Inhibitory Pathways Underlying Viral Persistence In Vivo

 描述(申请人提供)：慢性病毒感染困扰着全球超过5亿人。尽管在小鼠和人类持续的病毒感染过程中，刺激和抑制通路的改变有助于T细胞的抑制，但其潜在的机制尚不完全清楚。应该指出的是，人类持久性病毒(包括人类免疫缺陷病毒、乙肝和丙型肝炎病毒)仅限于人类和非人类灵长类动物，免疫系统的复杂性无法在体外准确重建。因此，研究慢性病毒感染期间的免疫调节需要使用适当的整个机体病毒系统。通过在小鼠体内感染慢性淋巴细胞性脉络膜脑膜炎病毒(LCMV)，我们和其他人对了解几种免疫调节因子在慢性病毒感染中的作用和动态做出了重大贡献。特别是，我们已经证明，与急性巨细胞病毒感染相比，转化生长因子β(β)的产生和信号在持续感染期间持续存在。最近，我们利用时间和细胞特异性控制的小鼠遗传学，观察到活化的CD4T细胞中的转化生长因子β信号对于慢性(但不是急性)巨细胞病毒感染后宿主早期存活是必不可少的，并在后期促进病毒复制。我们还发现，转化生长因子β在感染后早期抑制CD4T细胞的增殖和终末分化，并意外地抑制CD4T细胞的细胞毒程序，同时在后期抑制CD8T细胞的功能和积聚。重要的是，初步数据表明，转化生长因子β抑制病毒特异性细胞毒性CD4T细胞延伸到艾滋病毒感染的患者。了解转化生长因子β在体内持续病毒感染过程中复杂而关键的作用及其与相关细胞因子的相互作用的细胞和分子因素，对于操纵这一途径在不引起免疫病理的情况下增强抗病毒防御至关重要。为此，在目标1中，我们建议充分认识转化生长因子β在慢性巨细胞病毒感染后早期和晚期调节CD8T细胞和CD4T细胞反应、免疫病理学和病毒控制中的时间作用。我们还将评估转化生长因子β3和转化生长因子β激活分子整合素-β8在免疫反应和病毒持久性中的作用。在目标2中，我们将通过在慢性巨细胞病毒感染后不同时间去除Smad4并确定Smad4靶向基因启动子来确定转化生长因子β调节CD4T细胞反应的机制。我们还将评估转录因子(TF)在CD4T细胞中的作用，转录因子(TF)可能是SMAD-4的共同调节因子，我们发现这些转录因子受转化生长因子β的调节。最后，在第三个目标中，我们将研究白细胞介素12(IL-12)是否介导了慢性巨细胞病毒感染过程中转化生长因子β去除所诱导的表型，因为转化生长因子β缺陷的CD4T细胞表现出增强的IL-12反应。为此，我们将阻断转化生长因子β受体充足和缺陷小鼠的IL-12，并评估持续感染巨细胞病毒后对T细胞反应、免疫病理学和病毒控制的影响。这些实验将为未来旨在加强IL-12信号以克服慢性病毒感染期间转化生长因子β抑制的方法奠定基础。总而言之，我们的发现支持这样一种观点 转化生长因子β在体内慢性病毒感染过程中发挥重要作用，影响T细胞反应、宿主生存和病毒控制。虽然这暗示了免疫治疗的新机会，但进一步了解转化生长因子β的动态活性和潜在的分子因素对于有效和安全地操纵这一途径和缓解慢性病毒疾病是必要的。此外，鉴于LCMV本身不仅是一种具有重要医学意义的病原体，而且它也是Arenaviridae家族的原型成员，我们的工作将有助于解释致病ArenaV是如何抑制T细胞反应而导致人类LEH出血热的。最后，同样重要的是，由于转化生长因子β是在几种感染过程中产生的，而T细胞是抗病毒免疫的核心，我们的工作可能对多种传染病具有广泛的影响。

 DESCRIPTION (provided by applicant): Chronic viral infections afflict more than 500 million people worldwide. Although altered stimulatory and inhibitory pathways contribute to T cell suppression during persistent viral infections in mice and humans, the underlying mechanisms are not fully understood. It should be noted that human persistent viruses, (including human immunodeficiency virus, hepatitis B and C viruses) are restricted to human and nonhuman primates and that the complexity of the immune system cannot be accurately recreated in vitro. Thus, the study of immune-regulation during chronic viral infections requires the use of appropriate whole organism viral systems. By using in vivo chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we and others have significantly contributed to understanding the roles and dynamics of several immune-regulators during chronic viral infections. In particular, we have shown that transforming growth factor beta (TGFβ) production and signaling are sustained during persistent compared to acute LCMV infection. More recently, we have used temporally and cell-specifically controlled mouse genetics and observed that TGFβ signaling in activated CD4 T cells is essential for host survival early after chronic (but not acut) LCMV infection and promotes viral replication at later stages. We also found that TGFβ suppresses proliferation, terminal differentiation and (unexpectedly) a cytotoxic program in CD4 T cells early after infection while inhibiting CD8 T cell functions and accumulation at later stages. Importantly, initial data suggest that TGFβ suppression of virus-specific cytotoxic CD4 T cells extends to HIV-infected patients. Understanding the cellular and molecular factors underlying the complex but critical role of TGFβ and its crosstalk with contextual cytokines during an in vivo persistent viral infection is essential for manipulating this pathway to boost ani-viral defenses without causing immunopathology. To this end, in Aim #1 we propose to fully discern the temporal role of TGFβ in modulating CD8 and CD4 T cell responses, immunopathology and viral control early versus late after chronic LCMV infection. We will also evaluate the role of TGFβ3 and the TGFβ activating molecule, integrin-β8, in immune responses and viral persistence. In Aim #2 we will determine the mechanisms underlying TGFβ regulation of CD4 T cell responses by ablating SMAD-4 at different times after chronic LCMV infection and identifying SMAD-4 targeted gene promoters. We will also evaluate the role of transcription factors (TFs) that are putative SMAD-4 co-regulators and TFs that we found to be regulated by TGFβ in CD4 T cells. Last, in Aim #3 we will examine whether interleukin-12 (IL-12) mediates the phenotypes induced by TGFβ ablation during chronic LCMV infection, given that TGFβ deficient CD4 T cells show enhanced IL-12 responsiveness. For that, we will block IL-12 in TGFβR sufficient and deficient mice and evaluate the consequences for T cell responses, immunopathology and viral control after persistent LCMV infection. These experiments will establish the foundation for future approaches aiming at enforcing IL-12 signaling to overcome TGFβ suppression during chronic viral infection. In conclusion, our findings support the idea that TGFβ plays vital roles during a chronic viral infection in vivo, influencing T cell responses, hos survival and viral control. While this suggests promising novel opportunities for immunotherapies, further understanding the dynamic activity of TGFβ and the underlying molecular factors is necessary to effectively and safely manipulate this pathway and alleviate chronic viral diseases. Furthermore, given that LCMV is not only itself a pathogen of medical importance but it is also a prototypic member of the family arenaviridae, our work will help explaining how pathogenic arenaviruses suppress T cell responses to cause lethalhemorrhagic fevers in humans. Last, but not least, because TGFβ is produced during several infections and T cells are central for anti-viral immunity, our work could have broad-reaching implications for multiple infectious diseases.