Dendritic Cell-Based Biosensor System

描述（由申请人提供）：树突状细胞（DC）战略性地位于环境界面，作为免疫前哨和组织驻留抗原呈递细胞。当暴露于感染性微生物或“危险信号”时，DC产生促炎介质，经历成熟，并迁移到淋巴结以激活幼稚T细胞。因此，DC激活是连接先天免疫和适应性免疫的初始和关键事件。我们将使用基于DC的生物传感器系统来系统地研究DC激活过程并开发新型DC刺激剂。具体目标是：1）优化基于DC的生物传感器。我们已经开发了一个DC生物传感器的原型，通过用13个具有不同顺式增强子的荧光素酶报告基因转染XS 106 DC细胞系，并确定了各种微生物，生物，药理学和物理因子，激活DC中不同的转录调控途径。我们将通过分离永久表达EGFP报告构建体的DC克隆来优化该系统。2)系统验证。我们将通过测试目前已知的DC刺激剂和通过从NCI筛选化合物库（>105复杂度）来测试DC生物传感器的实用性。一旦得到验证，这项技术将被转移到该行业，以促进他们的药物发现工作。3)合成DC刺激肽。类肽（N-取代的甘氨酸寡聚物）在蛋白酶抗性、更高的稳定性和更高的多样性方面具有优于肽的优点。将以珠固定形式筛选以分裂/合并方式合成的类肽文库（>107复杂度）。“命中”化合物将转化成可溶性二聚体形式，并通过分子改组进行修饰，以获得DC刺激“先导”类肽。4)合成直流电刺激照明。使用可调照明设备，我们将通过测试>107种不同照明的影响来探索光的宇宙。我们还将测试的概念，DC激活是诱导的表面受体的光化学交联。5)确定不同试剂激活DC的生物学后果。将检查新鉴定的DC刺激剂对DC的基因表达谱、表面表型、细胞因子和趋化因子产生以及T细胞刺激能力的体外影响以及对朗格汉斯细胞的体内影响。我们将使用计算生物学工具分析experimenta数据，以定义控制DC激活过程的算法。我们的研究将为DC生物学提供新的见解，并可能导致新的佐剂和治疗剂的开发。

DESCRIPTION (provided by applicant): Dendritic cells (DC) are strategically located at the environmental interface, serving as immunological sentinel and tissue-resident antigen presenting cells. Upon exposure to infectious microorganisms or "danger signals", DC produce pro-inflammatory mediators, undergo maturation, and migrate to lymph nodes to activate naive T cells. Thus, DC activation is the initial and key event linking innate to adaptive immunity. We will employ the DC-based biosensor system to study DC activation processes systematically and to develop novel DC-stimulating agents. Specific aims are: 1) To optimize the DC-based biosensor. We have developed a DC biosensor prototype by transfecting XS106 DC line with 13 luciferase reporters with different cis-enhancers and identified various microbial, biological, pharmacological, and physical agents that activate distinct transcription regulatory pathways in DC. We will optimize this system by isolating DC clones permanently expressing EGFP reporter constructs. 2) System validation. We will test the utility of the DC biosensor by testing currently known DC-stimulating agents and by screening a chemical compound library (>105 complexity) from NCI. Once validated, this technology will be transferred to the industry to facilitate their drug discovery effort. 3) To synthesize DC-stimulating peptoids. Peptoids (N-substituted glycine oligomers) have advantages over peptides in protease resistance, higher stability, and higher diversity. Peptoid libraries (>107 complexity) synthesized in split/pool fashions will be screened in bead-immobilized forms. "Hit" compounds will be transformed into soluble dimeric forms and modified by molecular shuffling to obtain DC-stimulating "lead" peptoids. 4) To synthesize DC-stimulating illumination. Using a tunable illumination device, we will search the universe of light by testing the impact of >107 different illumination. We will also test the concept that DC activation is inducible by photochemical cross-linking of surface receptors. 5) To determine biological consequences of DC activation by different agents. Newly identified DC-stimulating agents will be examined for their in vitro impact on gene expression profiles, surface phenotype, cytokine and chemokine production, and T cell-stimulating capacity of DC and for in vivo impact on Langerhans cells. We will analyze experimenta data using computational biology tools to define algorithms that govern DC activation processes. Our study wil provide new insights into DC biology and may lead to the development of novel adjuvants and therapeutics.