Immune Defense: The Role of NKG2D/DAP10

描述(由申请人提供)：NK细胞提供对已感染病毒或经历肿瘤转化的细胞的第一线监测。在众多介导NK细胞识别靶细胞的细胞表面受体中，NKG2D是独一无二的，因为它识别各种I类相关分子，这些分子是感染或异常细胞的“旗帜”。体外研究表明，NKG2D仅依赖跨膜适配器DAP10进行信号传递。DAP10包含一个YxxM基序，它招募磷脂酰肌醇-3激酶。同样的对接基序在CD28中也有报道，CD28在幼稚T细胞中介导共刺激信号。因此，NKG2D/DAP10目前被认为是所有NK细胞和激活的细胞毒性T细胞上的共刺激复合体。NKG2D/DAP10和CD28之间在功能上的相似性提出了几个重要的问题：NK细胞是否需要共刺激信号来杀死它们的靶点和/或在体内增殖？NKG2D/DAP10是否与CD28类似，参与外周淋巴器官CD8+T细胞的启动和扩增？或者，NKG2D/DAP10主要增强CTL效应对外周组织中表达NKG2D配体的细胞的反应吗？ 为了解决这些关键问题，我们通过基因打靶产生了DAP10缺陷小鼠。我们的初步结果表明，在NK细胞中，NKG2D不仅可以与DAP10结合，还可以与适配器DAP12结合，后者通过不同的细胞内信号通路介导直接激活。相反，在T细胞中，NKG2D只与DAP10结合，因此仅限于YxxM“共刺激”途径。在这些结果的基础上，我们建议通过比较DAP10-/-小鼠和DAP12-/-小鼠以及正常小鼠的NK细胞功能，来剖析NKG2D/DAP10“共刺激”和NKG2D/DAP12“激活”通路在NK细胞生物学中的作用。此外，我们建议阐明NKG2D/DAP10“共刺激”通路在体内抗病毒和抗肿瘤CD8+T细胞反应中的作用。为此，我们将分析DAP10-/-CD8+T细胞在病毒感染和肿瘤移植模型中的激活、扩增和效应功能的获得。此外，我们还将研究DAP10在原发感染和肿瘤疫苗接种后建立和维持长期记忆CTL的需求。 虽然以前的研究表明，NKG2D配体在病毒感染和肿瘤细胞上的表达对于产生保护性免疫反应是重要的，但在反应过程中为什么以及在哪里需要NKG2D信号尚不清楚。因此，在分析DAP10-/-小鼠的基础上详细了解NKG2D/DAP10在体内的功能将是非常有价值的，特别是考虑到NKG2D-NKG2D-配体相互作用可能在肿瘤治疗和疫苗设计中的开发。

DESCRIPTION (provided by applicant): NK cells provide first line surveillance against cells that have been infected by viruses or undergo neoplastic transformation. Among the numerous cell surface receptors mediating NK cell recognition of target cells, NKG2D is unique in that it recognizes a variety of class I-related molecules that serve as "flags" for infected or abnormal cells. In vitro studies indicate that NKG2D depends solely on the transmembrane adapter DAP10 for signaling. DAP10 contains an YxxM motif that recruits Phosphatidyl Inositol-3 Kinase. The same docking motif has been reported in CD28, which mediates a costimulatory signal in naive T cells. Therefore, NKG2D/DAP10 is currently considered a costimulatory complex on all NK cells as well as on activated cytotoxic T cells. This functional parallel between NKG2D/DAP10 and CD28 raises several important issues: Do NK cells require a costimulatory signal to kill their targets and/or proliferate in vivo? Is NKG2D/DAP10 involved in priming and expansion of CD8+ T cells in peripheral lymphoid organs, similar to CD28? Or, does NKG2D/DAP10 mainly enhance CTL effector responses against cells expressing NKG2D ligands in peripheral tissues? To address these crucial questions we have generated DAP10-deficient mice by gene targeting. Our preliminary results indicate that in NK cells, NKG2D has the capacity to associate not only with DAP10 but also with the adapter DAP12, which mediates direct activation via a distinct intracellular signaling pathway. In contrast, in T cells, NKG2D associates only with DAP10 and therefore is limited to the YxxM "costimulatory" pathway. On the basis of these results, we propose to dissect the role of NKG2D/DAP10 "costimulatory" and NKG2D/DAP12 "activating" pathways in NK cell biology by comparing NK cell function in DAP10 -/- mice with that of DAP12 -/- and normal mice in vitro and in vivo. In addition, we propose to clarify the role of the NKG2D/DAP10 "costimulatory" pathway in anti-viral and anti-tumor CD8+ T cell responses in vivo. For this purpose, we will analyse activation, expansion and acquisition of effector function of DAP10 -/- CD8+ T cell in mouse models of viral infection and tumor engraftment. In addition, we will study the requirement of DAP10 for establishment and maintenance of long-term memory CTLs following primary infections and tumor vaccination. While previous studies have indicated that expression of NKG2D ligands on virally infected and tumor cells is important for generating a protective immune response, it is not understood why and where NKG2D signaling is required during the response. Therefore, detailed knowledge of NKG2D/DAP10 function in vivo based on the analysis of DAP10-/- mice will be of great value, particularly in view of the possible exploitation of NKG2D-NKG2D-ligands interactions in tumor therapy and vaccine design.