Project 3 - Modulation of Antiviral Immunity and T cell Exhaustion by Inhibitory Receptors

摘要 尽管PD1和LAG3等阻断检查点具有相当大的临床影响，但其机制仍然存在 人们对此知之甚少。剩下的一个空白是PD1和LAG3如何在早期调节TeX前体的形成 慢性感染和成熟的TeX亚群较晚。回答这个问题将确定分子机制， 将PD1和LAG3与预防或扭转疲惫和新的治疗机会联系起来。我们假设 来自PD1和LAG3单独和组合的信号在时间上截然不同，在治疗上相关 调节T细胞耗竭的机制将通过剖析它们之间的协同作用而被发现 T细胞耗竭不同阶段的途径。本项目将通过测试来解决这一假设： 目标1：来自PD1和/或LAG3的信号如何促进TeX前体的形成？在这里，我们将 揭示PD1和/或LAG3如何参与早期TeX形成的初始分子和细胞建立 并为防止精疲力竭的发展提供机会。我们假设PD1和/或LAG3是 在慢性病毒感染期间通过以下途径启动和暂时加强TeX的发展 涉及TCF1、NFAT和/或TOX的机制。我们将使用结构性或诱导性CD8T细胞固有的PD1 和/或LAG3缺乏伴抗体(Ab)阻断和新的力竭追踪小鼠 (即，来自核心B的Lag3CreERT2.Rosa26LSL.tdTomato或ToxCreerT2.Rosa26LSL.tdTomato)。因此，Aim 1将提供详细的 PD1和LAG3如何单独和共同调节TeX的初始形成的地图。 目的2：由暂时诱导的PD1或LAG3缺失引起的分子和表观遗传学事件是什么 在成熟的TeX子集中？尽管检查点封锁具有临床相关性，但Tex的潜在生物学 重振作用仍然知之甚少，特别是围绕与以下相关的早期分子事件 活体再生及其对不同纹理亚群的影响。我们假设早期的分子事件 在去除PD1和/或LAG3之后，对于不同的TeX亚集是不同的，赋予新的官能团， 转录和/或分化的变化，使我们能够识别新的分子靶点来逆转或 防止筋疲力尽。在这里，我们将询问大量和单细胞转录以及表观遗传学的变化。 在去除PD1和/或LAG3之后的高分辨率时间过程中的全部TeX和TeX子集。 这些发现将使用体内CRISPR/CAS9筛查和RV方法(核心C)进一步剖析。这些 数据将为在人类身上应用PD1和/或LAG3阻滞剂提供重要的见解。 PPG相互作用：因为衰竭的核心程序在慢性感染、肿瘤和 自动免疫，项目3将与项目1和项目2广泛联系，用于实验模型和机械 来自自身免疫或肿瘤的洞察力。我们还将与核心A协调交换数据和互动，核心 B用于获得小鼠，核心C用于RV方法和生物信息学，核心D用于免疫组织学。

SUMMARY Despite considerable clinical impact of blocking checkpoints such as PD1 and LAG3, the mechanisms remain poorly understood. One remaining gap is how PD1 and LAG3 regulate formation of TEX precursors early in chronic infection and mature TEX subsets later. Answering this question will identify molecular mechanisms that link PD1 and LAG3 to preventing or reversing exhaustion and new therapeutic opportunities. We hypothesize that individual and combined signals from PD1 and LAG3 engage temporally distinct, therapeutically relevant mechanisms to regulate T cell exhaustion that will be discovered by dissecting the synergy between these pathways at different stages of T cell exhaustion. This project will address this hypothesis by testing: Aim 1: How do signals from PD1 or LAG3 or both promote formation of TEX precursors? Here, we will reveal how PD1 and/or LAG3 are involved in initial molecular and cellular establishment of early TEX formation and provide opportunities for preventing development of exhaustion. We hypothesize that PD1 and/or LAG3 are necessary to initiate and temporally reinforce development of TEX during chronic viral infection through mechanisms that involve TCF1, NFAT, and/or TOX. We will use constitutive or inducible CD8 T cell-intrinsic PD1 and/or LAG3 deficiency together with antibody (Ab) blockade and novel exhaustion tracking mice (i.e.Lag3CreERT2.Rosa26LSL.tdTomato or ToxCreERT2.Rosa26LSL.tdTomato) from Core B. Thus, Aim 1 will deliver detailed maps of how PD1 and LAG3 separately and together regulate initial formation of TEX. Aim 2: What are the molecular and epigenetic events caused by temporally induced loss of PD1 or LAG3 in mature TEX subsets? Despite the clinical relevance of checkpoint blockade, the underlying biology of TEX reinvigoration remains poorly understood, particularly surrounding early molecular events associated with reinvigoration in vivo and its impact on different TEX subsets. We hypothesize that early molecular events following removal of PD1 and/or LAG3 are distinct for different TEX subsets, imparting novel functional, transcriptional, and/or differentiation changes that will enable us to identify new molecular targets to reverse or prevent exhaustion. Here, we will interrogate bulk and single-cell transcriptional as well as epigenetic changes in total TEX and TEX subsets over a high-resolution time-course following removal of PD1 and/or LAG3. Discoveries will be further dissected using in vivo CRISPR/Cas9 screening and RV approaches (Core C). These data will provide important insights for applying PD1 and/or LAG3 blockade in humans. PPG Interactions: Because the core program of exhaustion is conserved in chronic infections, tumors, and autoimmunity, Project 3 will connect extensively with Projects 1 and 2 for experimental models and mechanistic insights from autoimmunity or tumors. We will also coordinate with Core A to exchange data and interact, Core B to obtain mice, Core C for RV approaches and bioinformatics, and Core D for immunohistology.