【作者】 陈成；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2005， 硕士

【摘要】 树突状细胞(Dendritic cells,DCs)是目前已知的体内最重要的专职性APC,能摄取、加工及提呈抗原,刺激初始型T细胞的活化和增殖,是启动、调控并维持特异性免疫应答的中心环节。DCs介导的T细胞免疫应答在机体抗肿瘤免疫中发挥主导作用,包括对抗原特异性T细胞的诱导、激发、扩增和功能介导等。业已表明,DCs和Th细胞的极化诱导密切相关,不同的抗原致敏和致DCs成熟信号可能会赋予DCs不同的介导T细胞极化能力。凋亡肿瘤细胞可以提供DCs丰富的肿瘤抗原,能够提供多个MHC-Ⅰ和MHC-Ⅱ两类途径的抗原表位,因而采用凋亡肿瘤细胞作为抗原可以有效获得肿瘤特异性树突状细胞。CD40/CD40L信号被认为是DCs发育成熟至关重要的信号,CD4~+T细胞与DCs表面CD40L-CD40的相互作用对T细胞和DCs的功能均产生重要影响。利用CD40信号激发在体外可望获得表型和功能上成熟的树突状细胞。 本研究第一部分利用共聚焦显微镜观察了未成熟树突状细胞对凋亡肿瘤细胞的摄取,采用流式细胞术和~3H-TdR掺入实验研究了CD40和TNFR信号对凋亡肿瘤细胞负载的树突状细胞表型的促成熟作用和促T细胞增殖效应,采用胞内染色、流式细胞术和ELISA探讨了CD40配基化的肿瘤特异性树突状细胞对Th细胞的极化作用,发现CD40较TNFR信号更能增强树突状细胞介导Th细胞向Th1细胞极化的能力。 论文第二部分试图从共刺激分子的激发和调节性表达角度探讨DCs对Th细胞极化的作用。首先采用RT-PCR和流式细胞术分析了CD40信号对树突状细胞表面B7-H3分子表达的调节作用,发现了CD40信号能上调树突状细胞上B7-H3分子的表达,并进一步采用特异性抗B7-H3抗体阻断实验、~3H-TdR掺入实验和ELISA发现了B7-H3分子的上调性表达有利于树突状细胞介导Th1型免疫应答。同时还发现CD40信号促进凋亡肿瘤细胞负载的树突状细胞IFN-γ的分泌,推测CD40信号上调树突状细胞上B7-H3分子的表达可能和其介导IFN-γ更多还原

【Abstract】 Dendritic cells (DCs) are professional APC that are specialized for the initiation and regulation of T cell immunity. Critical determinants of T cell activation induced by DCs include the density of peptide-MHC ligand available for TCR engagement as well as the provision of costimulatory signals. DCs also play an important role in Th1 or Th2 polarization. In vitro, DCs can be derived from bone marrow precursor cells when the appropriate cytokine signals are provided. Previous studies have shown that apoptotic tumor cells could be efficiently phagocytosed by immature DCs via cell surface expression of αvβ3, αvβ5 and CD36. DCs process, cross-present tumor Ag and stimulate antigen-specific, class Ⅰ or Ⅱ-restricted CD8~+ T cells. The utilization of apoptotic tumor cells as tumor antigens raises the possibility that antitumor specific immune responses activated by DCs without identification of TAA or TSA. In order to induce DCs maturation and trigger their transitions from immature Ag-capturing cells to mature Ag-presenting cells, various cytokines or biological factors were applied, such as sCD40L, CD40L transfectants or agonist CD40mAb, tumor necrosis factor-α (TNF-α). Notably, studies demonstrated that CD40 triggering DCs were most potent to elicit antitumor immunity. Moreover, several reports suggested that CD40 activated mature DCs preferable induced Th1 immune response in vitro or in vivo. However, the rational mechanisms remain unknown.Although appreciated as being fundamental to T cell activation, costimulatory molecules have also been implicated in promoting Th1 and Th2 specificity. Among the most important costimulatory signals are those delivered by the B7 family. Like most other members of this family, B7-H3 was described as a type I membrane protein. Collectively, the as-yet-unidentified receptor is expressed on activated T cells,and binding to B7-H3 might participate in the regulation of cellular and humoral immune responses..In the present report, we generated mouse myeloid DCs from bone marrow precursor in vitro using GM-CSF and IL-4. Phagocytosis of apoptotic tumor cells by DCs was observed by Confocal Microscopy. Immature DCs loaded with apoptotic tumor cells were stimulated with CD40L transfectants or TNF-a to prepare mature DCs. 3H-thymidine incorporation test was used to detect the T cell proliferation stimulated by mature DCs. The concentration IFN-y in supernatants of MLR from dendritic cell-driven T cells activation was analyzed by ELISA. Intracellular staining and FCM were used to detect the percent of CD4+IFN-y+T cells and CD4+IL-4+T cells hi T cells activated by mature DCs. Our results demonstrated that allogeneic MLR responses induced by CD40 activated DCs generated more Thl effectors than the responses induced by TNF-a stimulated DCs in vitro. FCM showed that during the process of maturation, DCs up-regulated the expression of CD40, CD80, CD86, and no significant differences between CD40 activated and TNF-a stimulated DCs were found in these molecules(P>0.05). In further study, we examined the expression and function of mouse B7-H3 on dendritic cell. Expression of B7-H3 in DCs was detected by FCM, and mRNA of B7-H3 was detected by RT-PCR. 3H-TdR incorporation test was used to detect the T cell proliferation stimulated by DCs with or without blocking B7-H3 by mAb respectively. The concentrations of IFN-y in supernatants of MLR from distinct groups were analyzed by ELISA. The concentration of IFN-y in supernatant of DCs was determined by ELISA. The data showed that B7-H3 expression on dendritic cells appears to be up-regulated by CD40 ligation, and differential expression of B7-H3 was obvious on DCs stimulated by mCD40L-CHO cells or TNF-a. In the blockade assay, we found an antagonistic mAb to B7-H3 inhibited CD40 activated DCs mediated T cells proliferation and IFN-y production in vitro. CD40 ligation was a potent signal to enhance apoptotic tumor cells pulsing DCs to secrete IFN-y. We hypothesized that auto-secreting IFN-y contributed toup-regulating expression of B7-H3 in DCs ligated by CD40, which sequentially favored CD40 activated DCs to enhance Thl- potential and mediate Thl immune response.In conclusion, our studies substantially gave evidences for a precise balance of costimulatory molecules determining the outcome of a T cell response and expanded our knowledge of costimulatory molecules regulatory network. Furthermore, this study was performed to provide some guidance about the DCs preparation we might take into clinical studies. It might be anticipated that DCs triggered by CD40 signal in vitro will be entirely compatible with their preprogrammed function. Remarkable advances in our understanding of the role of CD40 activated tumor specific DCs in generation of effectors T lymphocyte had shed new light on the regulation of the immune responses. Once we understand what the ideal T lymphocyte responders require, this new knowledge will help develop better therapies for controlling the immune responses. Ultimately the clinical data and practical issues will decide which DCs preparation clinicians will select, and DCs activated by CD40 signal would have a promising tumor immunotherapeutic prospect.更多还原