【作者】 古涛；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 内科血液学， 2003， 博士

【摘要】 树突状细胞（dendritic cells，DCs）是已知体内功能最强的专职性抗原递呈细胞（antigen presenting cells，APC），DCs能有效摄取、加工及递呈抗原，并能刺激未致敏T细胞的增殖和活化，是启动、调控并维持特异性免疫应答的中心环节。制备肿瘤特异性DCs疫苗免疫肿瘤患者，可有效地激发特异性的、持久的肿瘤免疫应答。这已成为目前肿瘤免疫治疗一个热点，并在临床试验中取得一些令人振奋的结果。然而，DCs临床应用仍有诸多关键问题亟待解决：DCs体外诱导的最佳途径及成熟调控；DCs疫苗接种的数量、次数及免疫途径；DCs疫苗体内的免疫原性和迁移能力；有效佐剂的配合使用和毒副作用的评价及解决方案等。 本研究首先评价了独特型免疫球蛋白负载的DCs（idiotype immunoglobulin pulsedDCs，Id-DC）疫苗和凋亡肿瘤细胞负载的DCs（apoptotic tumor cells pulsed DCs，AP-DC）疫苗的主动免疫治疗作用，继而着重研究CD40配基化的DCs激发肿瘤特异性免疫应答的机制，率先提出负性共刺激分子PD-L1和PD-L2的适度表达对于激发和维持肿瘤特异性免疫应答有着至关重要的作用，并对相关信号转导机制进行探讨。 第一部分 独特型免疫球蛋白负载的树突状细胞在B细胞淋巴瘤免疫治疗中的作用 目的：评价Id-DC疫苗在B细胞淋巴瘤荷瘤小鼠中的主动免疫治疗作用。 方法：利用细胞融合技术制备并筛选获得分泌Id的阳性克隆；体内诱生腹水和体外无血清培养法制备Id，优球蛋白沉淀法结合凝胶Suphecray-200过滤纯化Id；对Id-KLH络和物负载小鼠骨髓前体细胞来源的DCs，分别采用mCD40L-CHO细胞、TNF-α、LPS或单纯培养基刺激48h，制备四种Id-DC疫苗。体外采用³H-TdR掺入试验和⁵¹Cr释放试验测定DCs体外刺激T细胞增殖和CTL细胞杀伤毒效应。同时C D40配基化的肿瘤特异性树突状细胞的免疫治疗作用及其相关机制研究中文提要应用四种Id一DC疫苗开展SB4和SBS荷瘤小鼠的主动免疫治疗，观察肿瘤生长情况及小鼠生存期;并检测血清抗Id抗体的分泌。结果:获得2株稳定分泌Id的Baib/c小鼠B细胞淋巴瘤细胞SB4和SBS;四种成熟的ld一DC疫苗，均可以抑制肿瘤生长，延长生存期，从总体上评价CD40配基化的Id一DC疫苗疗效最佳护<0.05)，但均未有肿瘤完全缓解（compl改e remission，cR）的荷瘤小鼠。Id一DC联合Id一 KLH治疗可以激发更强的体液免疫应答，但不同Id一DC疫苗组之间结果无显著性差异（P>认05）。结论:CD4O配基化的Id一DC疫苗可以有效地延长荷瘤小鼠的生存期、抑制肿瘤生长，但针对单一Id位点激发不能诱导有效抗肿瘤免疫应答，整体疗效不佳，未有CR的荷瘤小鼠。更多还原

【Abstract】 Overwhelming evidences demonstrated that dendritic cells (DCs), the most potent professional antigen presenting cells (APC), which expressed high levels of MHC class I and II and costimulatory molecules, were efficient in phagocytosing antigens, migrating to lymphoid organs, and presenting antigens to naive T cells. The unique ability of DCs to induce and sustain primary immune responses made them optimal candidates for vaccination protocols in cancer immunotherapy.DCs played an important role in initiating innate and adaptive immune responses, especially the potent antitumor immunity. Immature DCs had specialized antigen uptake and processing machineries, whereas mature DCs had an extraordinary capacity to present antigens and stimulate naive T cells. Functionally, mature DCs were 100 times more potent than macrophages in activating naive T cells in vitro. DCs loaded with appropriate tumor associated antigen (TAA) could induce protective/rejection immune responses in animal models and promising preliminary data are reported in human. Several systems had been used to deliver TAA to DCs, including (1) defined peptides of known sequences, (2) undefined acideluted peptides from autologous tumors, (3) whole tumor lysates, (4) retroviral and adenoviral vectors, (5) tumor cell-derived RNA, (6) fusion of DCs with tumor cells, and (g) exosomes derived from DCs pulsed with tumor peptides (subcellular structures containing high levels of MHC molecules and peptides).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 CD40 ligand (CD40L) on activated T cells, soluble CD40L, CD40L transfectants or agonist mAbCD40, tumor necrosis factor-a (TNF-a), IL-1b, lipopolysaccharide (LPS), unmethylated CpG DNA sequences, double-stranded RNA(dsRNA), nucleotides (ATP, UTP), and heat shock proteins (HSP). Notably, studies demonstrated that CD40 stimulated DCs (either cross-linked by agonists mAbCD40 or ligated by soluble CD40L and CD40L transfectants) were most potent to induce antitumor immunity. Moreover, several reports suggested that DCs could not stimulate cytotoxic T lymphocytes (CTL) unless they were firstly stimulated via CD40 on their surface. TNF-a, as a standard cytokine to induce DCs maturation, was inferior in conditioning DCs to activate tumor specific immune response, particularly CD8+ CTL, compared with CD40L. However, DCs-mediated induction of immunity represented a challenge, and several parameters needed to be considered to ensure the optimal outcome of DCs based vaccination protocols including the source of TAA, the methods for TAA preparation and loading, and the diversity of DCs subsets, et al. It was crucial and useful to discern the biological characteristics of CD40 ligation DCs or TNF-a stimulation DCs, as DCs vaccines were applied in many clinic trials.Part I : Roles of idiotype immunoglobulin pulsed dendritic cells in B cell iymphoma’s immunotherapyEach B lymphocyte expressed a clonal immunoglobulin (Ig) whose heavy and light chain variable regions (VH and VL, respectively) comprised a unique collection of antigenic determinants known as the idiotype immunoglobulin (Id). B cell malignancies, as low grade non-Hodgkin’s lymphoma (NHL), presented an opportunity of tumor specific immunotherapy because Id was maintained following B lymphocytes’ malignant transformation. Id containing sequences epitopes which could be recognized by antibodies, CD4+ and CD8+ T cells was regarded as a useful TAA of B cell lymphoma.Objective: Our study aimed to investigate the role of Id-DC vaccine in B cell Iymphoma’s immunotherapy.Methods: Balb/c mouse B cell lymphoma cell lines were obtained by fusing SP2/0 cells with Balb/c mouse spleen cells after immunization with pristane and incomplete Freund’s adjuvant (IFA) respectively. Enzyme-linked immunosorbent assay (ELISA) and flowcytometry (FCM) were used to screen the secreting Id clones and analysis the phenotypes更多还原