Development of novel PD1 agonist therapeutic strategies for multiple sclerosis

项目摘要/摘要 目前多发性硬化症(MS)的治疗方法不足以维持长期免疫 动态平衡和有效地重新校准患者的T辅助细胞失衡。仍然有一个未得到满足的临床 需要新的策略来恢复和维持多发性硬化患者的免疫耐受。 PD1信号在维持免疫耐受中起关键作用 。PDL1/PD1表达改变，和/或 阻断PD1信号，导致免疫耐受性的崩溃，并使小鼠和人类容易 自身免疫和组织炎症的发展。例如，封锁PD1及其配体可以 加重EAE，一种MS的小鼠模型事实上，我们最近发现Smad7，一种与MS有关的主要分子 自身免疫通过限制T细胞中的PD1和PD1配体维持小鼠的肠道和中枢神经系统炎症 DCs，从而抑制PD1诱导的Tregs。鉴于PD1信号在限制组织中的关键作用 炎症和自身免疫，PD1可能是MS的一个高度临床感兴趣的治疗靶点。 然而， 增强PD1信号对治疗EAE和MS的影响从未被测试过。 对于这一提议，我们开始探索人类T细胞中的PD1激动剂。我们发现痛苦的PD1是通过 免疫球蛋白融合蛋白PDL1-Fc或PDL2-Fc促进人新生Treg诱导并限制Treg 可塑性。有趣的是，我们还发现髓系细胞中的pd1受刺激抑制了炎性细胞因子。 已知可促进Th1/17的发育并破坏MS和EAE中Tregs的稳定。因此，我们将测试 我们的假设是，PD1激动剂单一疗法可以通过直接增强 在EAE和MS中抑制效应T细胞反应的Treg动态平衡基于我们令人兴奋的初步研究 小剂量IL-2与PD1激动剂联合应用对CD4+T细胞PD1的直接诱导作用 协同增强人类Treg诱导， 我们还将研究是否将小剂量IL-2与PDL1/2-FC结合 将协同提高多发性硬化症患者的Treg反应。 由于IL-2仍能促进效应者T细胞反应， 将PD1激动剂与小剂量IL-2联合使用也可能抑制小剂量IL-2对血管内皮细胞的任何不良直接影响 增强效应T细胞。此外，PD1激动剂可能通过靶向其他途径进一步补充低剂量IL-2 不受IL-2影响的重要细胞类型(例如髓系细胞)。在目标1中，我们将测试 通过体外治疗MS患者的免疫细胞和人源化的PD1敲打疗法来实现PD1激动剂的单一治疗 在体内感染EAE的小鼠中。在目标2中，我们将测试PDL1/2-FC和小剂量IL-2联合治疗 多发性硬化症患者免疫细胞的体外培养和人源化PD1/IL-2受体α(RA)双敲入小鼠的治疗 与体内的EAE，包括治疗小鼠免疫反应的单细胞RNA测序。总而言之，我们 将探索一种从未试验过的PD1激动剂/小剂量IL-2联合治疗策略在EAE和 毫秒至 解决围绕PD1激动剂如何促进人类免疫耐受的悬而未决的问题， PD1激动剂治疗策略的翻译潜力，以及如何在中枢神经系统自身免疫中实施。

Project Summary/Abstract Current multiple sclerosis (MS) therapeutic approaches are insufficient to maintain long-term immune homeostasis and effectively recalibrate T helper cell imbalances in patients. There remains an unmet clinical need for new strategies that restore and sustain immune tolerance in MS. PD1 signaling plays a critical role in the maintenance of immune tolerance . Altered PDL1/PD1 expression, and/or blockade of PD1 signaling, results in the breakdown of immune tolerance and predisposes mice and humans to the development of autoimmunity and tissue inflammation. For example, blockade of PD1 and its ligands can exacerbate EAE, a mouse model of MS. In fact, we recently found that Smad7, a major molecule implicated in autoimmunity, sustains intestinal and CNS inflammation in mice by limiting PD1 in T cells and PD1 ligands in DCs, thereby dampening PD1-induced Tregs. Given the critical role of PD1 signaling in limiting tissue inflammation and autoimmunity, PD1 could represent a therapeutic target of high clinical interest in MS. However, the impact of enhancing PD1 signaling for therapeutic benefit in EAE and MS has never been tested. For this proposal, we began exploring PD1 agonists in human T cells. We found agonizing PD1 via immunoglobulin fusion proteins PDL1-Fc or PDL2-Fc promotes de novo human Treg induction and limits Treg plasticity. Interestingly, we also found that agonizing PD1 within myeloid cells inhibits inflammatory cytokines that are known to promote Th1/17 development and destabilize Tregs in MS and EAE. Therefore, we will test our hypothesis that PD1 agonist monotherapy could effectively restore immune tolerance by directly enhancing Treg homeostasis while quenching effector T cell responses in EAE and MS. Based on our exciting preliminary finding that IL-2 directly induces PD1 in CD4+ T cells and that combining low-dose IL-2 with PD1 agonists synergistically boosts human Treg induction, we will also investigate if combining low-dose IL-2 with PDL1/2-Fc will synergistically boost MS patient Treg responses. Because IL-2 can still promote effector T cell responses, combining PD1 agonist with low-dose IL-2 may also restrain any undesired direct effect of low-dose IL-2 on boosting effector T cells. In addition, PD1 agonist might further complement low-dose IL-2 by targeting other important cell types unaffected by IL-2 (e.g. myeloid cells). In Aim 1, we will test the translational relevance of PD1 agonism monotherapy by treating MS patient immune cells in vitro, and by treating humanized PD1 knock- in mice with EAE in vivo. In Aim 2, we will test PDL1/2-Fc and low-dose IL-2 combination therapy by treating MS patient immune cells in vitro, and by treating humanized PD1/IL-2 receptor alpha (RA) double knock-in mice with EAE in vivo, including single-cell RNA sequencing of immune responses in treated mice. In summary, we will explore the efficacy of a never tested PD1 agonist/low-dose IL-2 combination therapy strategy in EAE and MS to address unanswered questions around how PD1 agonism promotes human immune tolerance, the translational potential of PD1 agonist therapeutic strategies, and how to implement them in CNS autoimmunity.