Apoptosis-Resistant Langerhans Cells

描述(由申请人提供)：基因工程的子集 树突状细胞(DC)家族，如朗格汉斯细胞(LC)，拥有巨大的 在DC疫苗开发中有望成为免疫治疗剂 治疗癌症和传染病。然而，工程树突状细胞很快就会进入细胞 体内免疫介导的凋亡导致的死亡，削弱了细胞的填充潜力 基于DC的疗法。我们认为，在DC中表达抗凋亡基因 会在体内抑制细胞凋亡，延长其寿命。因此，我们将测试 延长工程化DC的活体寿命将放大工程化DC 抗原特异性免疫反应。这些基因对人的寿命的影响 LC来源的DC细胞系及其调节免疫反应的能力将 利用成熟的3A9转基因动物模型进行研究 允许在体内检查DC和正常罕见的幼稚之间的相互作用 或被激活的抗原特异性T细胞。这项提议的具体目的是 研究结果如下：1.评价抗细胞凋亡基因的表达能力 体外树突状细胞对凋亡刺激的抵抗力。BCL-XL、CrmA和 杆状病毒p35阻断了生化分离的细胞凋亡途径。 其中一些与改变DC的寿命有关，但它们对DC的影响 都没有经过检查。因此，将抗凋亡基因导入树突状细胞。 将在体外测试基因及其抵抗凋亡刺激的能力。2. 评价抗细胞凋亡基因改变人卵巢癌寿命的作用 体内转染树突状细胞。体内研究将利用领养转移 将树突状细胞工程化为含有一定数量的CD4+T细胞的小鼠(从 3A9转基因小鼠)识别特定抗原-MHC II复合体 独家介绍了DC转染体。抗细胞凋亡的作用 在体内延长DC寿命的基因将被确定。3.评估 抗凋亡树突状细胞对体内免疫反应的影响。被转染者 抗凋亡DC诱导广谱免疫反应的能力或 移植给幼鼠后出现免疫紊乱及其对免疫功能的影响 将检查体内细胞的激活情况。4.量化DC之间的关系 长寿与DC工程免疫反应的规模有关。诱导性的 “自杀”基因(ICasp3)将作为超基因导入抗细胞凋亡的DC 以及野生型DC。小鼠DC过继转移后不同时间的变化 会通过将小鼠暴露在诱导剂中而在体内发生细胞凋亡。因此， 生成定义的免疫响应所需的最小DC寿命将为 下定决心。 此外，这些实验将为实现外源 控制工程DC基因的表达，在这种情况下，体内的寿命。

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.