【作者】 王龙信；

【导师】 王禾； 秦卫军；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 目的:细胞毒性T淋巴细胞(CD8+CTL细胞)是一类具有CD8+表面标志、受MHC I类分子限制性杀伤功能的T淋巴细胞,其重要功能是可以特异性地杀伤靶细胞,在肿瘤免疫中,体内杀伤肿瘤的最终效应细胞CD8+T淋巴细胞,但其杀伤肿瘤的能力经常被肿瘤分泌的免疫抑制因子所抑制,如转化生长因子-β(TGF-β)。在本研究中,我们应用TGF-β不敏感的CD8+T淋巴细胞过继性输注至肾癌个体化荷瘤免疫重建重度免疫缺陷(SCID)鼠体内,来评价其对肾癌的治疗效果。我们应用肿瘤裂解物负载的人树突状(DC)细胞,与CD8+T淋巴细胞共培养以激发其杀伤活性,用包含显性负相II型TGF-β受体(TβRIIDN)基因的逆转录病毒感染CD8+T淋巴细胞,使其对TGF-β信号通路不敏感。检测转染后CD8+T淋巴细胞表型变化及体外杀伤作用,将这种TGF-β不敏感的CD8+T淋巴细胞过继性输注至免疫重建后的个体化肾癌荷瘤SCID鼠体内,评价其抗肿瘤免疫治疗作用及机制。方法:1)构建含有显性负相TGF-βII型受体(TβRIIDN)质粒的逆转录病毒:含有TβRIIDN质粒的逆转录病毒转染293包装细胞,32℃下共转染12小时,10% DMEM培养基37℃下孵化过夜,PBS漂洗后同样条件再次转染293细胞24小时,收集上清,获得含有TβRIIDN重组逆转录病毒。2)人肾癌原代细胞系的建立:无菌条件下将新鲜切取的肾癌组织剪成1.0cm3小块,包埋于裸鼠双侧腋下,4周后肿瘤组织生长至2-3cm3大小时,连续传代3次以上后取组织块培养,用胶原酶II及胰酶消化,得到上皮样肿瘤细胞,连续传代50次以上,得到稳定传代的肾癌原代细胞系。3)SCID-beige鼠免疫重建(Hu-PBMC-SCID):取患者外周血5-10ml,用淋巴细胞分离液分离外周血单核细胞(PBMC),用PBS重悬成0.3ml细胞悬液,腹腔注射至6-8周龄SCID-beige鼠体内,注射前SCID鼠接受剂量为3.5 Gy的钴60照射,4周后尾静脉取血,酶联免疫吸附法(ELISA)检测血清中人IgG含量。4)检测肾癌组织及肾癌原代细胞系中TGF-β1表达:用免疫组织化学及免疫荧光方法检测肾癌手术标本、裸鼠成瘤组织以及肾癌细胞系中TGF-β1表达。5)肿瘤特异性CD8+T淋巴细胞的分离培养与激活:取肾癌患者外周血,分离获得PBMC细胞,加入含rhIL-4(1000U/ml)、GM-CSF(1000U/ml)的完全培养基,隔日半量换液,在第6d梯度离心收获非粘附细胞为非成熟的DC细胞。反复冻融法获得肾癌细胞裂解物,裂解物反复刺激冲击非成熟的DC细胞,同时用免疫磁珠法分离外周血获取CD8+T淋巴细胞,将负载肾癌抗原的DC细胞与CD8+T淋巴细胞,同时加入rhIL-2(500U/ml)共培养,获得肿瘤特异性CD8+T淋巴细胞。6)肿瘤特异性CD8+T淋巴细胞转染:获取TGF-β不敏感CD8+T细胞(TβRIIDN CD8+T细胞):含有TβRIIDN和GFP基因重组逆转录病毒转染负载肾癌抗原的CD8+T细胞5%CO2、37℃下孵化48小时,获得表达TGF-β不敏感CD8+T细胞,流式细胞仪检测CD8+T淋巴细胞表型CD27,CD45RA表达。7)TβRIIDN- CD8+T淋巴细胞体外杀伤实验:将未经活化的CD8+T淋巴细胞(na?ve CD8+T淋巴细胞)以及TβRIIDN CD8+T淋巴细胞与相关肾癌细胞系共培养,51Cr释放实验测定其细胞杀伤作用,人前列腺癌细胞系PC-3作为对照靶细胞。8)TβRIIDN- CD8+T淋巴细胞体内抗肿瘤评价:建立肾癌细胞系皮下荷瘤免疫重建SCID-beige鼠模型,免疫重建SCID-beige鼠15只,随机分为3组,每组5只,皮下注射肾癌细胞5×106个,注射14天后肿瘤长至2-3mm时,分别腹腔注射TβRIIDN- CD8+T细胞(1×107),na?ve CD8+T细胞(1×107),以及PBS(0.5ml),一周后重复注射一次;同时建立肺转移Hu-PBMC-SCID鼠模型,Hu-PBMC-SCID 15只,随机分为3组,每组5只,分别尾静脉注射肾癌细胞5×106个,于注射后第7天分别腹腔注射TβRIIDN- CD8+T细胞(1×107),na?ve CD8+T细胞(1×107),以及PBS(0.5ml),一周后重复注射一次,观察至第40天各组荷瘤小鼠的肿瘤体积,重量的改变及生存情况,同时用TUNEL法检测肿瘤组织凋亡情况。9)小鼠体内细胞因子检测:尾静脉取小鼠血清行酶联免疫吸附(ELISA)测定细胞因子IFN-γ变化情况。10)肿瘤组织凋亡检测:取肿瘤组织,用原位末端标记法(TUNEL)检测肿瘤凋亡结果:1)建立了5例肾癌原代细胞系:共取68例肾癌手术标本接种裸鼠,5例肿瘤标本在裸鼠体内生长并可在体外稳定传代超过100次,手术接种成功率7.3 %。2)肾癌组织及细胞系中TGF-β1表达:TGF-β1在肾癌组织及细胞系的胞浆及细胞膜中呈强表达。3)Hu-PBMC-SCID鼠血清中人免疫球蛋白(IgG)的含量:110只用患者PBMC进行免疫重建的SCID-beige鼠中,75只可以检测到IgG表达,免疫重建成功率68.2%,IgG表达水平在0.8-2.2mg/ml之间,各组表达水平无明显统计学差异(P>0.05)。4)流式细胞仪分析:免疫磁珠分离CD8+T淋巴细胞纯度为98%。对GFP荧光分析提示TβRIIDN和GFP的逆转录病毒转染效率分别为92.13%和91.95%。对两种CD8+T淋巴细胞表型分子CD27,CD45RA的分析提示TβRIIDN CD8+T细胞优势表达CD27+CD45RA-(78.6±6.7%),为效应性T细胞表型,而na?ve CD8+T细胞优势表达CD27+CD45RA+(60.5±16.2%),为未活化T细胞表型。5)51Cr释放实验测定显示TβRIIDN CD8+T淋巴细胞对肾癌细胞杀伤活性较na?veCD8+T细胞强,在E:T为100:1时,其杀伤活性为75.5%,而na?veCD8+T细胞仅为15.8%。两种CD8+T淋巴细胞对人前列腺癌细胞PC-3无明显杀伤作用,结果表明阻断的TGF-β信号通路能增加CD8+T细胞肿瘤特异性杀伤活性。6)在成功构建的皮下荷瘤的SCID鼠模型分别接种两种CD8+T细胞,TβRIIDN CD8+T细胞治疗组肿瘤组织重量和体积均较na?veCD8+T细胞治疗组有明显差异(p<0.001),而在小鼠肺转移模型中,TβRIIDN CD8+T细胞治疗组其肺转移灶明显少于na?veCD8+T细胞治疗组,其生存期也明显延长(p<0.05)。TUNEL检测发现TβRIIDN CD8+T细胞治疗组多数肿瘤细胞发生凋亡,而na?veCD8+T细胞治疗组并未发现明显凋亡。这些结果说明TGF-β不敏感的CD8+T细胞可以发挥其特异性抗肿瘤活性,提高肾癌皮下荷瘤及肺转移小鼠的生存率,降低肺转移率。7)接种两种CD8+T细胞的荷瘤小鼠体内可以测出IFN-γ基础水平,接种TβRIIDN CD8+T细胞其荷瘤小鼠体内IFN-γ水平升高更为明显(P<0.05)。结论:1)成功构建了含有TβRIIDN质粒的逆转录病毒;2)成功建立了5例肾癌原代细胞系,发现肾癌中存在有TGF-β1高表达。3)成功用患者外周血单核细胞(PBMC)对SCID-beige鼠进行人免疫系统重建,并可检测到人IgG表达。4)用肾癌细胞裂解产物作为抗原负载DC,与CD8+T淋巴细胞共培养,诱导出了肾癌特异性的CD8+T淋巴细胞;5)使用修饰后TβRII基因转染肾癌特异性的CD8+T淋巴细胞,使TβRII显性负相表达,阻断TGF-β信号通路;6)TβRIIDN CD8+T淋巴细胞呈效应性T细胞表型;7)TGF-β不敏感的CD8+T淋巴细胞能明显抑制肾癌皮下荷瘤小鼠肿瘤生长,并提高肺转移小鼠的生存率,诱导肿瘤凋亡。8)TGF-β不敏感的CD8+T淋巴细胞具有肿瘤特异性杀伤靶细胞作用。9) TGF-β不敏感的CD8+T淋巴细胞输注的荷瘤小鼠体内IFN-γ水平明显升高。更多还原

【Abstract】 ObjectiveCD8+ T lymphocyte cells are the T cells which have the CD8+ phenotype,MHC-I restricted cytotoxic function. In tumor immunotherapy,the functions of CD8+ T lymphocyte cells are often suppressed by the transforming growth factor-β(TGF-β) . TGF-βis a potent immunosuppressant. Adoptive transfer murine-derived, tumor-reactive, TGF-β-insensitive CD8+ T cells into tumor challenged mice has shown potent antitumor responses. The present study was conducted to a one-to-one adoptive transfer strategy to treat tumor-bearing severe combined immunodeficient (SCID/beige) mouse. The SCID/beige mice were humanized with PBMC from renal cell carcinoma (RCC) patients (Hu-PBMC-SCID) before adoptive transfer. Autologous CD8+ T cells were expanded ex vivo with using autologous patient’s dendritic cells (DCs) pulsed with the tumor-lysate and rendered TGF-β-insensitive by dominant-negative TGF-βtype II receptor(TβRIIDN). Flow cytometry analysis showed the TGF-β-insensitive CD8+ T cells were the effector CD8+ cells (CD27-CDRA+). Then, adoptive transfer autologous TGF-β-insensitive CD8+ T cell into tumor-bearing Hu-PBMC-SCID mice can induce powerful tumor-specific cytotoxic T lymphocyte (CTL) responses, induced tumor apoptosis, suppressed lung metastasis and prolonged survival times. This one-to-one adoptive transfer strategy provides a scientific rationale for expected clinical investigation in the treatment of renal cell carcinoma.Materials and methods1. Production of infectious TβRIIDN-GFP retrovirusTβRIIDN was excised from pcDNA3-TβRIIDN by BamHI/EcoRI digestion and inserted into the pMig-inteRIIDNl ribosomal entry sequence-green fluorescence protein (herein designated MSCV-GFP) vector by first linearizing pMig with EcoRI and ligating an EcoRI/BamHI adapter (5′-AATTGGATCCGCGGCCGCG-3′, 3′-CCTAGGCGCCGGCGCTTAA-5′). These clones were designated as MSCV-TβRIIDN and were screened by sequencing for correct orientation and insert numbers.Pantropic GP293 retroviral packaging cells (Clontech, San Diego, CA) were seeded at a density of 2.5×106 per T-25 collagen I-coated flask (Biocoat; BD Biosciences, Mountain View, CA) and incubated for 24 h before plasmid transfection in antibiotic-free 10% DMEM (Gibco, Grand Island, NY). A mixture of 2μg retroviral plasmid and 2μg vesicular stomatitis virus envelope G protein (VSV-G) envelope plasmid was cotransfected in serum-free DMEM by using Lipofectamine-Plus (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocols. Briefly, the cells were transfected for 12 h followed by the addition of an equivalent volume of 10% DMEM and incubation for an additional 12 h. The supeRIIDNtant was then aspirated, the cells were rinsed gently in PBS, and 3 ml fresh 10% DMEM was added to each flask. After 24 h, the virus-containing supeRIIDNtant was collected and used to infect target cells.2. Establish five renal cell carcinoma cell linesSpecimens were obtained from 68 patients who were diagnosed with RCC and underwent radical nephrectomy between September 2005 and December 2007.The study protocol was approved by the Ethics Committee of the Xijing Hospital, Fourth Military Medical University. Informed consent was obtained from all participants.Total of 68 fresh RCC tissues were obtained after operation and implanted subcutaneously to nude mice as described previously .Five kinds of RCC specimens remained engrafted successfully 1 month after transplantation. Histological section from each initial tissue and xenograft specimen was subjected to hematoxylin and eosin (H&E) analysis. Then, five kinds of human RCC cell lines were obtained from the xenograft transplanted in the nude mice. These RCC cell lines was maintained in a complete medium (CM) containing RPMI-1640 medium (HyClone, Logan, UT) supplemented with 10% heat-inactivated fetal bovine serum (FBS; GIBCO, Gaithersburg, MD), 2 mmol/L L-glutamine, 50μmmol/L 2-mercaptoethanol, 100 units/ml penicillin, and 100μg/ml streptomycin (Sigma, St Louis, MO).3. Establish the Hu-PBMC-SCID mice modelMale or female SCID/beige mice 6-8 weeks old were obtained from the Laboratory Animal Research Center of the Fourth Military Medical University and housed in sterile filter-top caged placed in a laminar backflow-cabinet under specific pathogen-free conditions. The SCID-beige mice were divided medially with five groups received PBMCs injection from five patients. One day before PBMCs injection, mice were sublethally irradiated with 3.5 Gy ([60Co] source Gammatron F 80S, Simens, Germany). Autologous PBMCs purified from each patient’s blood using a Ficoll-HyPaque (Pharmacia, New Jersey, USA) gradient after platelet depletion and washing, each mouse received 0.3 ml of the PBMCs (2×107cells) suspended in PBS via intra-peritoneal injection .4. Immunohistochemical and immunofluorescence analysis for TGF-β1 expression in RCC tumor tissues and cell linesFive kinds of patient’s RCC original and xenograft specimens were performed by immunohistochemical analysis for TGF-β1 expression. Briefly, Paraffin-embedded sections (4μm) were deparaffinized and rehydrated. After quenched endogenous peroxidase and blocking step performed, primary anti-TGF-β1 mAb and goat-anti-mouse second antibody (anti-TGF-β1 mAb, 1:100; second antibody 1:500; Abcam Biotechnology, Cambridge, UK) were incubated. Peroxidase substrate solution 3, 3’-diaminobenzidine was used for direct staining. Counter-staining was done with 10 % hematoxylin. Nonimmune murine antibody was used for negative control sections. For immunofluorescence analysis, cells were incubated with TGF-β1 mAb for 2h, stained with FITC-conjugated anti-mouse IgG (1:1000, Abcam Biotechnology) for 1h. The nuclei were stained with 100 ng/ml DAPI (4’,6-diamidino-2-phenylindole) and were examined by fluorescence microscopy (Nikon Corp., Tokyo, Japan).5. Generation of patient autologous tumor reactive TGF-β-insensitive CD8+ T cellsWith the use of CD8+ Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), patient’s CD8+ T cells were positively selected from whole blood with a purity of more than 98 %. CD8+ T cells was expanded with using autologous patient’s DCs pulsed with the tumor-lysate in the presence of recombinant human interleukin-2( IL-2;500 unit/ml, PeproTech, London, England).There were two types of CD8+ T cells:1) tumor-reactive TGF-β-insensitive CD8+ T cells which rendered insensitive to TGF-βby infection with TβRIIDN-green fluorescent protein (GFP)–containing retrovirus. 2) naive CD8+ T cells isolated from PBMC without any treatment.6. Flow cytometric analysis TGF-β-insensitive CD8+ T cell characterizationImmunophenotypical characterization of TGF-β-insensitive CD8+ T cell and naive CD8+ T cell were performed using fluorescein isothiocyanate (FITC)-conjugated anti-CD27mAb, anti-CD45RA mAb before administration to the mice. Cells stained with appropriate mAbs in PBS, 0.2% BSA, 50μM EDTA for 20 min at 4 0C and either directly analyzed or sorted into defined populations on a FACSVantae SE, using CellQuest software (BD Bioscience). 7. 51 Chromium release assaysThe two types of CD8+ T cells were subjected to a standard 51Chromium- release assay. RCC cell lines and another irrelevant cell line, human prostate carcinoma cell line, PC-3 cells were used as targets. Briefly, target cells were labeled with 100μCi 51Cr/105 cells. Different groups of CD8+ T cells were added to U-bottom plates containing 5,000 cells /well with various effectors to target (E/T) ratios ranging from 1:1 to 100:1. Equal volumes of RPMI-1640 and 1 mol/L HCl were added to other wells as the negative and positive controls, respectively. After a 4-hour incubation, 100μl of supernatants was harvested from each well and the 51Cr released was measured using a gamma-counter. The percent cell lysis was calculated according to the formula: percent specific 51Cr-Release= (Experimental Release– Spontaneous Release)×100/ (Maximum Release– Spontaneous Release)].8. Adoptive transfer TGF-β-insensitive CD8+ T cell in tumor bearing Hu- PBMC-SCID miceThe Hu-PBMC-SCID mice received an injection in the right flank of 5×106 RCC patient’s autologous tumor cell line (Day 0).Tumors developed approximately 2-3 mm in diameters 14 days later. At day 14, adoptive transfer with patient’s autologous TGF-β-insensitive CD8+ T cells were done in the tumor-bearing Hu-PBMC-SCID mice. Three groups (5 mice per group) received intraperitoneal injection with different types of adoptive transfer composed of TGF-β-insensitive CD8+ T cells (1×107), naive CD8+ T cells (1×107), PBS (0.5ml), respectively. The vaccination was repeated on day 21.Tumor growth and animal survival was monitored daily after vaccine.The mice pulmonary metastasis model was also prepared by a single injection of 5×106 RCC in the tail vein. On day 7,the tumor-bearing mice (n=5/group)were inoculated with two types of CD8+ T cells vaccines (1×107 cells) and PBS via intraperitoneal injection, respectively. Forty days after adoptive transfer, all mice were sacrificed and the tumors were isolated for evaluation of the volume (volume=length×width2×π/6), weight and histological analysis. Other tissues such as spleen, pulmonary were also harvested.9. ELISA assay for INF-γThe sera of the 3 above mentioned mouse groups were harvested. The serum levels of INF-γwere determined using an ELISA kit (R&D Systems, Minneapolis, MN) according to the protocol. Serum was stored at -70℃until the assay.10. TUNEL staining for tumor apoptosisParaffin-embedded tumor sections were used for apoptosis assay. The nuclear and terminaldeoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) apoptosis assay kit(R﹠D system, Minneapolis, MN) were done as described previously11. Statistical analysisNumerical data were expressed as mean±standard deviation (SD). ANOVA and chi-square tests were performed to determine the differences in the means among the various treatment groups. P < 0.05 was considered statistically significant. The SPSS 12.0 software package (SPSS Inc., Chicago, IL) was used for analysis. The Kaplan-Meier survival curve was analyzed by the log-rank test with the Graphpad Prism 4.02 software (Graphpad Software Inc., San Diego, CA).Results1. Establishment and characterization of RCC cell lines Five kinds of human RCC cell lines were established successfully from specimens inoculated nude mice. All RCC cell lines stably cultured more than 100 passages after cryopreserved and thawed. The histological analysis of xenograft in mice was similar to the histological evaluation of the original patient tissue specimen2. TGF-β1 expression in RCC xenograft in mice and cell linesA representative result of the immunohistochemistry for TGF-β1 in xenograft in mice is shown in Fig.2A-B.In these cancerous tissues, TGF-β1 was found strong expression either in the cytoplasm or on the plasma membrane of the neoplastic cell. This result was also confirmed by immunofluorescent staining in RCC cell lines.3. Appearance of human immunoglobulins in Hu-PBMC-SCID miceFour weeks after PBMCs injection, human immunoglobulins could be detected in 75 of 110 (68.2%) Hu-PBMC-SCID mice sera. The IgG levels of each group averaged between 0.8-2.2 mg/ml which in agreement with results of previous studies.There were no significant differences in the success rate of PBMC engraftment and the levels of IgG were similar between each group of mice (P>0.05). Furthermore, no severe xenogenic graft versus host disease (GVHD) was observed.4. Phenotypic analysis of TGF-β-insensitive and naive CD8+ T cellsFlow cytometry analysis showed that the expression of co-stimulatory molecules CD27 and CD45RA in two types of CD8+ T cells were different. In the TGF-β-insensitive CD8+ T cells, the dominant phenotype was the CD27+CD45RA-(78.6±6.7%),the effector CD8+ T cell phenotype. But in naive CD8+ T cells, most of the cells showed the CD27+CD45RA+ phenotype(60.5±16.2%), the unprimed CD8+ T cell phenotype.5. TGF-β-insensitive CD8+ T cells showed superior anti-tumor responses in vitroThe specific tumor-killing ability of the autologous TGF-β-insensitive CD8+ T cells was shown by the in vitro CTL assay. We found that the TGF-β-insensitive CD8+ T cells displayed 5-fold greater tumor-killing activity than the na?ve CD8+ T cells((75.5% vs 15.8% at an E:T cell ratio of 100:1,).When incubated with an irrelevant cell line, PC-3 cell, no apparent lytic activity was observed.6. TGF-β-insensitive CD8+ T cells showed superior anti-tumor responses in vivo In the group treated with TGF-β-insensitive CD8+ T cells ,the average tumor volumes and tumor weights was significantly decreased than the group treated na?ve CD8+ T cells and PBS group (P<0.05). Interestingly, in the mice with pulmonary metastasis, all the animals died in the PBS treated group before day 30, there were 4 of 5 mice died in the na?ve CD8+ T treated group before day 32 of the experiment due to poor health conditions, while all the mice survived in the TGF-β-insensitive CD8+ T cell treated group at the end of the experiment. According to the long-rank test, there were significant differences among three groups (p<0.05).7. TGF-β-insensitive CD8+ T cells induced high serum levels of INF-γThe serum levels of INF-γexhibited increases in the TGF-β-insensitive CD8+ T cells treated group, but in the na?ve CD8+ T cell and PBS treated group, the expression levels of INF-γwere negligible. The differences of INF-γserum levels between TGF-β-insensitive CD8+ T cells, na?ve CD8+ T cell group and PBS group were significant (Fig.7. p<0.01). The greater increase in levels of serum INF-γobserved in the TGF-β-insensitive CD8+ T cell treated group indicated that immune cells were most strongly activated in these hosts.8. TGF-β-insensitive CD8+ T cell induced tumor cell apoptosisThe TUNEL assay confirmed that autologous TGF-β-insensitive CD8+ T cell could induce tumor cell apoptosis in the TβRIIDN group. But in the other two groups, no apparent apoptotic cells were observed in tumor tissues.Conclusion1. We successfully constructed a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN).2. We successfully established five RCC cell lines and detected the high expression of TGF-β1 in RCC.3. Established the Hu-PBMC-SCID mouse models successfully,and found human immunoglobulins expression in these mice.4. Incubated with tumor lysate loaded-DCs pulsed with CD8+T cells can induce and activate RCC-specific CD8+T cells.5. RCC-specific CD8+T cells were rendered TGF-βinsensitive by infecting with a retrovirus containing dominant-negative TGF-βtype II receptor (TβRIIDN), leading to the blockade of TGF-βsignals to members of the Smad protein family.6. The TGF-β-insensitive RCC-specific CD8+T cells were the effector CD8+T phenotype .7. TGF-βinsensitive TP- CD8+T cells suppressed tumor growth ,induced tumor apoptosis,and increased survival rate of pulmonary metastases mice.8. The most potent CTL response was induced by the TGF-?-insensitive CD8+T cells in vitro (75.4% killing activity at an effector:target cell ratio of 100:1). No apparent lysis was observed against irrelevant PC-3 cells.9. TGF-βinsensitive TP- CD8+T cells induced higher IFN-γlevel in vivo.更多还原