【作者】 张文皓；

【导师】 侯健；

【作者基本信息】 第二军医大学 ， 内科学， 2012， 博士

【摘要】 多发性骨髓瘤是血液系统发病率第二的B细胞肿瘤，中国汉族人发病率达到1/10万，在发达国家发病率达到4/10万。美国2010年全年新发病人数大概在20000人。骨髓瘤占所有血液系统恶性肿瘤13.4%，占所有因血液系统恶性肿瘤死亡的19%及所有肿瘤死亡的2%。美国2010年有10,000名左右患者因骨髓瘤死亡。骨髓瘤患者的总体预后是不好的。接受常规治疗的患者中位生存为3-4年，而接受自体外周血造血干细胞移植患者中位生存被延长到5-7年，一些新药也已经与其他传统疗法联合应用于临床并可能进一步改善预后。但就目前结果提示，几乎所有患者最终都会复发。异基因造血干细胞移植是目前唯一一种能有希望治愈多发性骨髓瘤的治疗手段，但是选择异基因造血干细胞移植治疗的时机及这种治疗起到的作用尚存在争论。因为异基因造血干细胞移植后非复发死亡率在30%-50%之间，即使一部分患者能够长期生存，但目前过高的相关死亡率导致了这一治疗方式仍未被广泛接受。多发性骨髓瘤患者化疗/移植后复发的根源是机体内残存的微小残留病灶，我们迫切的需要一种治疗方式，它能够在患者完成造血干细胞移植后清除微小残留病灶。免疫治疗可以成为控制或消除微小残留病灶的一个选择，我们期待它能够巩固接受化疗或干细胞移植治疗患者的疗效。骨髓瘤细胞分泌的单克隆免疫球蛋白都带有独特的抗原决定簇,这可能成为一个肿瘤特异性抗原。有学者将患者血浆中的独特型免疫球蛋白提取并接种在患者体内进行免疫治疗，但是结果不尽如人意。部分原因是因为这种独特型免疫球蛋白免疫原性较弱，而且每位患者体内的这种独特型抗原都是独一无二的，其他患者无法从针对该独特型免疫球蛋白的疫苗或其介导的细胞毒性T细胞（CTL）抗骨髓瘤效应中受益。因此我们迫切的需要一种新的肿瘤相关抗原作为骨髓瘤免疫治疗靶点，这个靶点应当是大部分骨髓瘤细胞共有的一种抗原，并且可以在大部分患者中诱导出足够强的免疫反应。热休克蛋白就是具有这些特点的一类肿瘤特异性抗原，它包括热休克蛋白27、热休克蛋白70及热休克蛋白90，在很多肿瘤中优先的高表达，并且在某些肿瘤的高表达中常提示预后极差并对治疗耐受，同时热休克蛋白对于肿瘤细胞的生存至关重要。下调或者抑制热休克蛋白的表达会使骨髓瘤细胞显著凋亡。一种热休克蛋白90的抑制剂已经完成了I期临床试验。热休克蛋白的这些特性使它成为合适的新型候选肿瘤相关抗原。热休克蛋白在多发性骨髓瘤中高表达，而在正常组织中表达水平很低，并且它在骨髓瘤细胞的生存中起到不可替代的作用。有研究证明肿瘤细胞中热休克蛋白90复合物是活化并且具有高亲和力，而在正常组织中则处于失活状态，这也使得热休克蛋白成为肿瘤治疗的优良靶点。我们假设在骨髓瘤细胞中广泛高表达的热休克蛋白是骨髓瘤细胞的抗凋亡分子，并将其作为骨髓瘤免疫治疗的新靶点。本实验共分为三个部分：第一部分，筛选热休克蛋白来源HLA-A0201限制性CTL表位肽；第二部分，体外验证细胞毒性T细胞对骨髓瘤细胞的细胞毒作用；第三部分，建立原代多发性骨髓瘤SCID-rab动物模型并验证热休克蛋白特异性细胞毒性T细胞在动物体内的抗骨髓瘤作用。第一部分筛选热休克蛋白来源HLA-A0201限制性CTL表位肽并验证其免疫原性目的：筛选热休克蛋白来源HLA-A0201限制性表位肽并验证其免疫原性。方法：用肽积分系统（http://bimas.dcrt.nih.gov/molbio/hla_bind/）对热休克蛋白27、热休克蛋白70及热休克蛋白90α来源的HLA-A0201限制性CTL表位肽进行预测，用（http://syfpeithi.bmi-heidelberg.com）系统对预测结果确认。合成出积分最高的四条肽段，用HLA-A0201+T2杂交瘤细胞验证肽段的亲和力及稳定性，进一步筛选出亲和力及稳定性最佳的两条肽段用来诱导肽段特异性细胞毒性T细胞产生。合成HLA-A0201-肽段四聚体用以检测Hsp-CTLs细胞阳性率。最后，我们给HLA-A0201转基因小鼠接种所筛选的热休克蛋白肽段，两周后处死小鼠取脾脏细胞，检测是否有热休克蛋白特异性细胞毒性T细胞产生并验证这些细胞毒性T细胞的功能。结果：通过积分系统我们预测并挑选出积分最高的4条肽段，分别是热休克蛋白27相关肽段aa27，序列为RLFDQAFGL；热休克蛋白70相关肽段aa393，序列为LLLLDVAPL；热休克蛋白90相关肽段aa362，序列为KLYVRRVFI；热休克蛋白90α相关肽段aa670，序列为ALLSSGFSL。通过进一步T2细胞结合试验，我们筛选出结合力和稳定性最强的两条肽段aa27及aa670。我们通过四聚体检测健康供者及患者体内aa27及aa670特异性细胞毒性T细胞水平，发现患者体内aa27及aa670四聚体阳性细胞毒性T细胞水平较健康供者上升，证明筛选合成的肽序列为生理状态下产生的表位肽片段。结论：所筛选的热休克蛋白肽段aa27及aa670与HLA-A0201分子亲和力最高，结合后稳定性最强，并且在患者体内可以检测到以上肽段特异性细胞毒性T细胞。将我们筛选的热休克蛋白90肽段接种到HLA A0201转基因小鼠体内后我们检测到了热休克蛋白特异性细胞毒性T细胞，这提示我们筛选的肽段具有免疫原性。第二部分体外验证细胞毒性T细胞对骨髓瘤细胞的细胞毒作用并研究其特性目的：通过体外实验验证细胞毒性T细胞对骨髓瘤细胞的细胞毒作用并了解细胞毒性T细胞特性及细胞毒作用机制。方法：用HLA-A0201+健康供者外周血诱导出肽段特异性细胞毒性T细胞，用HLAA0201+四聚体检测所肽段特异性细胞毒性T细胞水平，用流式细胞学方法检测细胞毒性T细胞表型及其分泌的细胞因子，用流式细胞学及CCK-8方法验证细胞毒性T细胞的增殖，用乳酸脱氢酶释放试验及流式细胞学方法验证细胞毒性T细胞分别对U266、ARH-77、RPMI8226、LP1细胞株及骨髓瘤原代细胞、HLA-A0201+健康供者PBMC的杀伤作用，用流式细胞学方法检测细胞毒性T细胞功能。结果：将外周血单个核细胞与肽段冲击过的自体树突状细胞共培养四周后，在共培养的淋巴细胞中可以检测到比例逐渐上升的四聚体阳性的细胞毒性T细胞，其比例由共培养前的不足1%上升到超过20%，这些淋巴细胞的表型也从低表达CD45RO高表达CD45RA转换为高表达CD45RO低表达CD45RA，并且分泌IFN-γ。我们将肽段冲击过的DC与T淋巴细胞共培养并与未经过肽段冲击过的DC与T淋巴细胞共培养作为对照，用CCK-8测试发现与肽段冲击DC共培养的淋巴细胞明显增殖，而另一组没有增殖。我们将与肽段冲击过DC共培养的淋巴细胞分别与HLA-A0201+骨髓瘤细胞株及HLA-A0201-骨髓瘤细胞株共培养并设置空白对照，发现与ARH-77（HLA-A0201+）细胞共培养的T淋巴细胞发生明显增殖，而与LP1（HLA-A0201-）共培养及未与肿瘤细胞共培养的两组淋巴细胞无增殖现象。将这些淋巴细胞与不同细胞株、原代细胞或PBMC共培养后通过乳酸脱氢酶释放试验及流式细胞学检测，发现其对HLA-A0201+细胞株U266、ARH-77及HLA-A0201+骨髓瘤原代细胞有明显的细胞毒作用，而对HLA0201-的细胞株RPMI8226、LP1及来自健康供者的HLA0201+PBMC没有细胞毒作用，同时我们发现在杀伤过程中CD8+的CTL细胞表达CD107α水平明显上升，并且细胞毒作用通过perforin及granzyme B途径发挥作用。结论：热休克蛋白肽段aa27及aa670可以诱导出该肽段HLA0201+细胞毒性T细胞，并且具有细胞活性，对HLA-A0201+的骨髓瘤细胞株及骨髓瘤原代细胞具有细胞毒作用，并通过Perforin及granzyme B机制起作用。第三部分验证Hsp-CTLs在原代多发性骨髓瘤动物模型SCID-rab中的抗骨髓瘤效应目的：建立SCID-rab原代多发性骨髓瘤动物模型并验证Hsp-CTLs在动物体内的抗骨髓瘤作用。方法：给10只6-8周雄性CB.17-SCID小鼠植入4周龄新西兰兔四肢扁骨，4-6周后将当天分离好的骨髓瘤患者原代CD138+骨髓瘤细胞注射入已植入的骨腔中，每周留取小鼠血样并检测轻链水平，每周进行X线照射观察植入兔骨变化，判断是否成瘤及决定治疗时机。成瘤后即开始按照分组情况通过鼠尾静脉注射Hsp-CTLs细胞。肿瘤注射起开始计算生存期，至肿瘤面积达到225mm2算作死亡。用脱颈方法处死肿瘤达到225mm2小鼠，取瘤组织行病理学免疫组化检测。小鼠全部死亡后计算生存期。结果：在注射CD138+原代骨髓瘤细胞的9只SCID-rab小鼠中，4只成瘤，成瘤率44.4%。成瘤小鼠生存时间为49-70天，较未成瘤小鼠明显缩短，其差别有统计学意义p<0.05。接受Hsp-CTLs细胞治疗后，HLA-A0201+荷瘤小鼠生存期延长，但因例数较少，尚无统计学意义。结论：原代多发性骨髓瘤细胞可以在SCID-rab模型中形成肿瘤。更多还原

【Abstract】 Multiple myeloma is the second most common hematologic cancer.The incidence variesfrom1per100000people in China, to about4per100000people in most developedcountries. The American Cancer Society estimates that20,180new cases of Multiplemyeloma will be diagnosed in the United States in the year2010. Median survival afterconventional treatments is3–4years; high-dose treatment followed by autologous stem-celltransplantation can extend median survival to5–7years. Novel drugs, for use alone and incombination with existing treatments, are increasingly being assessed for ability to furtherimprove survival. But the vast majority of these patients are certain to develop diseaserecurrence after SCT or novel drug therapy.Allogeneic hematopoietic stem celltransplantation (SCT) is a potentially curative approach in patients with multiple myeloma，it result in long-term progression-free survival (PFS), with a plateau in survival curvessuggesting possible cure. However, the role and timing of allogeneic SCT in the diseasecourse are still controversial. And it was associated with excessively high rates ofnonrecurrence mortality (NRM), reaching30%to50%, leading to a nearly completeabandonment of this approach.The origin of recurrence is the minimal residual disease(MRD)exist in vivo. Additional measures are required urgently after transplantation to eliminateminimal residual disease. Immunotherapy is an appealing option for this purpose toconsolidate the curative effect after stem cell transplantation.Heat shock proteins (Hsps) may belong to this category of tumor-associated antigens.Hsps, including Hsp27, Hsp70and Hsp90, are abundantly and preferentially expressed invarious cancers，including MM，and their (over)expression in some cancers correlates withpoor prognosis and resistance to therapy. Moreover, these proteins have emerged as being ofprime importance for the survival of cancer cells. Downregulating or inhibiting Hspexpression results in significant apoptosis in cancer cells, including myeloma cells. One ofthe inhibitors for Hsp90was studied in recently completed phase I testing, and it was welltolerated. Owing to these special properties, Hsps have become a novel and valid target for cancertherapeutics. Hsps over expressed in myeloma cells and low expressed in normalcells,meanwhile hsps took prime importance for the survival of cancer cells.The observationthat tumor cells contain Hsp90complexes in an activated, high-affinity conformationwhereas Hsp90in normal cells is in a latent, uncomplexed state, allows a selective targetingof the molecules in cancer cells.We hypothesize that the broad expression of Hsps in myeloma, together with theirfunctional role as anti-apoptotic molecules in cancer cells, makes them a new ideal target forimmunotherapy in MM.We have make three aims in this experiment:1. Identify HLA-A0201restricted epitopes on Hsps and validate its immunogenicity;2. Examine in vitro cytolyticeffects of the CTLs on myeloma cells, including cell lines and primary myeloma cells frompatients, and on normal hematopoietic cells;3. Construction a novel myeloma-SCID-rab host.Part Ⅰ Identify HLA-A0201restricted epitopes on Hsps and validate its immunogenicity.Objective:To identify HLA-A0201restricted epitopes on Hsps and validate itsimmunogenicity.Methods:The sequence of Hsp27, Hsp70, and Hsp90α have been reviewed for peptidesthat potentially bind to the HLA-A0201molecule using a peptide-motif scoring system(http://bimas.dcrt.nih.gov/molbio/hla_bind/). We also used another system, the SYFPEITHIdatabase (http://syfpeithi.bmi-heidelberg.com) to confirm the results. Four peptides withthe highest scores and/or with shared sequences among the Hsps will be synthesized, andtheir affinity for HLA-A0201will be confirmed in a functional peptide-binding assay usingthe HLA-A0201+T2hybridoma (11). The two best peptides from each of the Hsps will beused to generate peptide-specific CTLs from HLA-A0201+blood donors.HLA-A-0201-peptide tetramers will be constructed to monitor the T cells.Results: Four peptides from each of the Hsps based on the highest scores obtained with theNIH/BIMAS system,are aa27from Hsp27with the sequence of RLFDQAFGL,aa393fromHsp70with the sequence of LLLLDVAPL,aa362from Hsp90α with the sequence ofKLYVRRVFI,aa670from Hsp90α with the sequence of ALLSSGFSL, which were also (in most of the cases) the best-scored peptides by the SYFPEITHI system.These peptides arebeing synthesized. We have used the functional peptide-binding assay to determine two ofthe four peptides,aa27and aa670should be used to generate CTLs.We also have found thehigh level of Hsp27-CTLs and Hsp90α-CTLs in patient by HLA-A0201-peptidetetramers,which confirmed that the aa27and the aa670exist in physiological state.Conclusion:The two best peptides from each of the Hsps are aa27from Hsp27and aa670from Hsp90α,and we have detected the Hsp-CTLs in patient with multiple myeloma byHLA-A0201-peptide tetramers. Part II Examine in vitro cytolytic effects of the CTLs on myeloma cells, including cell linesand primary myeloma cells from patients, and on normal hematopoietic cells.Objective:To examine in vitro the cytolytic effects of the CTLs on myeloma cells, includingcell lines and primary myeloma cells from patients, and on normal hematopoietic cells.Methods: The two best peptides from each of the Hsps will be used to generatepeptide-specific CTLs from a HLA-A0201+blood donor, Peptide-pulsed autologousdendritic cells (DCs) will be used as antigen-presenting cells (APCs). HLA-A0201-peptidetetramers will be constructed to monitor the T cells. Hsp-CTLs will be tested for theircytolytic activity against a panel of myeloma cell lines and primary myeloma cells isolatedfrom patients, whether or not they are HLA-A0201+. Other properties of these T cells, suchas the cytokine-section profile, MHC restriction, and surface markers, will also be analyzed.We used T-cell proliferation assays to monitor a specific T-cell response.Results: Mature DCs were generated as described in the Methods, and a27-or a670pulsedDCs were cocultured with autologous T cells from one HLA-A0201donor. We havecultured T cells with peptide-pulsed DCs for4weeks and we found the percentage oftetramer increasing from less1%to more and more than20%,also these hsp-CTLs display adistinct phenotype from unstimulated T cells recognized by high level of CD45RO and thelow level of CD45RA.These Hsp-CTLs show specific cell proliferation in response to HLA-A0201+myeloma cell line or HLA-A0201+primary myeloma cells.However,theHsp-CTLs did not proliferate in response to the MHC mismatched LP1cell line.TheHsp-CTLs can also induce specific lysis of HLA-A0201+myeloma cell lines and theHLA-0201+primary myeloma cells but have no effect on the HLA-A0201+normal PBMC orHLA-0201-myeloma cell lines.The Hsp-CTLs which incubated with HLA-A0201+myeloma cell line ARH-77displayed increased CD107a expression on CD8+T cells.Incontrast,unstimulated T cells from donor showed no expression of CD107a on CD8+cells inresponse to stimulation with the HLA-A0201+myeloma cell line ARH-77.The Hsp-CTLsexpressed high levels of perforin in response to HLA-A0201+myeloma cell line U266.Conclusion:The two Hsp-derived peptides can be used to generate specific CTLs fromhealthy blood donor.The Hsp-CTLs showed CTL-mediated cytotoxicity to MHC matchedprimary myeloma cells or myeloma cell lines.The Hsp-CTLs killed the MHC matchedmyeloma cells via the perforin pathway,because they expressed high levels of perforin. PartⅢ Determine the in vivo effects of the CTLs on primary myeloma cells using amyeloma-SCID-rab host system.Objective: Determine the in vivo effects of the CTLs on primary myeloma cellsMethods: CB.17/ICr-SCID mice(6-8-week old) were obtained from Vital Rive and pregnantNew Zealand rabbits from Sheng Wang.The femora and tibiae of3-4-week-old rabbits werecut inserted subcutaneously through a small incision.The engraftment of the bones wasallowed to tak place for6-8weeks.3-10×106purified PCs in100ul of phosphate-bufferedsaline(PBS) were injected directly into the implanted rabbit bone.At least two mice wereused for each experimenting.Mice were periodically took X-ray images and bled from thetail vein to judge the time tumor begin to grow. The treatment consisted of three weeklyinjections of10×106CTL cells. The survival was calculating when injected PCs and gets theend point when the tumor reached225mm2. Mice were deeply anesthetized and euthanizedby cervical dislocation,the tumor was taken to have immunohistochemistry andhistochemistry research. Results：Four SCID-rab mouse in nine burden myeloma tumor after3-4weeks.Tumor burdenSCID-rab mouse have shorter survival comparing with the tumor free SCID-rab mouse.Conclusion: The purified primary multiple myeloma cells can form tumor in the SCID-rabmouse.更多还原