Regulatory T-Cells and Chronic Colitis

描述(由申请人提供)：已知自然产生的调节性T细胞(nTregs；CD4+CD25+Foxp3+)通过几种不同的机制抑制广泛的免疫反应，包括产生调节性细胞因子、竞争基本细胞因子和接触依赖机制。由于nTregs已被证明在体内抑制Th1和Th17自身免疫反应，因此这些细胞可能用于治疗慢性炎症性疾病，如克罗恩病(CD)，引起了极大的兴趣。研究使用nTregs治疗CD的一个主要限制是这些抑制细胞相对较少，因为它们构成了小鼠外周CD4+T细胞群的10%和人类CD4+T细胞群的1-2%。为了绕过这一重大限制，我们开发了一种新的体外方法，可以产生大量表达Foxp3的Tregs，可以用来评估它们在慢性结肠炎小鼠模型中的治疗效果。我们提供的初步数据表明，在IL-2、转化生长因子β和全反式维甲酸存在的情况下，常规CD4+Foxp3-的多克隆激活可以诱导这些T细胞90%转化为表达Foxp3的Tregs(称为iTregs)，并在体外培养4天后使增殖增加4-5倍。此外，我们发现肠归巢黏附分子A4？7的表面表达增强，表明通过我们的转换/扩展方案对iTregs进行组织特异性印迹。最后，我们提供的初步数据表明，iTregs在体外显著更有效地抑制应答者T细胞的激活，在体内逆转已建立的结肠炎方面与nTregs同样有效。这项建议的总体目标是确定Treg在活动性肠炎期间在哪里发挥抑制活性，并确定这些淋巴细胞如何运输到这些组织。假设：我们认为，体外产生的iTregs利用CD62L(L-选择素)和/或A4？7运输到炎症的Peyer‘s斑块(PP)、肠系膜淋巴结(MLN)和/或结肠固有层(LP)，在那里它们抑制Th1/Th17效应细胞的功能，从而逆转/减轻活动期疾病。为了验证我们的假设，我们建议：a)确定PPS和/或MLN是否是iTreg介导的先前存在的结肠炎逆转/抑制所必需的，以及b)确定iTreg介导的活动期结肠炎的缓解所需的特定T细胞相关黏附分子。从这些探索性/发展性(R21)研究中获得的数据将通过提供有关Treg抑制先前存在的肠道炎症的解剖位置(S)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黏附分子是归巢到这些组织以抑制疾病所必需的。此外，本申请中概述的研究将提供必要的初步数据，用于启动更详细的调查(R01)，探索如何操纵体外产生的Tregs，以更具体地将它们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的哪里以及如何发挥保护作用是至关重要的。公共卫生相关性：本申请中提出的工作旨在提高我们对体外产生的调节性T细胞(Tregs)在哪里以及如何减轻先前存在的肠道炎症的理解。从这些研究中获得的数据可能为开始开发基于Treg的治疗炎症性肠病患者的治疗策略提供必要的数据。

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.