Inhibitory Pathways Underlying Viral Persistence In Vivo

 描述（由申请人提供）：慢性病毒感染困扰着全世界5亿多人。尽管在小鼠和人类持续性病毒感染期间改变的刺激和抑制途径有助于T细胞抑制，但其潜在机制尚未完全了解。应该注意的是，人类持久性病毒（包括人类免疫缺陷病毒、乙型肝炎B和丙型肝炎病毒）仅限于人类和非人类灵长类动物，并且免疫系统的复杂性无法在体外准确重现。因此，慢性病毒感染期间免疫调节的研究需要使用适当的全生物体病毒系统。通过在小鼠体内使用慢性淋巴细胞性脉络丛脑膜炎病毒（LCMV）感染，我们和其他人对理解慢性病毒感染期间几种免疫调节剂的作用和动力学做出了重大贡献。特别是，我们已经表明，与急性LCMV感染相比，转化生长因子β（TGFβ）的产生和信号传导在持续性感染期间持续存在。最近，我们使用了时间和细胞特异性控制的小鼠遗传学，并观察到活化的CD 4 T细胞中的TGFβ信号传导对于慢性（但非急性）LCMV感染后早期宿主存活至关重要，并促进后期病毒复制。我们还发现TGFβ在感染后早期抑制CD 4 T细胞的增殖、终末分化和（出乎意料地）细胞毒性程序，同时在后期抑制CD 8 T细胞功能和积累。重要的是，最初的数据表明，TGFβ对病毒特异性细胞毒性CD 4 T细胞的抑制延伸到HIV感染患者。了解TGFβ复杂但关键作用的细胞和分子因素及其在体内持续病毒感染期间与背景细胞因子的串扰对于操纵该途径以增强抗病毒防御而不引起免疫病理学至关重要。为此，在目标#1中，我们提出充分辨别TGFβ在慢性LCMV感染后早期与晚期调节CD 8和CD 4 T细胞应答、免疫病理学和病毒控制中的时间作用。我们还将评估TGFβ3和TGFβ活化分子整合素β8在免疫应答和病毒持久性中的作用。在目标#2中，我们将通过在慢性LCMV感染后的不同时间消融SMAD-4并鉴定SMAD-4靶向基因启动子来确定TGFβ调节CD 4 T细胞应答的潜在机制。我们还将评估转录因子（TF）的作用，这些转录因子是公认的SMAD-4辅助调节因子，我们发现TF在CD 4 T细胞中受TGFβ调节。最后，在目标#3中，我们将检查白细胞介素-12（IL-12）是否介导慢性LCMV感染期间TGFβ消融诱导的表型，因为TGFβ缺陷的CD 4 T细胞显示增强的IL-12应答性。为此，我们将在TGFβR充足和缺乏的小鼠中阻断IL-12，并评估持续LCMV感染后T细胞应答、免疫病理学和病毒控制的后果。这些实验将为未来旨在加强IL-12信号传导以克服慢性病毒感染期间TGFβ抑制的方法奠定基础。总之，我们的研究结果支持这样的观点， TGFβ在体内慢性病毒感染过程中发挥重要作用，影响T细胞应答、宿主存活和病毒控制。虽然这为免疫疗法提供了有希望的新机会，但进一步了解TGFβ的动态活性和潜在的分子因子对于有效和安全地操纵这一途径并缓解慢性病毒性疾病是必要的。此外，鉴于LCMV本身不仅是一种具有医学重要性的病原体，而且也是沙粒病毒科的原型成员，我们的工作将有助于解释致病性沙粒病毒如何抑制T细胞反应，从而导致人类致死性出血热。最后但并非最不重要的是，由于TGFβ在几种感染过程中产生，而T细胞是抗病毒免疫的核心，我们的工作可能对多种感染性疾病产生广泛的影响。

 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.