T CELLS ON THE FUNCTIONAL LIFE-SPAN OF DENTRITIC CELLS

描述（由申请人提供）：树突状细胞（DC）活化后成为专职抗原呈递细胞，能够刺激初始和记忆T细胞并启动免疫应答。DC活化后能存活多久并有效地将Ag呈递给T和B淋巴细胞仍不清楚。免疫应答的成功调节取决于DC存活足够长的时间以刺激足够数量的Ag特异性T细胞的能力，以及当应答不再需要时停止呈递的能力，例如当病原体已被清除时。本提案的目标是研究树突状细胞的存活和与应答T细胞相互作用后死亡的机制，以及它们对这种相互作用的抵抗力取决于它们的分化和活化状态。该项目的目标将在体外和体内实验小鼠中进行。在体外，将使用不同的凋亡测定法来研究与T细胞共孵育后树突细胞的死亡时间，从而确定静息和活化T细胞对培养的树突细胞活力的影响。在体内，以类似于使用DC作为疫苗的方案的方式注射到小鼠中的可追踪的树突状细胞的命运将在引流淋巴结中，在不存在或存在初次或二次免疫应答的情况下进行免疫组织化学和流式细胞术研究。体外和体内研究MHC-Ag限制性T细胞对DC的作用，将揭示免疫应答是否可以影响旁观者树突状细胞。从Fas细胞死亡受体功能缺陷的野生型或lpr自身免疫小鼠生长的DC的存活率的比较将确定在免疫应答期间DC的寿命调节中对该死亡受体的需求。在体外和体内测试处于不同分化状态并由不同活化剂刺激的DC，可能有助于发现对T细胞作用具有抗性的DC。微阵列分析和广泛的分子研究，这样处理的DC将有助于确定参与他们的生存/死亡的因素。这些候选因子将被测试以创建新的方案，以获得具有更长寿命和/或对T细胞诱导的凋亡具有抗性的DC。该项目的结果可能对疫苗开发产生重大的实际影响。DC已被用作疫苗载体以提高免疫力（例如肿瘤的免疫治疗）。减少或延长DC存活的因素的鉴定可用于增加传统佐剂以及使用DC作为疫苗的方案的功效。它可能会给我们提供新的线索，免疫病理学的发病机制，如自身免疫性疾病，这种调节可能会改变。本研究为进一步研究DCs在自身免疫性疾病发病机制中的作用，验证DCs可能通过凋亡缺陷或延迟诱导/维持自身免疫的假说提供了基础。阐明DCs在自身免疫中的作用将有助于了解哪种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.