【作者】 刘振华；

【导师】 郭坤元；

【作者基本信息】 第一军医大学 ， 血液病学， 2001， 博士

【摘要】 多发性骨髓瘤（MM）为浆细胞恶性增殖性疾病，其特征是浆细胞合成分泌单克隆的独特型Id（idiotype）。迄今，应用常规化疗（马法兰，强的松等）患者中位生存期仍然徘徊在3年左右。大剂量化疗结合自体造血干细胞移植可延长中位生存期，但仍非治愈性。同种异体骨髓移植（BMT）在部分病例中归因于免疫性GVM效应，可达到满意效果。 资料表明，MM患者可产生针对Id的T淋巴细胞反应和相应抗抗体生成，体外试验和动物模型证实，基于Id的免疫治疗可致浆细胞瘤消退。树突状细胞（dendritic cells，DC）是体内功能最强抗原提呈细胞，可诱导特异性细胞毒T淋巴细胞（cytotoxic T lymphocyte，CTL）生成。在MM患者单核来源DC（MDDC）有正常活性和功能。负载Id后免疫治疗，部分患者出现显著免疫反应，考虑到MM患者经过传统治疗易达微小残存疾病状态（MRD），使用DC及CTL免疫治疗有助延长生存期，并延迟复发。 细胞杀伤抑制受体（killer inhibition receptor，KIR）主要表达于自然杀伤细胞（NK），也表达于部分T细胞。一般认为，TCR介导T细胞活化和增殖，而KIR分子介导免疫抑制性反应。迄今，未见MM患者CTL与KIR表达相关研究报道。 目的 本研究通过DC呈递自体抗原途径体外诱导MM患者CTL，并探讨其免疫生物学特征，为临床过继免疫治疗MM打下基础。研究内容分为四个部分：（一）MM患者外周血DC的分离、培养和鉴定；（二）MM患者外周血CTL的诱导（in vitro）；（三）MM患者CTL细胞KIR表达研究；（四）MM患者CTL体内免疫效能观察（in vivo）。 方法与结果 第一部分：用塑料贴壁的PBMNCs来源或者免疫磁珠阴性选择分离途径，建立体外培养技术，从MM患者外周血中获取DCs。在含GM-CSF和IL-4的无血清培养基（AIM-V）中，37℃，5％CO₂培养箱中培养5～7天，可获得具有典型树突状细胞形态学特征的细胞。两种分离途径得率统计学比较无差异（n=5）。同种混合淋巴细胞反应及自体抗原呈递试 第一军医大学博土学位论文验证实，MM患者外周血来源DC具有很强的刺激同种异体淋巴细胞增殖能力及呈递自体抗原的能力。外周血来源DC培养5～7天后，流式细胞仪分析表明，部分细胞表达成熟DC特征性标记CDI。，部分表达共刺激分子 CD80及 CD86，高表达 HLA－DR分子，几乎不表达单核细胞特异性标记 CD14。rhIL－3或／及rhSCF加入培养体系并不增加 MM患者外周血DC得率。兔疫表型也无明显改变。提示DC来源于PBMNCS中单核细胞，而非CD34”造血祖细胞，本研究为获取DC进行深入研究及临床免疫治疗奠定实验基础。 第二部分：从MM患者外周血或尿液中，以盐析、层析等生化方法提取纯化自体Id蛋白(包括IgG，IgA及人人SDS－PAGE电泳鉴定为单－条带纯，免疫磁珠阴性选择法分离患者外周血T淋巴细胞，建立体外CTL诱导体系。在CTL诱导过程中最适的自体抗原负载浓度为 10ug／ml，只有在DC纯化后，加入自体抗原，T淋巴细胞达到最大程度增殖。利用有限稀释方法，从5例MM患者外周血中，有3例可体外诱导出Id特异性 CTL克隆。例 2（初治患者，血清 IgG75．4g／L）及例 5（尿人轻链疾病）未能诱导出。并分析各自CTL前体频率相应为1％，5％和7％。三例MM患者CTL细胞免疫表型分析表明，为非均一性细胞群体，以CDS”CTL为主，包括少量CD4”及部分CDS”CD28”、CD3－CD56”CD16”及Y6“CTL等亚群。本研究建立了通过DC呈递自体抗原诱导CTL细胞生成的体外无血清培养体系，该途径可望为MM患者过继免疫治疗提供崭新的手段。 第三部分：测定了3例MM患者T细胞及CTL细胞KIR分子表达率。结果显示KIR表达有多型性特点，在体外抗原激活后CTL以表达P58．2分子为显著特征．利用单抗荧光标记抗体结合有限稀释方法，体外p分选培养出MR”CTL及mR”CTL两型细胞。体外杀伤试验（E：T为 10：l）证实，两型细胞对Daudi细胞均有一定杀伤活性，而以抗KIR抗体阻断KIR 分子表达后，KIR”CTL杀伤活性提高。KIR”CTL几乎不杀伤RPMI8226细胞，但阻断相应以R分子或靶细胞MHC－I分子后，杀伤活性得到一定程度恢复。两型细胞均可体外杀伤负载自体抗原的DC细胞，阻断DC表面MHC．I分子后，杀伤活性下降。证实诱导的CTL为抗原特异性。Elispot试验结果表明，KIR－CTL以分泌r二FN为主，参与Thl ．4． 第一军医大学博士学位论文 反应，而mR“CTL主要分泌L－4，可能介导 ThZ反应。利用 RT－PCR 方法从三例MM患者KIR”CTL细胞中都可以扩增出300hp大小的 P58二CDNA特征性条带。条带荧光强度与KIR分子表达率相一致。电镜 观察和 AO／EB染色可观察到 KIR”CTL凋亡细胞的显著形态学特征， DNA电泳有典型的“ Ladder”梯形条带，A。exin－Vo1双染证头mR” CTL早期凋亡事更多还原

【Abstract】 ABSTRACT Multiple myeloma is a malignant disease involving plasma cells, usually accompanied by the production of an Ig paraprotein of a defined idiotype. Until recently, it was clear that optional management of the disease involved relatively low-dose chemotherapy (eg. Melphalan and prednisone), resulting in a median survival of approximately 3 years. It has become clear that more intensive chemotherapy, followed by autologous stem cell transplantation, prolongs median survival, but is unlikely to be curative. Allogeneic BMT in suitable candidates can achieve a satisfactory outcome, with complete remissions and prevention of disease progression, attesting to an immunological graft-versus-myeloma effect. The Ig paraprotein provides a natural TAA for multiple myeloma immunotherapy. MM patients are capable of generating T lymphocytes and antibody-mediated responses to Ig idiotype. In vitro studies suggest that plasma cells are targets for cytotoxic I lymphocytes and animal models show that vaccination strategies, based on Ig idiotype, eradicate plasmacytoma. Some studies of DC biology in MM patient have begun. Dentritic cells are known to be the most competent APCs. They initiate T cells, especially na飗e T cells to proliferate, and induce the generation of cytotoxic T lymphioctyes (CTL) both in vitro and in vivo. MDDC (monocyte-derived dentritic cells)presented from MM patients have been reported to have normal activities. MDDC pulsed with idiotype administered to individual patients induced a T lymphocyte proliferative response and an anti-idiotype antibody response. Because MM patients are highly applicable to the MRD (minimal residual disease) state being achieved after autologous stem cells transplantation, DC-based vaccination strategies, or Id-Specific CTL infusion after transplantation may prolong disease-free survival and postpone the inevitable relapse. KIRs (killer inhibition receptors) expressed mainly on natural killer cells, and also on I cells. Such subsets of T cells have two different types of -6- MHC-recognizing recepors on their surface-TCRs and KIRs. TCR recognizes antigen-MHC complex and induce positive signals for activities proliferation whereas KIRs mediates the opposite negative, signals for effector function and cell growth. Until recently, there is no published data demonstrating KIR expression on CTLs with MM patient. Objective The present study aimed at the in vitro induction of CTLs with MM and its immunobiological characteristics, including the following 4 sections: 1. the isolation, culture and characterization of DCs from peripheral blood with MM patients; 2. In vitro induction of CTLs with MM patients; 3. The study of KIR expression on CTLs with MM patients; 4. The in vivo immunological effect of CTLs with MM patients. Method and Results 1. To set up an in vitro culture procedure obtaining DCs from peripheral blood of MM patients. Plastic-adherent peripheral blood mononuclear cells (PBMNCs) or sorted cells by immmunomagenetic separation method were cultured in AIM-V serum-free medium containing GM-CSF and IL-4 at 37C, 5%C02 humidified atmosphere.After 5?7 days of culture, a large quantities of cells exhibiting a typical characteristic dentritic cell morphology were collected. No significant difference in yield existed between two separation met更多还原