The mechanism of NK cell defects in human NEMO deficiency

描述（由申请人提供）：由NF-？B必需修饰子（NEMO）基因损害多种免疫功能并导致严重的感染易感性。由这些突变（NEMO- id）引起的疾病是由于NEMO不能有效地发挥其激活NF-？关键免疫受体结扎后的B转录因子。NEMO-ID患者易受巨细胞病毒感染，这使我们确定他们具有NK细胞毒性缺陷。NK细胞是先天免疫系统的淋巴细胞，在形成免疫突触（immune synapse， IS）后，能够通过定向分泌溶解颗粒到靶细胞上的过程杀死目标细胞。NK细胞在防御巨细胞病毒和其他利用策略逃避适应性免疫的病毒方面特别有用。其他几种影响NK细胞的原发性免疫缺陷也强调了这一点。虽然NEMO-ID中的许多免疫缺陷在机制水平上被理解，但NK细胞的细胞毒性却不是。通过本申请中提出的工作，我们将确定NEMO-ID中NK细胞缺陷的机制。这是相关的，因为NK细胞缺乏会对NEMO-ID患者的健康产生不利影响，了解其机制可能会提出专门克服它的治疗策略。明确NEMO-ID中NK细胞缺乏的机制也将为细胞溶解细胞和IS的功能提供意想不到的新见解。在我们对NEMO-ID患者NK细胞的研究中，我们发现这种缺陷是特异性的，并且可以在IL-2刺激后被绕过。因此，NK细胞不太可能发育异常，NEMO在NK细胞毒性中起急性作用的机制可能是存在的。通过体外初步研究，我们发现nemo依赖性NF-？NK细胞活化受体连接后可快速诱导B通路。这导致NF-？b依赖基因的转录和新蛋白质的合成在与细胞溶解过程一致的时间框架内。使用几种方法，我们已经确定了有限数量的NEMO-和NF-？b依赖性蛋白在NK细胞中快速合成，并在细胞毒性和裂解性IS中发挥作用。因此，我们的长期目标是进一步建立NK细胞中NEMO功能的范例，并确定NEMO依赖的快速合成蛋白在is形成和细胞毒性中的作用。具体来说，我们将：1)定义NK细胞IS的阶段，这取决于NEMO功能和NF-？B激活;2)确定快速合成的nemo依赖蛋白在NK细胞功能中的作用；3)确定IL-2是否能恢复关键的NEMO/NF-？NF-?阻断NK细胞中的B依赖性蛋白B信号。总的来说，这些研究将确定NEMO-ID中NK细胞缺乏的机制，确定调节细胞溶解功能的新途径，并为NEMO-ID患者提供潜在有用的治疗方法。公共卫生相关性：我们的提案，“人类NEMO缺乏症中NK细胞缺陷的机制”，旨在了解为什么NEMO基因亚型突变的患者具有缺陷的NK细胞功能。我们认为答案是NEMO在快速转录和蛋白质合成中起着令人惊讶的作用，从而使NK细胞具有细胞毒性。我们的工作将验证这一假设，并具有广泛的相关性，因为它将定义一种新的机制，通过这种机制可以调节免疫功能，并更具体地了解NEMO缺乏症中的NK细胞，从而可能允许合理的治疗。

DESCRIPTION (provided by applicant): Human primary immunodeficiency caused by hypomorphic mutation of the NF-?B essential modifier (NEMO) gene impairs several immunological functions and leads to grave infectious susceptibilities. The disease resulting from these mutations (NEMO-ID) arises from an inability of NEMO to effectively perform its typical function enabling activation of the NF-?B transcription factor after ligation of key immunoreceptors. Patients with NEMO-ID are vulnerable to infection with cytomegalovirus, which has led us to identify their having a defect of NK cell cytotoxicity. NK cells are lymphocytes of the innate immune system that are capable of killing through the process of directed secretion of lytic granules onto a target cell after forming an immunological synapse (IS). NK cells are especially useful in defense against cytomegalovirus and other viruses that employ strategies to evade adaptive immunity. This is underscored by several other primary immunodeficiencies affecting NK cells. Although a number of the immunological deficits in NEMO-ID are understood at a mechanistic level, that of NK cell cytotoxicity is not. Through the work proposed in this application we will determine the mechanism of the NK cell defect in NEMO-ID. This is relevant as NK cell deficiency adversely affects the health of patients with NEMO-ID and understanding its mechanism will likely suggest therapeutic strategies to specifically overcome it. Defining the mechanism of NK cell deficiency in NEMO-ID will also provide unexpected and novel insight into the function of cytolytic cells and the IS. In our studies of NK cells from NEMO-ID patients we have found that the defect is specific and can be circumvented after IL-2 stimulation. Thus it is unlikely that the NK cells are developmentally abnormal and a mechanism in which NEMO serves an acute role in NK cell cytotoxicity is likely. Through in vitro preliminary studies we have found that the NEMO-dependent NF-?B pathway is rapidly induced in NK cells after activation receptor ligation. This leads to rapid NF-?B-dependent gene transcription and synthesis of new proteins in a timeframe consistent with the cytolytic process. Using several approaches we have identified a limited number of NEMO- and NF-?B-dependent rapidly synthesized proteins in NK cells and are pursuing their role in enabling cytotoxicity and the lytic IS. Thus, our long term objective is to further establish this paradigm of NEMO function in NK cells and define a role for NEMO-dependent rapidly synthesized proteins in formation of the IS and cytotoxicity. Specifically we will: 1) define the stage of the NK cell IS that depends upon NEMO function and NF-?B activation; 2) determine the role of rapidly synthesized NEMO-dependent proteins in NK cell function; 3) determine if IL-2 can restore the expression of critical NEMO/NF-?B dependent proteins in NK cells with blocked NF-?B signaling. Cumulatively, these studies will determine the mechanism of NK cell deficiency in NEMO-ID, define a novel pathway of regulating cytolytic function and provide insight into potentially useful therapy for NEMO-ID patients. PUBLIC HEALTH RELEVANCE: Our proposal, "The Mechanism of NK Cell Defects in Human NEMO Deficiency", is aimed at understanding why patients with hypomorphic mutations in the NEMO gene have defective NK cell function. We believe the answer is that NEMO serves a surprising role in rapid transcription and protein synthesis to enable NK cell cytotoxicity. Our work will test this hypothesis and be of broad relevance in that it will define a novel mechanism by which immunological function can be regulated as well as more specific insight into NK cells in NEMO deficiency that may allow for rational therapies.