Regulatory T-Cells and Chronic Colitis

描述（由申请人提供）：已知天然存在的调节性T细胞（nT细胞; CD 4 + CD 25 + Foxp 3+）通过几种不同的机制抑制广泛的免疫应答，包括调节性细胞因子的产生、对必需细胞因子的竞争和接触依赖性机制。由于nTcB已被证明在体内抑制Th 1和Th 17自身免疫应答，因此关于这些细胞治疗慢性炎性疾病如克罗恩病（CD）患者的可能用途已经产生了很大的兴趣。在研究使用nT细胞治疗CD中的一个主要限制是这些抑制细胞的相对缺乏，因为它们构成小鼠外周CD 4 + T细胞群的<10%和人类CD 4 + T细胞的1-2%。为了规避这一重大限制，我们已经开发了一种新的离体方法，该方法产生大量表达Foxp 3的TcR，其可用于评估其在慢性结肠炎小鼠模型中的治疗功效。我们目前的初步数据表明，在IL-2，TGF β和全反式视黄酸存在下，常规CD 4 + Foxp 3-的多克隆活化诱导这些T细胞转化为Foxp 3表达的T细胞（称为iT细胞）>90%，以及在体外孵育4天后增殖增加4-5倍。此外，我们发现肠道归巢粘附分子a4 <$7的表面表达增强，表明通过我们的转换/扩增方案，iTdR具有组织特异性印迹。最后，我们提供的初步数据表明，iTsalt在体外抑制应答T细胞活化方面显著更有效，在体内逆转已建立的结肠炎方面与nTsalt同样有效。该提案的总体目标是确定Tcl 4在活动性肠道炎症期间发挥其抑制活性的位置，并确定这些淋巴细胞如何运输到这些组织。假设：我们提出，离体产生的iT细胞利用CD 62 L（L-选择素）和/或α 4 β 7运输到发炎的派伊尔集合淋巴结（PP）、肠系膜淋巴结（MLN）和/或结肠固有层（LP），在那里它们抑制Th 1/Th 17效应细胞功能，从而逆转/减轻活动性疾病。为了检验我们的假设，我们提出：a）确定PP和/或MLN是否是iTreg介导的先前存在的结肠炎的逆转/抑制所需的，和B）鉴定iTreg介导的活动性结肠炎的减弱所需的特异性T细胞相关粘附分子。从这些探索性/发展性（R21）研究中获得的数据将通过提供关于TGFAP抑制既存肠道炎症的解剖位置的新的和重要的机制信息来推进该领域，并将确定哪些不同的Treg粘附分子是归巢到这些组织以抑制疾病所需的。此外，本申请中概述的研究将提供必要的初步数据，这些数据将用于启动更详细的研究（R 01），探索如何操作离体产生的TdR，使其更特异性地靶向发炎的淋巴组织和肠道。如果基于Treg的过继免疫疗法要成为治疗CD（或溃疡性结肠炎）的可行治疗方法，那么更好地了解这些调节细胞在体内何处以及如何发挥其保护作用至关重要。公共卫生相关性：本申请中提出的工作旨在提高我们对离体产生的调节性T细胞（TCFs）在何处以及如何减弱预先存在的肠道炎症的理解。从这些研究中获得的数据可以为开始开发基于Treg的治疗策略以治疗炎症性肠病患者提供必要的数据。

DESCRIPTION (provided by applicant): Naturally occurring regulatory T-cells (nTregs; CD4+CD25+Foxp3+) are known to suppress a wide range of immune responses via several different mechanisms including production of regulatory cytokines, competition for essential cytokines and contact-dependent mechanisms. Because nTregs have been shown to suppress Th1 and Th17 autoimmune responses in vivo, a great deal of interest has been generated regarding the possible use of these cells to treat patients with chronic inflammatory diseases such as Crohn's disease (CD). A major limitation in investigating the use of nTregs for the treatment of CD is the relative paucity of these suppressor cells as they constitute <10% of the peripheral CD4+ T-cell population in mice and 1-2% of the CD4+ T-cells in humans. In an attempt to circumvent this significant limitation, we have developed a novel ex vivo method that generates large numbers of Foxp3-expressing Tregs that can be used to evaluate their therapeutic efficacy in a mouse model of chronic colitis. We present preliminary data demonstrating that polyclonal activation of conventional CD4+Foxp3- in the presence of IL-2, TGF¿ and all-trans retinoic acid induces >90% conversion of these T-cells to Foxp3-expressing Tregs (termed iTregs) as well as a 4-5-fold increase in proliferation following a 4 day incubation period in vitro. Furthermore, we find enhanced surface expression of the gut-homing adhesion molecule a4¿7 suggesting tissue-specific imprinting of iTregs by our conversion/expansion protocol. Finally, we provide preliminary data demonstrating that iTregs are significantly more potent at suppressing responder T-cell activation in vitro and equally effective as nTregs at reversing established colitis in vivo. The overall objective of this proposal is to identify where Tregs exert their suppressive activity during active gut inflammation and define how these lymphocytes traffic to these tissues. Hypothesis: We propose that ex vivo generated iTregs utilize CD62L (L-selectin) and/or a4¿7 to traffic to the inflamed Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and/or colonic lamina propria (LP) where they suppress Th1/Th17 effector cell function thereby reversing/attenuating active disease. In order to test our hypothesis we propose to: a) Determine whether PPs and/or MLNs are required for iTreg-mediated reversal/suppression of preexisting colitis and b) Identify the specific T-cell-associated adhesion molecules that is/are required for iTreg-mediated attenuation of active colitis. Data obtained from these Exploratory/ Developmental (R21) studies will advance the field by providing new and important mechanistic information on the anatomic location(s) where Tregs suppress preexisting gut inflammation and will identify which of the different Treg adhesion molecules is/are required for homing to these tissues to suppress disease. In addition, the studies outlined in the current application will provide the necessary preliminary data that will be used to initiate a more detailed investigation (R01) exploring how ex vivo-generated Tregs may be manipulated to more specifically target them to the inflamed lymphoid tissue and gut. If Treg-based adoptive immunotherapy is to become a viable therapeutic approach for the treatment of CD (or ulcerative colitis), then it is crucial to better understand where and how these regulatory cells exert their protective effects in vivo. PUBLIC HEALTH RELEVANCE: The work proposed in this application is designed to improve our understanding of where and how ex vivo-generated regulatory T-cells (Tregs) attenuate preexisting gut inflammation. Data obtained from these studies may provide the necessary data to begin to develop a Treg-based therapeutic strategy for the treatment of patients with inflammatory bowel disease.