T CELLS ON THE FUNCTIONAL LIFE-SPAN OF DENTRITIC CELLS

描述(申请人提供)：树突状细胞(DC)激活后成为专业的抗原提呈细胞，能够刺激幼稚和记忆T细胞并启动免疫反应。树突状细胞在激活后还能存活多久，并能有效地向T和B淋巴细胞递送AGS，目前尚不清楚。免疫反应的成功调节取决于DC能否存活足够长的时间以刺激足够数量的抗原特异性T细胞，并在不再需要反应时停止呈现，例如当病原体被清除时。这项建议的目的是研究树突状细胞与应答T细胞相互作用后的存活和死亡机制，以及它们对这种相互作用的抵抗力取决于它们的分化和激活状态。该项目的目标将在体外和体内通过实验小鼠来实现。体外测定静息和活化T细胞对培养的树突状细胞活性的影响，并用不同的细胞凋亡率来研究树突状细胞与T细胞共同孵育后的死亡时间。在体内，可追踪的树突状细胞以类似于使用树突状细胞作为疫苗的方式注射到小鼠体内，在没有或存在初级或次级免疫反应的情况下，将在引流淋巴结中进行免疫组织化学和流式细胞术研究。在体外和体内研究MHC-Ag限制T细胞对树突状细胞的作用，将揭示免疫反应是否可以影响旁观者树突状细胞。从野生型和LPR自身免疫小鼠中培养的DC的存活率的比较，将确定在免疫反应过程中对DCs寿命的调节中对这种死亡受体的需求。在体外和体内检测不同分化状态和不同激活剂刺激下的DC，可能有助于寻找对T细胞效应具有抵抗力的DC。微阵列分析和广泛的分子研究将有助于确定与其生存/死亡相关的因素。这些候选因素将被测试以创建新的方案，以获得寿命更长的DC和/或抵抗T细胞诱导的凋亡的DC。该项目的成果可能会对疫苗开发产生很强的实际影响。DC已被用作疫苗载体以提高免疫力(如肿瘤的免疫治疗)。识别减少或延长DC存活的因素可用于提高传统佐剂以及使用DC作为疫苗的方案的疗效。这可能会给我们提供免疫病理学发病机制的新线索，例如自身免疫性疾病，在这些疾病中，这种调节可能会改变。本项目获得的实验证据将为进一步研究树突状细胞在自身免疫性疾病发病机制中的作用提供基础，以验证树突状细胞可能由于缺乏或延迟凋亡而诱导/维持自身免疫的假说。阐明树突状细胞在自身免疫中的作用将有助于了解哪些细胞是治疗自身免疫性疾病的正确靶细胞。

DESCRIPTION (provided by applicant): Upon activation Dendritic Cells (DCs) become professional Antigen Presenting Cells, able to stimulate naive and memory T cells and initiate an immune response. How long DCs can survive after activation and efficiently present Ags to T and B lymphocytes is still unclear. Successful regulation of the immune response depends on the capacity of DCs to survive for a period of time long enough to stimulate a sufficient number of Ag-specific T cells, and to cease presenting when the response is no longer necessary, for example when the pathogen has been cleared. It is the goal of this proposal to study the survival of dendritic cells and the mechanisms involved in their death after interaction with responding T cells, and their resistance to the effects of such interactions depending on their state of differentiation and activation. The goals of the project will be approached in vitro and in vivo in experimental mice. In vitro the effects of resting and activated T cells on the viability of cultured dendritic cells will be determined, using different apoptotic assays to study the time of death of dendritic cells after co-incubation with T cells. In vivo the fate of traceable dendritic cells, injected into mice in a manner resembling the protocols that use DCs as vaccines, will be followed by immunohistochemistry and flow cytometry studies, in the draining lymph nodes, in the absence or the presence of primary or secondary immune responses. Studies in vitro and in vivo of the MHC-Ag-restriction of the T cell effects on DCs, will reveal whether an immune response can influence bystander dendritic cells. The comparison of survival of DCs grown from wild type or lpr autoimmune mice, which are deficient in the function of the Fas cell-death receptor, will determine the requirement for this death receptor in the regulation of the life span of DCs during an immune response. Testing DCs both in vitro and in vivo in different states of differentiation and stimulated by different activators, may help to find DCs that are resistant to the effects of T cells. Micro-Array analysis and extensive molecular studies of so treated DCs will help to identify the factors involved in their survival/death. These candidate factors will be tested to create new protocols to obtain DCs with a longer life span and/or that are resistant to T cell-induced apoptosis. The results of this project may have a strong practical impact in vaccine development. DCs have been used as vaccine carriers to improve immunity (e.g. immunotherapy of tumors). The identification of factors that reduce or prolong DC survival can be used to increase the efficacy of traditional adjuvants as well as protocols that use DCs as vaccines. It may give us new clues into the pathogenesis of immunopathologies, such as autoimmune diseases, where this regulation may be altered. The experimental evidence obtained from this project will provide the foundation for future investigations concerning the role of DCs in the pathogenesis of autoimmune diseases, to test the hypothesis that DCs may induce/maintain autoimmunity because of deficient or delayed apoptosis. The clarification of the role of DCs in autoimmunity will help to understand which is the right cell to target in the therapy of autoimmune diseases.