Molecular Pathways Regulating Tissue-resident Memory T cells in the Gut

摘要 肠粘膜具有丰富的免疫微环境，能够对入侵的病原体迅速作出反应 同时维持对肠道微生物群和膳食蛋白质的调节的稳态。组织驻留 记忆性T细胞基于其快速应答的能力对于粘膜表面的免疫稳态是关键的 入侵的病原体。CTLA-4和PD-1受体抑制T细胞功能，并已成为主要的 用于增强癌症患者中T细胞介导的免疫的治疗靶点。然而，针对这些 抑制性受体经常诱导炎性不良事件，结肠炎是最常见的， 由检查点抑制（CPI）诱导的严重炎症不良事件。我们最近报道了一个深入的 CPI结肠炎患者和健康受试者粘膜中免疫细胞的单细胞分析。该分析 显示了在小鼠中具有高度增殖和细胞毒性状态的CD 8 T细胞群的急剧积累， 所有研究的CPI结肠炎患者。我们使用CD 8 T细胞的T细胞受体（TCR）序列来研究CD 8 T细胞的免疫应答。 这些克隆扩增的T细胞的起源。有趣的是，我们发现大部分结肠炎相关的 细胞毒性CD 8 T细胞具有与组织驻留记忆CD 8 T细胞相同的TCR序列。因此我们 假设CTLA-4和PD-1抑制性受体将组织驻留记忆T细胞（Trm）保持在 检查，这些抑制信号的丢失可以诱导Trm的大规模克隆扩增，这是一个主要的 CPI结肠炎的发展步骤。抗体阻断CTLA-4和PD-1受体的个体， 因此，免疫学上正常的粘膜提供了对这些受体的生理功能的了解， 人类在目的1中，我们将研究CTLA-4和PD-1受体如何调节Trm细胞的功能， 在人类和小鼠模型中。我们将研究炎症通路的靶向是否可以逆转 T细胞从高度增殖，细胞毒性状态回到Trm状态，通过执行深入的单细胞 分析了来自CPI结肠炎患者的结肠活检，这些患者参加了一项单独资助的随机2期临床试验， 一项评估TNFα阻滞剂与皮质类固醇的试验。我们的单细胞分析表明， CPI结肠炎的TNF基因表达特征，回顾性临床数据表明，TNFα阻断是 对CPI结肠炎有效。我们还将在小鼠模型中研究这一假设， CTLA-4和PD-1抗体对肠粘膜中CD 8 T细胞的活化和细胞毒性状态的影响。在目标2中 我们将研究肠道中T细胞群与免疫细胞、上皮细胞和基质细胞的空间相互作用。 使用CODEX技术对组织进行高度多重免疫荧光分析， 使用DNA条形码化的mAb组进行切片。这种高维成像方法提供了一个机会， 研究健康肠粘膜和CPI结肠炎中的细胞-细胞相互作用，包括来自 目标1中描述的临床试验。因此，该项目解决了一个基本的问题是粘膜免疫学。 1

Abstract The intestinal mucosa has a rich immune microenvironment capable of responding rapidly to invading pathogens while maintaining a regulated homeostatic state to commensal microbiota and dietary proteins. Tissue-resident memory T cells are critical for immune homeostasis at mucosal surfaces based on their ability to rapidly respond to invading pathogens. The CTLA-4 and PD-1 receptors inhibit T cell function and have become major therapeutic targets for boosting T cell-mediated immunity in cancer patients. However, targeting of these inhibitory receptors frequently induces inflammatory adverse events, and colitis is one of the most common and severe inflammatory adverse events induced by checkpoint inhibition (CPI). We recently reported an in-depth single cell analysis of immune cells in the mucosa of CPI colitis patients and healthy subjects. This analysis demonstrated dramatic accumulation of CD8 T cell populations with highly proliferative and cytotoxic states in all studied CPI colitis patients. We used the T cell receptor (TCR) sequences of CD8 T cells to investigate the origin of these clonally expanded T cells. Interestingly, we discovered that a large fraction of colitis-associated cytotoxic CD8 T cells had the same TCR sequences as tissue-resident memory CD8 T cells. We therefore hypothesize that the CTLA-4 and PD-1 inhibitory receptors hold tissue-resident memory T cells (Trm) in check, and that loss of these inhibitory signals can induce massive clonal expansion of Trm, a major step in the development of CPI colitis. Antibody blockade of CTLA-4 and PD-1 receptors in individuals with immunologically normal mucosa thus provides insight into the physiological function of these receptors in humans. In Aim 1, we will investigate how the CTLA-4 and PD-1 receptors regulate the function of Trm cells, both in humans and murine models. We will investigate whether targeting of inflammatory pathways may revert T cells from highly proliferative, cytotoxic states back into a Trm state by performing an in-depth single cell analyses on colon biopsies from CPI colitis patients enrolled in a separately funded randomized phase 2 clinical trial that evaluates TNFα blockade versus corticosteroids. Our single cell analysis demonstrated upregulated TNF gene expression signatures in CPI colitis, and retrospective clinical data indicate that TNFα blockade is efficacious in CPI colitis. We will also investigate this hypothesis in murine models by evaluating the impact of CTLA-4 and PD-1 antibodies on the activation and cytotoxic states of CD8 T cells in the gut mucosa. In Aim 2 we will study the spatial interactions of T cell populations with immune, epithelial and stromal cells in the intestinal mucosa using the CODEX technology that enables highly multiplexed immunofluorescence analysis of tissue sections using panels of DNA-barcoded mAbs. This high-dimensional imaging approach affords an opportunity to study the cell – cell interactions in the healthy gut mucosa and in CPI colitis, including biopsy specimens from the clinical trial described in Aim 1. This project thus addresses a fundamental question is mucosal immunology. 1