Regulation of NK cell antiviral responses through alternative splicing

通过选择性剪接调节 NK 细胞抗病毒反应

• 批准号: 10550262
• 负责人: Daniel Ram
• 金额: $ 17.58万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-15 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550262
• 关键词: Abnormal Cell; Acceleration; Acute; Affect; Alternative Splicing; Antigens; Antisense Oligonucleotides; Antiviral Response; Area; Cancerous; Candidate Disease Gene; Cell Separation; Cell physiology; Cells; Cellular biology; Characteristics; Chronic; Classification; Cryopreservation; Cytomegalovirus; Data; Development; Disease; ES04; Effector Cell; Epigenetic Process; Event; Evolution; Exhibits; Exons; Family; Family member; Female; Flow Cytometry; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Goals; HIV; HIV Infections; HIV therapy; Human; Immune; Immune response; Immunity; Immunology; Impairment; Inactivated Vaccines; Individual; Infection; Infection Control; Innate Immune Response; Innate Immune System; Laboratories; Lead; Macaca; Macaca mulatta; Malignant Neoplasms; Mediating; Memory; Modification; NUP214 gene; Natural Killer Cells; Output; Pattern; Peptides; Peripheral Blood Mononuclear Cell; Persons; Play; Population; Property; Protein Isoforms; Publishing; RNA; RNA Splicing; Regulation; Reporting; Research; Resistance; Role; SIV; Sampling; Small Interfering RNA; Sorting; Spliced Genes; TNFRSF6 gene; Testing; Therapeutic Intervention; Third Generation Sequencing; Tissues; Training; Transcript; Vaccination; Variant; Viral; Viral Vaccines; Viral reservoir; Virus; Virus Diseases; Work; acute infection; adaptive immunity; antiretroviral therapy; chronic infection; cohort; environmental change; epitranscriptomics; gene product; immunosenescence; improved; in vivo; influenza virus vaccine; insight; interest; male; member; novel; peripheral blood; post-doctoral training; pre-doctoral; protein complex; receptor; response; stem; transcription factor; transcriptome; transcriptome sequencing; transcriptomics; tumor; vaccine response; vaccine strategy

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.

项目总结/摘要 自然杀伤（NK）细胞已被证明在调节病毒感染和免疫方面发挥着重要作用。 消灭癌细胞传统上，NK细胞被归类为先天免疫系统的成员 这是因为它们能够快速且非特异性地对感染或异常细胞作出反应。有趣的是， 新兴的证据表明NK细胞也可能具有肽特异性记忆反应（以前仅 这可归因于适应性免疫），特别是在包括HIV/SIV和CMV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， 这也包括研究传统的先天效应器如NK细胞的训练免疫。是否自适应NK 细胞反应是表观遗传修饰的结果，如在训练的免疫中观察到的，或者是前 形成多态性受体，如与MHC分子相互作用的KIR或NKG 2家族成员，或 尚未发现的受体有待确定。在这里，我们建议研究NK细胞转录组， 可以提供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 感染后可变基因剪接的变化如何导致NK细胞功能障碍或 适应性NK细胞反应。选择性剪接是细胞利用的一种机制，以产生多个 从单个基因的基因产物，通过剪接外显子在一起，在各种组合，有时导致 不同功能的基因产物。基因剪接的调控是复杂的，但已经表明， 在病毒感染或疾病期间所看到的环境变化可以改变基因剪接模式。 利用从急性SIV感染队列中分选的NK细胞获得的初步数据，我们已经确定了几种 在SIV感染后表现出差异同种型表达的候选可变剪接基因。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， NKp 30同种型表达在离体刺激后或甚至在恒河猴中接种后改变 猕猴和人NK细胞样品中的细胞。在这里，我建议确定新的候选基因，并评估其作用， 的bHLH TF剪接变异体和其他候选基因在恒河猴和人类NK细胞。通过这个 我的工作旨在确定特定基因亚型表达是否影响正常NK细胞功能， 恒河猴在急性和慢性SIV感染期间体内适应性NK细胞应答的形成。 本提案中概述的方法将使我能够联合收割机结合我在NK细胞生物学方面的博士后培训， 通过我的博士前培训，我评估了选择性剪接在体内的作用，以确定 几个候选基因在逆转录病毒感染期间对正常或适应性NK细胞应答没有影响。我们的最终 目的是鉴定和靶向特异性同种型，以在体内操纵NK细胞功能， 目前的疫苗反应或引发针对HIV的靶向NK细胞特异性疗法。