Generation and characterization of intestinal CD8+ regulatory T cell lines

描述（由申请人提供）：本资助申请涉及广泛的挑战领域（04）：临床研究和特定的挑战主题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有力的初步数据，这些数据记录了IBD（克罗恩病和溃疡性结肠炎）患者体外CD 8 + T细胞活化的缺陷，以及令人兴奋的新数据，这些数据详细说明了CD粘膜（肠）的体外缺陷性CD 8 + T细胞。在这项提案中，我们将表征正常的人CD 8+调节性T细胞系（来自固有层），定义其抑制机制，并开发方法来扩大这些细胞的前体从外周血。我们的初步数据表明，正常LP来源的CD 8+调节性T细胞是免疫反应的有效抑制剂，而来自克罗恩病患者的类似细胞系则未能抑制免疫反应。如果我们能够从CD患者的外周血前体产生Treg系，我们将开发出一种用于治疗CD的新型细胞基治疗剂。第一个目标将集中于定义正常固有层调节性CD 8 + T细胞系的表型和功能特征。这将包括针对参与介导抑制效应子功能的表面分子的单克隆抗体的开发。该mAb还将允许鉴定PB中的前体细胞。第二个目的是比较和对比克罗恩病和溃疡性结肠炎患者与正常对照的LP CD 8 + T细胞系。这一目标的一个重要特征将是微阵列分析，其不仅将识别CD与正常细胞系之间的特定差异，而且还将增加对正常细胞系抑制机制的不同观点。最后，第三个目标将集中在识别PB中CD 8 + T细胞前体的努力，并制定体外扩增这些细胞的策略。最终目标是利用外周血来源的细胞系作为治疗IBD的新疗法。对这些研究的资助将使我们能够雇用另外两个人（一名博士后研究员进行抑制效应功能所涉及的细胞表面分子的生化分析，一名技术人员参与免疫组织化学，细胞分选和分析。目标2中的微阵列研究将在贝勒免疫学研究所进行，该研究所需要额外的技术人员。这些研究强调了西奈山和BIIR之间的新联盟，该联盟提供了使能技术，将这些研究的结果转化为用于临床试验的新型细胞疗法。 公共卫生相关性：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43.6/100，000。IBD的特征在于胃肠道的慢性和复发性炎症，其代表肠中免疫应答控制的缺陷。我们认为，CD 8+调节细胞是参与控制肠道局部免疫反应的重要细胞群，并建议研究该细胞亚群并开发工具，以允许将该细胞群用作CD的潜在疗法。

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.