Project 2 - Modulation of Anti-Tumor Immunity by Inhibitory Receptors

项目摘要 细胞表面抑制性受体（IR）PD 1和LAG 3控制细胞信号传导和关键细胞内在效应物 T细胞功能和分化。然而，这些IR也在耗尽的肿瘤内T细胞上表达，限制了免疫应答。 疾病清除IR对于维持免疫控制至关重要，但也是有效免疫的主要障碍。 抗肿瘤免疫PD 1和LAG 3在多种疾病（包括癌症）中具有协同作用，并且许多CD 8+和LAG 4+细胞在多种疾病中具有协同作用。 CD 4+肿瘤浸润淋巴细胞（TIL）表达PD 1/LAG 3。事实上，我们发现肿瘤免疫疗法 靶向PD 1/LAG 3可以诱导小鼠肿瘤几乎完全缓解，临床证据现在支持 单一（抗PD 1）或双重（抗PD 1/LAG 3）IR阻断的益处。这个项目将测试中心假设 “PD 1和LAG 3协同作用，使用重叠和 在CD 4+和CD 8+效应T细胞中独特的细胞内在和外在功能”。由于耐受性的潜力， 肿瘤微环境（TME），小鼠模型代表了研究IR机制的杰出系统， 与自身免疫（项目1）和慢性病毒环境（项目3）中的过程进行比较。我们将追求三个目标： 目的1：PD 1和LAG 3在限制初始CD 8 + T细胞增殖中的相对和协同作用是什么？ 细胞抗肿瘤效应反应？我们假设“PD 1和LAG 3不启动，但暂时重新启动， 通过限制群体动力学、分化、代谢和免疫调节， 健康和多功能性”。我们将使用四AT系统，共转移肿瘤特异性CD 8 + T细胞， 是野生型或缺乏PD 1和/或LAG 3，用于在相同的微环境中进行功能和转录分析。 目的2：PD 1和LAG 3在限制初始CD 4 + T细胞转化中的相对作用和协同作用是什么？ 抗肿瘤效应反应？我们假设“PD 1/LAG 3对CD 4 + Tconv细胞具有协同作用， 通过差异调节它们的功能和CD 8 + T细胞帮助的能力”。我们将审问 使用我们的新型CreERT 2系删除CD 4 + T细胞中的等位基因，但不删除CD 8 + T细胞中的等位基因， T. 目的3：PD 1/LAG 3对抗肿瘤T细胞记忆和维持的持久性有何影响 系统免疫力？我们假设“PD 1和/或LAG 3通过以下方式限制抗肿瘤免疫的持久性： 影响Tex再生和Tconv细胞记忆的发展和维持，减弱他们的回忆反应 和限制全身免疫”。我们将提出2个问题：（A）PD 1/LAG 3损失对Tex和 他们的复兴？(B)PD 1/LAG 3对维持T细胞抗肿瘤记忆和回忆的影响 回应？ PPG相互作用：项目2将与项目1合作，比较PD 1/LAG 3丢失对CD 4+的影响 自身免疫与肿瘤环境中的T细胞功能;与项目3比较诱导的CD 8 + T细胞耗竭 通过肿瘤或慢性病毒感染;并与核心A交换数据，核心B获得小鼠，核心C 转录分析和CRISPR/sgRNA序列，以及用于免疫组织学分析的核心D。

PROJECT SUMMARY Cell surface inhibitory receptors (IRs) PD1 and LAG3 control cellular signaling and critical cell intrinsic effector T cell function and differentiation. Yet, these IRs are also expressed on exhausted intratumoral T cells, limiting disease clearance. IRs are critical to maintain immune control, but also represent a major barrier to effective anti-tumor immunity. PD1 and LAG3 are synergistic in multiple diseases, including cancers, and many CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs) express PD1/LAG3. Indeed, we showed that tumor immunotherapy targeting PD1/LAG3 can induce almost complete remission of tumors in mice, and clinical evidence now supports the benefit of single (anti-PD1) or dual (anti-PD1/LAG3) IR blockade. This project will test the central hypothesis that “PD1 and LAG3 synergize to enforce tumor-induced tolerance using a combination of overlapping and unique cell intrinsic and extrinsic functions in CD4+ and CD8+ effector T cells”. Due to potency of tolerance in the tumor microenvironment (TME), mouse models represent an outstanding system to study IR mechanisms to compare with processes in autoimmune (Project 1) and chronic viral settings (Project 3). We will pursue 3 Aims: AIM 1: What are the relative and synergistic contributions of PD1 and LAG3 in limiting the initial CD8+ T cell anti-tumor effector response? We hypothesize that “PD1 and LAG3 do not initiate but temporally re- enforce progression toward CD8+ T cell exhaustion by limiting population dynamics, differentiation, metabolic health, and polyfunctionality”. We will use a Quad AT system that co-transfers tumor-specific CD8+ T cells that are wild-type or lack PD1 and/or LAG3 for functional and transcriptional analysis in the same microenvironment. AIM 2: What are the relative and synergistic contributions of PD1 and LAG3 in limiting initial CD4+ Tconv anti-tumor effector response? We hypothesize that “PD1/LAG3 have synergistic effects on CD4+ Tconv cells by differentially modulating their function and capacity for CD8+ T cell help”. We will interrogate the role of PD1/LAG3 on CD4+ T cells using our novel CreERT2 line to delete alleles in CD4+ Tconv but not CD8+ T cells or T regs . AIM 3: What is the impact of PD1/LAG3 on the durability of anti-tumor T cell memory and maintenance of systemic immunity? We hypothesize that “PD1 and/or LAG3 limit durability of anti-tumor immunity by impacting Tex reinvigoration and Tconv cell memory development and maintenance, blunting their recall response and limiting systemic immunity”. We will ask 2 questions: (A) What is the impact of PD1/LAG3 loss on Tex and their reinvigoration? (B) What is the impact of PD1/LAG3 on maintenance of T cell anti-tumor memory and recall response? PPG Interactions: Project 2 will collaborate with Project 1 to compare the impact of PD1/LAG3 loss on CD4+ T cell function in autoimmune versus tumoral settings; with Project 3 to compare CD8+ T cell exhaustion induced by tumors or chronic viral infections; and with Core A to exchange data, Core B to obtain mice, Core C for transcriptional analysis and CRISPR/sgRNA sequences, and Core D for immunohistological analysis.