Generation and characterization of intestinal CD8+ regulatory T cell lines

描述(申请人提供)：这项资助申请涉及广泛的挑战领域(04)：临床研究和特定挑战主题04-AI-101：开发新方法并解决粘膜免疫学中的关键问题。该提案来自我们实验室的强大初步数据，这些数据记录了IBD(克罗恩病和溃疡性结肠炎)患者CD8+Tregs体外激活的缺陷，以及令人兴奋的新数据，详细描述了CD粘膜(肠道)体外缺陷的CD8+Tregs。在这项建议中，我们将描述正常人CD8+调节性T细胞系(来源于固有层)，确定它们的抑制机制，并开发从外周血中扩增这些细胞的前体细胞的方法。我们的初步数据表明，正常的LP来源的CD8+Tregs是免疫反应的有效抑制因子，而来自克罗恩病患者的类似株则无法抑制。如果我们能够从CD患者的外周血前体细胞中产生Treg细胞系，我们将开发出一种基于细胞的治疗CD的新方法。第一个目标将集中于确定正常固有层调节性CD8+T细胞株的表型和功能特征。这将包括开发针对参与抑制效应器功能的表面分子的克隆抗体。这一单抗还将允许识别外周血中的前体细胞。第二个目的是比较和对比来自克罗恩病和溃疡性结肠炎患者的LP CD8+T细胞系与正常对照组。这一目标的一个重要特征将是微阵列分析，它不仅将识别CD与正常细胞系之间的具体差异，而且还将从不同的角度研究正常细胞系抑制的机制。最后，第三个目标将集中在努力识别外周血中CD8+Tregs的前体细胞，并开发体外扩增这些细胞的策略。最终目标将是利用外周血源性细胞系作为治疗IBD的新疗法。对这些研究的资助将使我们能够再聘请两名人员(一名博士后对参与抑制效应功能的细胞表面分子进行生化分析，一名技术人员参与免疫组织化学、细胞分类和分析。AIM 2中的微阵列研究将在贝勒免疫学研究所进行，那里将需要更多的技术人员。这些后一项研究突出了西奈山和BIIR之间的新联盟，该联盟提供了使这些研究结果能够转化为用于临床试验的新型细胞疗法的技术。 与公共卫生相关：克罗恩病(CD)和溃疡性结肠炎(UC)是炎症性肠病(IBD)的主要形式，在美国是一个日益严重的健康问题，据报道患病率高达每10万人中有43.6人。IBD的特征是慢性和复发性的胃肠道炎症，表现为肠道免疫反应控制的缺陷。我们认为CD8+调节细胞是参与控制肠道局部免疫反应的重要细胞群，并建议研究这一细胞亚群并开发工具，使其能够用作CD的潜在治疗方法。

DESCRIPTION (provided by applicant): This grant application addresses broad Challenge Area (04): Clinical Research and specific challenge topic 04-AI-101: Develop novel methods and address key questions in mucosal immunology. The proposal draws from strong preliminary data from our laboratory documenting defects in the activation of CD8+ Tregs in vitro in patients with IBD (Crohn's disease and ulcerative colitis) and exciting new data detailing defective CD8+ Tregs ex vivo from CD mucosa (intestine). In this proposal we will characterize normal human CD8+ regulatory T cell lines (derived from the lamina propria), define their mechanism of suppression and develop approaches to expand precursors of these cells from the peripheral blood. Our preliminary data suggest that normal LP derived CD8+ Tregs are potent inhibitors of immune responses while similar lines derived from patients with Crohn's disease fail to suppress. If we can generate Treg lines from peripheral blood precursors from CD patients, we will have developed a novel cell based therapeutic for the treatment of CD. The first aim will focus on defining phenotypic and functional characteristics of normal lamina propria regulatory CD8+ T cell lines. This will include the development of monoclonal antibodies against surface molecules involved in mediating the suppressor effector function. This mAb will also allow for the identification of precursor cells in the PB. The second aim will compare and contrast LP CD8+ T cell lines from Crohn's and ulcerative colitis patients vs. normal controls. An important feature of this aim will be a microarray analysis that will not only identify specific differences in CD vs normal cell lines but also add a different perspective on the mechanism of suppression by normal lines. Finally, the third aim will focus on efforts to identify precursors of CD8+ Tregs in the PB and develop strategies to expand these cells ex vivo. The ultimate goal will be to utilize the peripheral blood derived cell lines as novel therapeutics in the treatment of IBD. Funding for these studies will allow us to hire two additional individuals (one post-doctoral fellow to perform the biochemical analysis of the cell surface molecule involved in suppressor effector function and one technician involved in the immunohistochemistry, cell sorting and analysis. The microarray studies in aim 2 will be performed at the Baylor Institute for Immunology Research where additional technicians will be required. These latter studies highlight a new alliance between Mount Sinai and the BIIR that provides enabling technologies to translate the results from these studies into novel cell based therapeutics for clinical trials. PUBLIC HEALTH RELEVANCE: Crohn's disease (CD) and ulcerative colitis (UC), the major forms of Inflammatory bowel disease (IBD), are a growing and serious health concern in the United States with reported prevalence rate as high as 43.6 per 100,000. IBD is characterized by chronic and relapsing inflammation of the gastrointestinal tract representing defects in the control of immune responses in the intestine. We believe that CD8+ regulatory cells are an important cell population involved in controlling local immune responses in the gut and propose to study this cell subset and develop tools that will allow for the use of this cell population as a potential therapy for CD.