Apoptosis-Resistant Langerhans Cells

抗凋亡朗格汉斯细胞

• 批准号: 6479790
• 负责人: Laura Timares
• 金额: $ 26.72万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2005-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6479790
• 关键词: BCL2 gene /protein; Langerhans' cell; antigen antibody reaction; apoptosis; cell age; cell line; genetically modified animals; histocompatibility antigens; immunogenetics; immunoregulation; laboratory mouse; leukocyte activation /transformation; longevity; passive immunization; reporter genes; transfection

DESCRIPTION (provided by the applicant): Genetically engineered subsets of the dendritic cell (DC) family, such as Langerhans cells (LC), hold tremendous promise as immuno-therapeutic agents in the development of DC-based vaccines for cancer and infectious diseases. However, engineered DC quickly undergo cell death by immune-mediated apoptosis in vivo, crippling the fill potential of DC-based therapies. We propose that expressing anti-apoptotic genes within DC will inhibit apoptosis and extend their life span in vivo. Thus we will test if extending the in vivo life span of engineered DC will amplify the engineered antigen-specific immune response. The effect of these genes on the life span of an LC-derived DC cell line and their ability to modulate immune responses will be investigated utilizing the well-established 3A9 transgenic animal model that permits in vivo examination of interactions between DC and normally rare naive or activated antigen-specific T cells. The specific aims of this proposal are as follows: 1. To evaluate the ability of anti-apoptotic genes to confer resistance to apoptotic stimuli within DC in vitro. Bcl-XL, CrmA and baculovirus p35 block steps in biochemically separate pathways of apoptosis. Some have been implicated in altering DC longevity, but their effects on DC have not been examined. Therefore, DC will be transfected with anti-apoptotic genes and their ability to resist apoptotic stimuli in vitro will be tested. 2. To evaluate the effect of anti-apoptotic genes on altering the life span of transfected DC in vivo. In vivo studies will utilize adoptive transfer of engineered DCs into mice containing defined numbers CD4+ T cells (obtained from 3A9 transgenic mice) that recognize specific antigen-MHC II complexes exclusively presented on the DC transfectants. The effect of anti-apoptotic genes on extending DC life span in vivo will be determined. 3. To assess the impact of apoptosis-resistant DC on immune responses in vivo. The transfected anti-apoptotic DC abilities to induce a broad-spectrum of immune responses or to develop immune disorders upon transfer to naive mice and their impact on I cell activation in vivo will be examined. 4. To quantify the relationship of DC longevity to the magnitude of the DC-engineered immune response. An inducible "suicide" gene (iCasp3) will be super-transfected into anti-apoptotic DC as well as wild-type DC. At different times after adoptive transfer into mice DC will undergo apoptosis in vivo by exposing mice to the inducing agent. Thus, the minimal DC life span required to generate a defined immune response will be determined. Moreover, these experiments will lay the foundation toward achieving exogenous control of engineered DC gene expression and, in this case, longevity in vivo.

描述（由申请人提供）：基因工程的子集 树突状细胞（DC）家族，例如Langerhans细胞（LC），拥有巨大的 在基于直流的疫苗开发中作为免疫治疗剂的承诺 用于癌症和传染病。但是，工程的DC迅速经历了单元 免疫介导的体内凋亡死亡，使填充潜力削弱 基于DC的疗法。我们建议在DC内表达抗凋亡基因 将抑制凋亡并在体内延长其寿命。因此，我们将测试是否 延长工程DC的体内寿命将放大工程的 抗原特异性免疫反应。这些基因对生命的影响 LC来源的DC细胞系及其调节免疫反应的能力将 使用公认的3A9转基因动物模型进行研究 允许体内检查DC和通常罕见的天真的相互作用 或活化的抗原特异性T细胞。该提议的具体目的是 如下：1。评估抗凋亡基因赋予的能力 在体外DC内对凋亡刺激的抗性。 Bcl-XL，CRMA和 杆状病毒p35块步骤在生化分开的凋亡途径中。 有些与改变DC的寿命有关，但它们对DC的影响 尚未检查。因此，DC将用抗凋亡转染 将测试基因及其抵抗凋亡刺激的能力。 2。 评估抗凋亡基因对改变寿命的影响 转染的直流体内。体内研究将利用 设计的DC进入包含定义数字CD4+ T细胞的小鼠（从 3A9转基因小鼠）识别特定抗原MHC II复合物 专门呈现在直流转染物上。抗凋亡的影响 将确定延长DC寿命的基因。 3。评估 抗凋亡DC对体内免疫反应的影响。转染 抗凋亡DC能力引起广泛的免疫反应或 转移到天真小鼠时会发展免疫疾病及其对I的影响 将检查体内细胞激活。 4。量化DC的关系 寿命至DC工程免疫反应的大小。诱导 “自杀”基因（ICASP3）将被超级转染为抗凋亡DC 以及野生型DC。在收养转移到小鼠DC之后的不同时间 将通过将小鼠暴露于诱导剂中的体内凋亡。因此， 生成定义的免疫反应所需的最小直流寿命将为 决定。 此外，这些实验将为实现外源性奠定基础 控制工程的直流基因表达，在这种情况下是体内寿命。