Immune Defense: The Role of NKG2D/DAP10

描述（由申请方提供）：NK细胞提供针对已被病毒感染或发生肿瘤转化的细胞的一线监测。在介导NK细胞识别靶细胞的众多细胞表面受体中，NKG 2D的独特之处在于它识别各种I类相关分子，这些分子作为感染或异常细胞的“标志”。体外研究表明，NKG 2D仅依赖于跨膜衔接子DAP 10进行信号传导。DAP 10含有募集磷脂酰肌醇-3激酶的YxxM基序。已经在CD 28中报道了相同的对接基序，其介导幼稚T细胞中的共刺激信号。因此，NKG 2D/DAP 10目前被认为是所有NK细胞以及活化的细胞毒性T细胞上的共刺激复合物。NKG 2D/DAP 10和CD 28之间的这种功能平行性提出了几个重要问题：NK细胞是否需要共刺激信号来杀死其靶点和/或在体内增殖？NKG 2D/DAP 10是否与CD 28相似，参与外周淋巴器官中CD 8 + T细胞的引发和扩增？或者，NKG 2D/DAP 10是否主要增强针对外周组织中表达NKG 2D配体的细胞的CTL效应子应答？ 为了解决这些关键问题，我们通过基因靶向产生了DAP 10缺陷小鼠。我们的初步结果表明，在NK细胞中，NKG 2D不仅能够与DAP 10结合，还能够与衔接子DAP 12结合，后者通过不同的细胞内信号传导途径介导直接激活。相反，在T细胞中，NKG 2D仅与DAP 10结合，因此仅限于YxxM“共刺激”途径。基于这些结果，我们建议通过在体外和体内比较DAP 10-/-小鼠与DAP 12-/-和正常小鼠的NK细胞功能，来剖析NKG 2D/DAP 10“共刺激”和NKG 2D/DAP 12“激活”途径在NK细胞生物学中的作用。此外，我们建议阐明NKG 2D/DAP 10“共刺激”通路在体内抗病毒和抗肿瘤CD 8 + T细胞应答中的作用。为此，我们将分析在病毒感染和肿瘤移植的小鼠模型中DAP 10-/-CD 8 + T细胞的激活、扩增和效应功能的获得。此外，我们将研究在初次感染和肿瘤疫苗接种后，DAP 10对建立和维持长期记忆CTL的需求。 虽然先前的研究表明，NKG 2D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应答很重要，但尚不清楚在应答期间需要NKG 2D信号传导的原因和位置。因此，基于DAP 10-/-小鼠分析的NKG 2D/DAP 10体内功能的详细知识将具有重要价值，特别是考虑到NKG 2D-NKG 2D-配体相互作用在肿瘤治疗和疫苗设计中的可能开发。

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.