Generation and Regulation of Anti-Viral CD4 T Cells with Cytolytic Potential

描述（由申请人提供）：由于病毒突变率高、缺乏明确的免疫相关保护和疫苗无效，甲型流感病毒（IAV）和人类免疫缺陷病毒（HIV）等新出现的大流行感染仍然是全球主要的健康问题。CD4 T细胞在病毒感染的免疫反应中是必不可少的，因为它们能够增强抗体的产生并帮助维持CD8 T细胞的记忆反应。最近，CD4细胞在抗病毒反应中发挥了更直接的作用，许多报道表明CD4 T细胞可以获得穿孔素介导的细胞毒性，这可能对降低病毒载量很重要。然而，诱导细胞溶解性CD4细胞（CD4 CTL）分化的信号以及这些细胞在抗病毒免疫反应中的生理影响仍然不清楚。这些研究的长期目标是了解CD4 CTL的发展及其在体内的作用，以便为针对传染性疾病的靶向细胞介导免疫的疫苗策略提供信息。本研究的目的是鉴定细胞因子和转录因子信号诱导CD4 CTL效应和记忆反应，从而导致长期的保护性抗病毒反应。我们的中心假设是：II类受限，穿孔素介导的细胞毒性通过IL-2信号传导和Th1极化条件发展，在记忆群体中存活并有助于异亚型保护。我们基于先前的研究和初步数据提出了这一假设，这些数据表明Th1极化的CD4效应物在IAV感染时获得穿孔素介导的细胞毒性，并且这些细胞的群体可以在记忆阶段被识别。此外，IL-2通过高亲和IL-2R¿信号传导促进STAT5磷酸化和T-bet表达，与GrB和穿孔素介导的细胞毒性相关。这一假设将通过追求三个特定目标来验证：1)确定CD4 CTL发育所需的细胞因子信号，2)确定驱动CD4 CTL分化的转录因子，以及3)确定CD4 CTL在记忆池和对流感感染的回忆反应中的作用。在第一个目标中，将利用细胞因子和细胞因子受体敲除小鼠，建立IL-2和炎症细胞因子信号在CD4 CTL发育中的作用。在目标2中，候选转录因子如STAT5、T-bet和Eomes，以及通过微阵列鉴定的独特转录因子，将被测试它们与穿孔蛋白启动子结合并控制CD4 CTL分化的能力。最后，目标3，

DESCRIPTION (provided by applicant): Emerging pandemic infections such as influenza A virus (IAV) and human immunodeficiency virus (HIV) remain major global health concerns due to high viral mutation rates, lack of defined immune correlates of protection and ineffective vaccines. CD4 T cells are essential in the immune response to viral infections because of their ability to enhance antibody production and help maintain CD8 T cell memory responses. Recently, a more direct role for CD4 cells during anti-viral responses has been appreciated, with many reports indicating CD4 T cells can acquire perforin mediated cytotoxicity that may be important for decreasing viral load. However, the signals that induce the differentiation of cytolytic CD4 cells (CD4 CTL) and the physiological impact of these cells in anti-viral immune responses remain obscure. The long-term goal of these studies is to understand the development of CD4 CTL and their role in vivo in order to inform vaccine strategies that target cell mediated immunity against infectious diseases. The objective of this proposal is to identify cytokine and transcription factor signals inducing CD4 CTL effector and memory responses leading to long term, protective anti- viral responses. Our central hypothesis is: class II restricted, perforin mediated cytotoxicity develops via IL-2 signaling and Th1 polarizing conditions, survives in a memory population and contributes to heterosubtypic protection. We formulated this hypothesis based on previous studies and preliminary data demonstrating that Th1 polarized CD4 effectors acquire perforin mediated cytotoxicity in response to IAV infection and a population of these cells can be identified at the memory stage. Further, IL-2 signaling via high affinity IL-2R¿ pro- motes STAT5 phosphorylation and T-bet expression, correlating with GrB and perforin mediated cytotoxicity. This hypothesis will be tested by pursuing three specific aims: 1) Identify the cytokine signals required for full CD4 CTL development, 2) Identify the transcription factors driving CD4 CTL differentiation, and 3) Establish a role for CD4 CTL in the memory pool and recall responses to influenza infection. In the first aim, the role of IL-2 and inflammatory cytokine signals in CD4 CTL development will be established using cytokine and cytokine receptor knockout mice. In aim 2, candidate transcription factors such as STAT5, T-bet and Eomes, as well as unique transcription factors identified by microarray, will be tested for their ability to bind to the perforin promoter and control CD4 CTL differentiation. Finally, aim 3, will monitor the development of CD4 CTL memory responses and their ability to promote heterosubtypic immunity by using viruses that differ in outer coat proteins in CD8 deficient mice or using viruses that lack dominant CD8 epitopes. This research is innovative because it uses a unique in vitro activation model, as well as an in vivo approach to interrogate the requirements for differentiation of a novel subset of CD4 T cells with cytolytic activity. The proposed research is significant because it is the first step in understanding the mechanisms that drive CD4 CTL differentiation that has the potential to inform novel vaccine strategies targeting T cell immunity against emerging pathogens.