Dendritic Cell-Based Biosensor System

描述(申请人提供)：树突状细胞(DC)位于环境界面的战略位置，作为免疫学前哨细胞和组织驻留抗原呈递细胞。当DC暴露在感染性微生物或“危险信号”下时，会产生促炎介质，经历成熟，并迁移到淋巴结以激活幼稚的T细胞。因此，DC的激活是与获得性免疫相关的初始和关键事件。我们将利用基于DC的生物传感器系统系统地研究DC的激活过程，并开发新型的DC刺激剂。具体目标是：1)优化直流电生物传感器。我们用13个含有不同顺式增强子的荧光素酶报告基因转染XS106 DC细胞，建立了DC生物传感器的原型，并鉴定了多种微生物、生物、药理和物理因子激活DC中不同的转录调控通路。我们将通过分离永久表达EGFP报告结构的DC克隆来优化这个系统。2)系统验证。我们将通过测试目前已知的DC刺激剂和从NCI筛选化合物库(&gt；105复杂性)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刺激性类肽。类肽(N-取代甘氨酸低聚物)与多肽相比，具有更高的稳定性、更高的耐酶活性和更高的多样性。以分裂/混合方式合成的类肽文库(&gt；107复杂性)将以珠状固定化形式进行筛选。“Hit”化合物将转化为可溶的二聚体形式，并通过分子洗牌进行修饰，以获得DC刺激的“铅”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&gt；107不同照明的影响来搜索光的宇宙。我们还将测试DC激活可以通过表面受体的光化学交联来诱导这一概念。5)确定不同试剂激活DC的生物学后果。新发现的DC刺激剂将在体外对DC的基因表达谱、表面表型、细胞因子和趋化因子的产生、DC的T细胞刺激能力以及体内对朗格汉斯细胞的影响进行检测。我们将使用计算生物学工具分析实验数据，以定义管理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药物的开发。

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.