与CTL不同，在CTL中，关键的识别事件驱动激活是由单个受体介导的，该特定细胞上表达的唯一AB TCR，NK细胞必须通过整合一组复杂的信号来发挥作用，这些信号来自于在询问靶细胞时参与的不同的激活和抑制NK受体阵列。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、ULBPs、RAE-l)和复杂的晶体结构，用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以及潜在结构和功能的决定因素的理解。在目标1中，我们建议用特定的突变来完成这些研究，以确认我们对唯一发散的配体MIC-A*004和ULBP4的识别模型、结晶学和SPR分析，并通过突变和基于细胞的激活分析来确定NKG2D信号转导的结构约束。接下来，我们建议将这些研究扩展到NKG2x-CD94受体家族的其余成员，已经分析了它们的配体(HLA-E)的额外结构以及其热稳定性、表达和亲和力之间的相关性。在目标2中，我们建议对NKG2x-CD94-HLA-E相互作用的结构、识别机制、亲和力和动力学进行结晶学和连续的SPR分析，同时基于细胞研究各种激活和抑制的CTLD NK受体在驱动激活过程中传递的信号的相对强度。完成目标1和2将导致完全表征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，在目标3中，我们建议研究其他与MICs相互作用的受体：V81Y8TCR。在表达了三种不同MIC响应的Y8 TCR的可溶性形式后，我们建议进行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)和结晶学分析来表征这些化合物。

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.