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

总结 尽管阻断PD 1和LAG 3等检查点具有相当大的临床影响，但这些机制仍然存在。 不太了解。剩下的一个缺口是PD 1和LAG 3如何在细胞生长早期调节TEX前体的形成。 慢性感染和成熟TEX亚群。探讨这个问题将确定分子机制， 将PD 1和LAG 3与预防或逆转衰竭和新的治疗机会联系起来。我们假设 来自PD 1和LAG 3的单个和组合信号在时间上不同， 调节T细胞耗竭的机制，将通过解剖这些之间的协同作用来发现。 在T细胞耗竭的不同阶段。本项目将通过检验来解决这一假设： 目的1：来自PD 1或LAG 3或两者的信号如何促进TEX前体的形成？在这里，我们将 揭示PD 1和/或LAG 3如何参与早期TEX形成的初始分子和细胞建立 并提供防止衰竭发展的机会。我们假设PD 1和/或LAG 3是 在慢性病毒感染期间，通过以下途径启动和暂时加强TEX的发展是必要的： 涉及TCF 1、NFAT和/或TOX的机制。我们将使用组成型或诱导型CD 8 T细胞-内源性PD 1 和/或LAG 3缺陷以及抗体（Ab）阻断和新型耗竭追踪小鼠 （即Lag3CreERT2.Rosa26LSL.tdTomato或ToxCreERT2.Rosa26LSL.tdTomato）。因此，目标1将提供详细的 PD 1和LAG 3如何分别和一起调节TEX的初始形成。 目的2：PD 1或LAG 3的暂时性缺失导致的分子和表观遗传事件是什么 在成熟的TEX子集中？尽管检查点阻断具有临床相关性，但TEX的潜在生物学特性 复苏仍然知之甚少，特别是围绕与 在体内恢复活力及其对不同TEX亚群的影响。我们假设早期的分子事件 在去除PD 1和/或LAG 3后，不同TEX亚群的PD 1和/或LAG 3是不同的，赋予新的功能， 转录和/或分化的变化，这将使我们能够确定新的分子靶点，以逆转或 防止疲劳。在这里，我们将询问散装和单细胞转录以及表观遗传变化 在除去PD 1和/或LAG 3之后的高分辨率时间过程中的总TEX和TEX子集中。 将使用体内CRISPR/Cas9筛选和RV方法进一步剖析发现（核心C）。这些 数据将为在人体中应用PD 1和/或LAG 3阻断提供重要的见解。 PPG相互作用：由于耗竭的核心程序在慢性感染、肿瘤和 自身免疫，项目3将广泛连接项目1和2的实验模型和机制 从自身免疫或肿瘤中获得的见解。我们也会和Core A进行协调，进行数据交换和互动，Core 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.