Regulation of NK cell antiviral responses through alternative splicing

项目摘要/摘要 自然杀伤(NK)细胞已被证明在调节病毒感染以及在 消除癌细胞。传统上，NK细胞被归类为天然免疫系统的成员 因为它们能够对感染或异常的细胞做出快速和非特异性的反应。有趣的是， 越来越多的证据表明，NK细胞也可能具有多肽特异性的记忆反应(以前只有 可归因于适应性免疫)，特别是在包括艾滋病毒/SIV和巨细胞病毒在内的几种病毒感染的情况下。 NK细胞如何形成这些适应性反应是免疫学研究中一个新兴领域的主题， 这也包括研究NK细胞等传统先天效应器的训练有素的免疫力。适应性NK是否 细胞反应是在训练免疫中观察到的表观遗传修饰的结果，或者是前 形成多态受体，如KIR或NKG2家族成员，与MHC分子相互作用，或 尚未发现的受体还有待确定。在这里，我们建议研究NK细胞转录组 可以提供关于感染或疾病状态如何调节NK细胞功能的见解，特别是通过评估 感染后选择性基因剪接的变化如何导致NK细胞功能障碍或形成 适应性NK细胞反应。选择性剪接是细胞利用的一种机制，以产生 来自单个基因的基因产物，通过以各种组合将外显子拼接在一起，有时导致 到功能迥异的基因产品。基因拼接的调控是复杂的，但它已经被证明 在病毒感染或疾病期间看到的环境变化可以产生基因剪接模式。 使用从急性SIV感染队列中分离的NK细胞的初步数据，我们已经识别了几个 候选剪接基因在SIV感染后表现出不同的异构体表达。的 特别令人感兴趣的是bHLH转录因子(TF)家族的成员，因为它们与 记忆反应的形成和NK细胞的发育。此外，在一项概念验证研究中，我们已经表明 在恒河猴中，NKp30亚型的表达在体外刺激后甚至在疫苗接种后发生变化 猕猴和人类NK细胞样本中。在这里，我建议识别新的候选基因并评估其作用 恒河猴和人类NK细胞中bHLHTF剪接变异体和其他候选基因的研究。通过这件事 工作I旨在确定特定的基因亚型表达是否影响正常的NK细胞功能或 急、慢性SIV感染猕猴体内适应性NK细胞反应的形成。 这份提案中概述的方法将使我能够将我在NK细胞生物学方面的博士后培训结合起来 在我的博士前训练中，我评估了体内选择性剪接的作用，以便确定 在逆转录病毒感染期间，有几个候选基因对正常或适应性NK细胞反应有影响。我们的终极 目标是识别和靶向特定的异构体，以在体内操纵NK细胞的功能，以达到改善 目前的疫苗反应或引发针对HIV的有针对性的NK细胞特异性治疗。

Project Summary/Abstract Natural killer (NK) cells have been shown to play a significant role in regulating viral infections as well as in eliminating cancerous cells. Traditionally, NK cells are classified as members of the innate immune system because of their ability to respond rapidly and non-specifically to infected or abnormal cells. Interestingly, burgeoning evidence suggests NK cells may also possess peptide-specific memory responses (previously only attributable to adaptive immunity), especially in the context of several viral infections including HIV/SIV and CMV. How NK cells can form these adaptive responses is the subject of an emerging area in immunology research, which also includes studying trained immunity of traditionally innate effectors like NK cells. Whether adaptive NK cell responses are the result of epigenetic modifications as observed in trained immunity, or the product of pre- formed polymorphic receptors like KIRs or members of the NKG2 family which interact with MHC molecules, or yet to be discovered receptors is to be determined. Here, we propose that investigating the NK cell transcriptome can provide insights into how infection or disease states can modulate NK cell function particularly by assessing how changes in alternative gene splicing following infection result in NK cell dysfunction or the formation of adaptive NK cell responses. Alternative splicing is a mechanism utilized by cells in order to produce multiple gene products from an individual gene, by splicing exons together in various combinations, sometimes leading to gene products with very different functions. Regulation of gene splicing is complicated, but it has been shown that environmental changes as are seen during viral infection or disease can gene splicing patterns. Using preliminary data from sorted NK cells in an acute SIV-infection cohort we have already identified several candidate alternatively spliced genes that exhibit differential isoform expression following SIV infection. Of particular interest are members of the bHLH transcription factor (TF) family due to their association with the formation of memory responses and NK cell development. Further, in a proof-of-concept study we have shown that NKp30 isoform expression is altered following ex vivo stimulation or even following vaccination in rhesus macaque and in human NK cell samples. Here, I propose to identify novel gene candidates and assess the roles of the bHLH TF splice variants and other candidate genes in rhesus macaque and human NK cells. Through this work I aim to determine whether specific gene isoform expression impacts normal NK cell functions or the formation of adaptive NK cell responses in vivo in rhesus macaques during acute and chronic SIV infection. The approaches outlined in this proposal will allow me to combine my postdoctoral training in NK cell biology with my predoctoral training assessing the role of alternative splicing in vivo in order to determine the roles that several gene candidates have on normal or adaptive NK cell responses during retroviral infection. Our ultimate goal is to identify and target specific isoforms to manipulate NK cell function in vivo for the purposes of improving current vaccine responses or eliciting targeted NK cell-specific therapies for HIV.