摘要
据统计2006年美国约40000例肾肿瘤的新发病例被确诊，大约13000例患者(包括成人和儿童)死于肾肿瘤。肾细胞癌是肾肿瘤中最常见的类型，占肾脏恶性肿瘤的90％以上，晚期的转移性肾癌对放疗与化疗均不敏感，一小部分患者对IL-2和IFN-α治疗有效，但药物的毒副作用十分明显。研究表明，肿瘤的发生及发展与患者体内的T淋巴细胞功能缺陷有关，进一步的研究发现，导致此缺陷的原因在于机体对成熟树突状细胞(Dendritic cell，DC)的功能下调。在体外，很多实验证实即使是晚期肿瘤患者外周血分离出的淋巴细胞对多种外界刺激仍然保持着很好的反应能力。因此，通过体外培养DC，诱导特异高效的细胞毒性T淋巴细胞(CTL)，回输免疫患者，有望成为治疗肿瘤的绝佳疗法。而目前最需解决的问题是：如何在已知靶抗原的前提下，获得良好活性、足够数量的特异性CTL?
DC是人体内功能最强大的抗原递呈细胞。如能将肿瘤抗原成功加载DC，可诱导抗原特异的CTL。目前，采用粒细胞一巨噬细胞集落刺激因子(Granulocyte-macrophage colony stimulating factor，GM-CSF)和白介素-(Interleukin-4，IL-4)已能成功从外周血单个核细胞培养出DC。但是，如何能够有效地将抗原导入DC?又如何有效利用DC诱导T淋巴细胞产生CTL?
研究表明：不同的抗原加载方式，对DC的活化效率不同，产生CTL的活性也不一致。目前抗原加载存在着多种手段，常用的策略有：抗原肽致敏DC及病毒载体携带抗原基因转染DC两种方式。
采用肿瘤抗原肽致敏DC虽有抗原比较特异，可多次免疫等优点，但存在的主要问题是：这类抗原肽往往半衰期较短，因此需要多次免疫方可获得良好活性的CTL，而且转导效率低下。
病素载体携带抗原肽基因转染DC，可使基因在DC体内稳定表达，从而使DC获得持续而大量的抗原肽刺激，有望解决单纯抗原肽转导存在的问题。目前转染DC常用的病毒载体有：逆转录病毒，腺病毒和慢病毒等。腺病毒载体(Ad)是常用的病毒载体之一。腺病毒载体是5型腺病毒基因重组而成的，由于其E1区的缺失，致使病毒的复制缺陷，不会引起宿主细胞的损伤或病毒扩散。展望与其它病毒载体的不同，腺病毒载体具有许多独特的优点：①宿主范围广，对人致病性低；②在增殖和非增殖细胞中感染和表达基因；③能有效进行增殖，滴度高；④不整合到染色体，无插入突变性；⑤能同时表达多个基因。由于腺病毒载体具有诸多优点，因此在研究中越来越多地被采用。
在前期研究的基础上，设计了以下三部分研究：(1)以肾肿瘤相关抗原G250蛋白冲击DC诱导特异的CTL，研究其对肾癌细胞株的杀伤情况。(2)以Ad为载体，将G250基因转染DC诱导CTL，研究其对肾癌细胞株的杀伤情况，并与G250蛋白诱导组作比较分析。(3)转染Ad／G250的人树突状细胞诱导CTL抑制人肾癌细胞系786—0裸鼠移植瘤发生与生长。通过以上三部分研究，探讨以Ad为载体，将肾癌抗原基因转染DC并诱导CTL，用于治疗肾癌的可行性，在此基础之上，优化制备工艺，为临床试用于抗原明确的肿瘤治疗奠定实验室基础。
第一部分G250蛋白致敏的DC瘤苗体外诱导特异性抗肾癌免疫
1研究方法：
1．1人外周血树突状细胞的分离培养及致敏
取健康者外周血50ml，肝素抗凝，用淋巴细胞分离液(1.077 Ficoll)分离获得单个核细胞(PBMC)，用AIM-V培养基制成细胞悬液，加入6孔培养板(1ml／L)中，在37℃，5％CO_2条件下培养4～6h，收集悬浮细胞(冻存备用)。在培养板中加入AIM-V+GM-CSF(终浓度为800U／ml)，每孔3ml，隔日半量换液，加入IL-4(终1000U／ml)，于第5d将DC分为2组，一组加入G250蛋白(浓度为750ug／L)，每孔100ul，另一组加PBS，两组同时加入TNF-α(终200U／ml)，37℃，5％CO_2继续培养3d。
1．2 DC表面标志的表达检测
将培养第8d的两组DC收集，计数。取两组DC，5×10~5／组，FITC-CD1a、PE=CD83、FITC-CD40、FITC-CD80、PE-CD86、PE-HLA-DR两两组合放入一管中标记DC，细胞用PBS洗涤2次，然后加入单抗10ul，4℃冰箱中避光反应30min，加入PBS 0.5ml，震荡悬浮细胞后上流式细胞仪分析。
1．3混合淋巴细胞反应的检测
取培养第8d的两组DC(一组为DC-G250；一组为DC-N)行混合淋巴细胞反应实验。将DC用30ug／ml的丝裂霉素，37℃作用30min，用PBS洗3次。冻存的自体T淋巴细胞用含IL-2(100U／L)和10％的人AB血清的RPMI-1640稀释成1×10~6／ml，加入96孔培养板(100ul／孔)。按1：5、1：10、1：20效靶比分别加入上述两组DC，同时设对照组(加入RPMI-1640培养液)，每组3个复孔，37℃，5％CO_2培养箱中孵育5d；然后加入5mg／ml的MTT 20ul／孔继续培养4h，加入二甲基亚砜(DMSO)100ul／孔。在酶标仪上测570nm波上的OD值。
1．4细胞毒性T淋巴细胞杀伤活性试验
取培养第8d的两组DC：一组为G250蛋白致敏组(DC-G250)，另一组为未致敏组(DC-N)。两组DC分加与自体淋巴细胞按1：20比例混合，以含人IL-2(终10U／ml)、GM-CSF(终800U／ml)、IL-4(终1000U／ml)、IL-7(终20ng／ml)的RPMI-1640培养液培养，5d后即获得两组CTL。收集共培养5d的两组CTL(第一组为T+DC-G250，第二组为T+DC-N)作为效应细胞，取两组的部分T(5×10~6／组)与FITC、PE、CY标记的单抗染色，流式细胞仪行细胞表型分析；分别以786-0(人肾透明细胞癌细胞系)和A549(肺癌细胞系)为阳性和阴性靶细胞，加入效应细胞，组内设定不同的效靶比观察孔，效靶比为5：1、10：1、20：1、40：1；每种效靶比设3个复孔，继续在37℃，5％CO_2培养箱中孵育44h，然后加入5mg／ml的MTT 20ul／孔，混匀，继续培养4h，每孔加入二甲基亚砜100ul，在酶标仪上测570hm处OD值。通过所测OD值，以及单纯效应细胞和单纯靶细胞为阴性对照所测OD值，利用公式算得CTL的杀伤活性。CTL杀伤活性=靶细胞对照组OD值-(实验组OD值-效应细胞对照组OD值)／靶细胞对照组OD值。
1．5统计学处理采用SPSS13.0软件进行分析，统计方法采用析因设计资料方差分析，LSD法进行多重比较，数据以均数±标准差表示，P≤0.05被认为具有统计学意义。
2研究结果
(1)．G250蛋白致敏组与未致敏(对照)组均高表达CD80、CD83、CD86、CD40、HLA-DR，较低表达CD1a。(2)．两组DC均能刺激T淋巴细胞的增殖，DC-G250组和DC-N组之间差异有统计学意义(F=83.545，P=0.000)；DC-G250组刺激T淋巴细胞增殖能力强于DC-N组；不同浓度效靶比之间有显著性差异，效靶比1：5时刺激增殖能力最强。(3)．DC-G250组和DC-N组所诱导的CTL表面标志CD3+和CD8+／CD3+差异有统计学意义(t=9.589 P=0.01；t=11.806 P=0.000)，DC-G250组高于DC-N组。(4)．DC-G250组所诱导的CTL对786-0杀伤率高于DC-N组所诱导的CTL，两组间差异有显著性意义(F=29.483，P=0.000)；两组效应细胞对A549的杀伤率无显著性差异。
3研究结论
肾癌相关抗原6250蛋白致敏的DC瘤苗体外可有效的诱导出具有良好抗肿瘤活性的CTL，说明了以G250蛋白冲击DC所获瘤苗具有较好的可行性和临床应用前景。为进一步以腺病毒为载体的G250基因转染DC诱导CTL治疗肾癌的研究打下了基础。
第二部分Ad／G250转染Dc诱导CTL治疗肾癌的研究
1研究方法：1．1 Ad／G250转染DC并诱导CTL
取健康人外周血，采用密度梯度离心的方法分离外周血单个核细胞(DC前体细胞)，培养于6孔板，贴壁4～6h，轻轻洗去悬浮细胞。将贴壁细胞分为三组：基因转染组、蛋白致敏组及对照组。基因转染组感染携带G250基因的重组腺病毒Ad／G250，感染5h后更换为新鲜AIM-V培养基和GM-CSF(终浓度为800U／ml)，其余两组用AIM-V培养基和GM-CSF培养。隔日半量换液，加入IL-4，终浓度为1000U／ml。第5d除加GM-CSF和IL-4外，蛋白致敏组加入G250蛋白(浓度为750ug／L)，每孔100ul，对照组加PBS溶液100ul／孔，三组同时加入TNF-α(终200U／ml)，37℃，5％CO_2继续培养3d。第8d，收集细胞，计数。取新分离的自体T淋巴细胞与培养所得的DC按DC：T=1：20比例混合，以AIM-V为培养基，同时加入GM-CSF(800U／ml)、IL-4(1000U／ml)、IL-2(10U／ml)及IL-7(20ng／ml)，共育5d，诱导获得三组细胞毒性T淋巴细胞(CTL)。
1．2 Ad／G250基因表达及DC表面标志检测
DC培养第8d，收集悬浮细胞(成熟DC)，显微镜观察细胞形态。基因转染组DC以RT-PCR扩增G250 mRNA产物，以对照组DC做对照。
流式细胞仪分析DC表面标志，表面标志选择了代表DC特征的：CD1a、CD80、CD83、CD86、HLA-DR等。
1．3 DC激发活的G250抗原特异性CTL抗肿瘤活性检测
激活的CTL细胞表面标志分析：在培养第13d，以带荧光标记的PE-anti-CD4、FITC-anti-CD8和PE-anti-CD56标记三组CTL，以流式细胞仪检测，从而得出三组CTL中CD8／CD4和CD8／CD56的比值。
MTT法检测CTL杀伤靶细胞的抗原特异性：以G250阳性及HLA-A2阳性的人肾癌细胞系786-0做为靶细胞，所有供血者的HLA-A2均与786-0是相匹配的；取G250阴性的人肺癌细胞株A549为靶细胞做阴性对照，采用MTT法检测所诱导的CTL对各类靶细胞的杀伤效率，分析CTL杀伤的抗原特异性。
2研究结果：Ad／G250高效转染DC，G250蛋白在DC内成功表达：采用RT-PCR成功在基因转染组DC中扩增出G250产物；Ad／G250转染的DC表面标志CD40、CD80、CD83、CD86、HLA-DR与其它两组相比有显著性差异，Ad／G250组明显高表达，表明Ad／G250基因转染组可上调DC表面标志表达；Ad／G250组诱导的CTL CD8／CD4、CD8／CD56的比值高于G250蛋白刺激组及对照组；Ad／G250组诱导的CTL对G250阳性的786-0靶细胞有很好的细胞毒杀伤效应，此杀伤明显优于G250蛋白致敏组及对照组。
3研究结论：
以Ad为载体介导抗原基因转染DC，并诱导特异的CTL，从技术上是可行的，而且所诱导的CTL杀伤活性好于采用G250蛋白冲击致敏DC所诱导的CTL，有望成为一种肿瘤免疫治疗的理想方法。此研究方法也说明了以G250为靶点的免疫治疗可能是。肾透明细胞癌一种理想治疗模式。
第三部分Ad／G250转染DC抑制786—0裸鼠移植瘤发生与生长
1、研究方法
1．1人外周血树突状细胞的分离培养及致敏
取健康者外周血50ml，肝素抗凝，用淋巴细胞分离液(1.077 Ficoll)分离获得单个核细胞(PBMC)，用AIM-V培养基制成细胞悬液，加入6孔培养板(1ml／L)中，在37℃，5％CO2条件下培养4～6h，轻轻洗去悬浮细胞。将贴壁细胞分为两组(基因转染组、蛋白致敏组)，基因转染组每孔加入Ad／G250病毒液100ul(病毒活性单位5.6×10~9 IU／ml)，感染5h后更换为新鲜AIM-V培养基和GM-CSF(终浓度为800U／ml)，蛋白致敏组用AIM-V+GM-CSF培养。分别于第1d和第3d，加入GM-CSF(终浓度为800U／ml)和IL-4(终1000U／ml)，隔日半量换液，第5d，两组中除加入GM-CSF和IL-4外，蛋白致敏组加入G250蛋白(浓度为750ug／L)，每孔100ul，两组同时加入TNF-α(终200U／ml)，37℃，5％CO_2继续培养3d。
1．2诱导细胞毒性T淋巴细胞(CTL)第8d，收集两组DC细胞，计数。以AIM-V为培养基，将两组DC与自体T细胞按DC：T=1：20的比例混合，除应用GM-CSF(终浓度800u／L)、IL-4(1000U／ml)，还加入IL-2(10U／ml)及IL-7(20ng／ml)，共育5d，获得两组细胞毒性T淋巴细胞(CTL)：一组为基因转染组(CTL-DC-Ad／G250)；另一组为G250蛋白冲击致敏组(CTL-DC-Pro／G250)。
1．3荷瘤裸鼠模型的建立
取裸鼠15只，收集对数生长期的786-0细胞，接种于裸鼠一侧肩颈部皮下，2×10~6／只，观察肿瘤发生时间及生长情况。肿瘤大小由肿瘤最大径与最小径的乖积(mm~2)来表示。
1．4 DC激活的CTL预防裸鼠人肾癌细胞系786—0移植瘤发生：
另外取15只裸鼠，随机分成三组，每组5只：第一组裸鼠一侧颈背部皮下注射基因转染DC诱导的CTL(2×10~6／只)；第二组裸鼠一侧颈背部皮下注射蛋白致敏DC诱导的CTL(2×10~6／只)，第三组裸鼠颈背部皮下注射生理盐水(200ul／只)作对照。3d后取对数生长期786—0细胞(2×10~6／只)接种于上述15只裸鼠另一侧颈背部皮下；每3d观察一次裸鼠移植瘤发生情况，直到接种后的第30d。
1．5 DC激活的CTL抑制裸鼠人肾癌细胞系786—0移植瘤生长：
1．3中接种786-0的裸鼠，接种后第16d，12只长出移植瘤，3只无移植瘤生长。接种后第21d，将成瘤裸鼠中肿瘤较大的两只和未成瘤的3只剔除本实验之外，入选的10只成瘤裸鼠移植瘤大小为8.6±1.2mm~2。将上述10只成瘤裸鼠随机分成两组：治疗组和对照组，每组5只。在治疗组，每只裸鼠另一侧背部皮下注射Ad／G250转染DC所诱导的CTL(2×10~6)作治疗；对照组注射生理盐水200ul。每3d观察一次移植瘤生长情况，肿瘤大小由肿瘤最大径与最小径的乖积(mm~2)来表示，直至接种后45d。
2结果
2．1 DC激活的CTL预防肿瘤发生基因转染DC诱导的CTL和蛋白致敏DC诱导的CTL预防裸鼠移植瘤发生实验中，在接种786-0肿瘤细胞后30d内两组裸鼠中均无移植瘤发生；而对照组于接种后第15d即可见移植瘤发生，观察至30d，5只中4只有移植瘤发生。
2．2 DC激活的CTL抑制移植瘤的生长
以Ad／G250转染DC诱导的CTL治疗成瘤裸鼠，因经费及时间所限，至接种后的第45d结束实验。接种后的第45d对照组、治疗组移植瘤大小分别为143.37±11.6 mm~2、42.55±5.62mm~2。结果显示Ad／G250转染DC所诱导的CTL对已生长的786-0移植瘤确有治疗作用，与对照组相比，治疗组移植瘤生长受到明显抑制(t=2.355，P=0.047＜0.05)。
3研究结论：
DC诱导的抗肿瘤免疫不仅能预防裸鼠人肾癌细胞系786-0移植瘤的发生，而且能抑制移植瘤生长。提示经G250基因转染制备的DC和G250蛋白冲击致敏的DC作为一新概念上抗肿瘤疫苗可能在肾肿瘤的预防及治疗中发挥重要作用。
Approximately 40,000 new cases of kidney cancer will be diagnosed and about 13,000 people, including adults and children, will die from this disease in 2006 in the United States. Renal cell carcinoma (RCC), the most common type of kidney cancer, accounts for more than 90% of malignant kidney tumors. Metastatic RCC (mRCC) has historically been refractory to conventional chemotherapy and chemotherapy. A minority of patients has shown some response to cytokine therapy with interleukin-2 (IL2) or interferon (IFN)-alpha, but associated toxicities can be severe. The studies demonstrated the occure and development of the cancer was related with the functional defection of T lymphocyte, and the further studies found the reason of which was the down-regulation of the mature dendritic cells (DC) by the body. In vitro, many experiments verified that the lymphocyte isolated from the advance stage cancer patient's peripheral blood still maintained very good reaction to the outside stimulation. So through culturing the DC in vitro and inducing the specific and high biology activity cytotoxic T lymphocyte (CTL), the injected back into the patient, which maybe becoming excellent therapy for the cancer. Therefore, if we know the target antigen for the cancer, how can we get the good biology activity and large amount specific CTL?
DC is the most effective Antigen Presenting Cell at activating naive T cells, which can activate antigen specific CTL with the antigen successful loading. At present, granulocyte-macrophaen colony stimulating factor (GM-CSF) and Interleukin-4 (IL-4) can promote the generation of large numbers of Dendritic Cells from peripheral blood monocytes. However, the focus point of the protocal in pulsing DC and inducing CTL in vitro is how to loading the antigen effectively?
The study showed: with different antigen loading technic, the DC activating efficiency is different. The CTL activity is also different. There are several ways to load the antigen, the popular strategy are: the antigen peptide transducing DC and virus vector carrying the antigen gene transfecting the DC.
Tuansducing DC with antigen peptide has advantages such as antigen peptide is specific and which can immunize times, but which also has one main problem is the half-life of this kind of antigen peptide usually us very short, so only after several times of immunization can we get CTLwith good activity. Moreover, the transduction efficiency is low.
Transfection DC with virus vector carrying antigen gene can make the DC produce a constant and large amount of antigen peptide, which can resolve the problem of antigen peptide transducing DC directly. At present, the common virus vectors for the DC transfection are: retrovirus (Retrov), adenovirus(Ad), et al. Ad vectors are the most currently used in clinical trails. Ad vectors are the Ad type 5 recombinant vectors which E1 region are removed. As we know, genes in the E1 region are necessary for activation of viral promoters and expression of both early and late genes. Thus, removal of the E1 coding sequence results in viruses that are severely impaired in their ability to replicate. Many features of adenovirus make it well suited for gene delivery, including the ability to grow recombinant viruses to high titers, a relatively high capacity for transgene insertion, and efficient transduction of both quiescent and actively dividing cells, usually without incorporation of viral DNA into the host cell genome. These characteristics, as well as the development of many methods for manipulating the viral genome, have made adenovirus a popular choice as a gene delivery vehicle. This is evidenced by the fact that adenovirus is currently being used in roughly one-quarter of all gene therapy clinical trials, making it second only to the use of retroviralvectors.
Three parts of gene therapy study using Ad as vector were designed on the base of pre-studies. The first part is: An in vitro study of specific antitumor immunity induced by dendritic cells pulsed with kidney tumor association antigen G250 protein. The second part is: Generating the CTL by G250 gene transfecting DC to treat kidney tumor. The third part is: Immue response induded by DC transfected by G250 gene in vitro inhibit growth of implanted tumor and reject challenge of tumor cells in nudes. Through them to study the possibility of kidney TAA G250 protein pulsing DC and G250 antigen gene transducing DC generating the CTL in kidney tumor treatment. Furthermore, we will optimize the protocol and make it as the experiment base for the future clinical trial in some kinds of cancer with clear antigen.
The first part: An in vitro study of specific anti-kidney cancer immunity induced by dendritic cells pulsed with G250 protein.
1. Methods
1.1 Generation of monocyte-derived DCs from peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Paque density gradient centrifugation of heparinized blood obtained from healthy donors and washed twice in PBS. The PBMCs were inoculated into six well culture plates at 37℃under a 5% CO_2 atmosphere for 4～6 hours, the adherent cells were incubated in AIM-V Medium containing rhIL-4 (final concentration of 1000IU/ml)and rhGM-CSF (final concentration of 800IU/ml), and the nonadherent cells was frozen as effective cell for use at a later date. On day 5, when they were defined as iDC, the adherent cells were divided into two groups: one was protein pulsed group, the other was control group. The protein pulsed group was pulsed with G250 protein (750 mg/ml). The control group was pulsed with PBS solution. At the same time, cytokines TNF-αat a final concentration of 200IU/ml was include in the medium in both groups throughout the culture. Both groups were cultured for a further 3 days. Half of the medium was replaced with fresh medium every other day. DCs were harvested on day 8, and they were defined as mDC.
1.2 Phenotypic analysis of DCs by flow cytometry.
On day 8, the phenotypic analysis of DCs was carried out by immunofluorescence staining of dendritic-monocyte differentiation and activation markers. Cells were labelled with human monoclonal antibodies (mabs) conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The following FITC-conjugated mabs were used: anti-HLA-DR, anti-CD83, anti-DC40. The following mabs were used as PE-conjugated reagents: anti-CD86, anti-CD80. anti-CD1a. PE/FITC isotype-matched mouse immunoglobulins were used as negative controls. Cells were washed once with PBS and incubated with the appropriate mab for 30 min on ice. Then cells were washed once again and fixed in PBS/1% paraformaldehyde. Samples were acquired and analyzed by flow cytometry.
1.3 Isogenic mixed lymphocyte reaction
After the two different group DCs were incubated with mitomycin C (30ug/ml) for 30 minutes, and then the DCs were washed with PBS solution. Lymphocytes were added into a 96-well culture plate. as responder cells at a concentration of 1x10~5/ml. DCs were collected in each group as stimulator cells at various ratios of stimulator cells to responder cells( 1: 5、1: 10、1: 20). The total terminal volume was 200μl per well. Each group was set to triple wells. After co-culture for 5 days, Add 20μl MTT (5g·L~(-1)) to each well of multiwell culture plate, incubate for 4 hours, then add 100μL DMSO, mixed about 10 min until the crystal completely dissolved. The absorption value (OD value) of each well was immediately read by automatic enzyme linked detector at 570nm wavelength.
1.4 Analysis of CTL for cytotoxicity essays
On day 8, nonadherent PBMCs from the same healthy donors were washed and added to protein-pulsed or mock-treated DCs (ratio of 20:1, responders: dendritics) in six-well culture plates. The RPMI-1640 cultures were supplemented with recombinant human GM-CSF(800U/ml), recombinant human IL-2(10U/ml), and IL-7(20ng/ml). After 5 days of coculture (day 13), the cells were collected for the CTL activity assay.
Then, we made three group effective CTLs: In group 1, CTLs were T+Ad/G250-DC; In group 2, CTLs were T+Protein/G250-DC CTLs; In groups 3, CTLs were T+N-DC. Part of the three group CTLs were carried out for Phenotypic analysis by flow cytometry. The Cells were labelled with human monoclonal antibodies (mabs) FITC or PE. Each group of CTLs as stimulator cells and 786-0 or A549 as responder cells were coculmred in a 96-well culture plate for 48 hours at various ratios of effector cells to target cells( 5: 1、10: 1、20: 1、40: 1); The total terminal volume was 200μl per well. Each group was set to triple wells. Add 20μl MTT (5g·L~(-1)) to each well of multiwell culture plate, incubate for 4 hours, then add 100μL DMSO, mixed about 10 min until the crystal completely dissolved. The absorption value (OD value) of each well was immediately read by automatic enzyme linked detector at 570nm wavelength.
The killing activity was measured by MTT and the killing rate was calculated by the follow formula:
Killing rate=[1-ODexperiment group/ODresponder cells group+ODtarget cells group]x100%.
1.5 Statistical analysis
The data were analyzed by the SPSS13.0 statistical software. Statistical method used analysis of variance of factorial design date, and the interblock difference compare applied LSD. P≤0.05 was considered to be statistically significant.
2. Results
(1). There were high expression of CD40, CD80, CD83, CD86 and HLA-DR, and low expression of CD1a in both the groups. (2). The ability to elicit lymphocyte proliferation in the DC pulsed with G250 protein(DC-G250)group was strong (F=83.545, P=0.000), whereas in the DC pulsed with PBS(DC-N) as control group it was low. At different ratio of effector cells to target cells, a significant difference was observed between the two groups (F=23.273, P=0.000). (3). The percentage of CD3+CD56+ and CD8+ cells in total CTL cells significantly increased in DC-G250 group. Phenotype of CTLs induced by DC-G250 higher than DC-N group (t=9.589 P=0.01; t=11.806 P=0.000). (4). In vitro the cytotoxicity to 786-0 of T lymphocytes activated by G250 protein pulsed DCs were greater than those of T lymphocytes in the control group (F=29.483, P=0.000). The cytotoxicity to A549 of T lymphocytes activated by G250 protein pulsed DCs were no significant difference than those of T lymphocytes in the control group.
3 Conclusions
The kidney tumor association antigen G250 pulsed DC vaccines can induce an effective and specific anti-renal carcinoma effect. The TAA G250 antigen may have a major impact on the adoptive immunotherapeutic protocols for patients with kidney cancer.
The second part: Study on Generating the CTL by G250 gene transfecting DC using Ad as vector to treat kidney cancer.
1. Methods
1.1 Ad/G250 transfecting the DC and generating the CTL
Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Paque density gradient centrifugation of heparinized blood obtained from healthy donors and washed twice in PBS. The PBMCs were inoculated into six well culture plates at 37℃under a 5% CO_2 atmosphere for 4～6 hours. After the removal of the nonadherent cells, the adherent cells were divided into three groups; one was gene transfer group; one was protein pulsed group; another one was control group. The gene transfer group was infected with Ad/G250 virus. After 5 hours of incubation, the medium/virus solution was removed, and the fresh AIM-V medium was added. GM-CSF at a final concentration of 800IU/ml was included in the medium in three groups throughout the culture. Human IL-4 at 1000IU/ml was added at day 3. On day 5, the protein pulsed group was pulsed with G250 protein (750mg/ml). At the same time, the control group was pulsed with PBS solution., cytokines TNF-αat a final concentration of 200IU/ml was include in the medium in the three groups throughout the culture. The three groups were cultured for a further 3 days. On day 8, they were defined as mDC. On day 8, nonadherent PBMCs from the same healthy donors were washed and added to Ad/G250-loaded, protein-pulsed or mock-treated DCs (ratio of 20:1, responders: dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF(800U/ml), recombinant human IL-2(10U/ml), and IL-7(20ng/ml). After 5 days of co-culture (day 13), the cells were collected for the CTL activity assay.
1.2 The G250 mRNA test, the DC surface marker analysis.
On day 8, collected the suspend cells(Mature DC), observed the morphologic features of DC with light microscope. G250 mRNA expression was detected in transduced DCs using RT-PCR amplification, and the unpulsed-DC served as control. The resulting G250 RT-PCR products were observed. Cell surface marker analysis of DCs was by flow cytometry. For the characterization of DCs, a panel of surface marker was used: CD83、CD80、CD86、HLA-DR、CD1a.
1.3 The immunity activity testing of G250-specific CTLs
Cell surface marker analysis of stimulated T cells: The primed T-cell populations were analyzed for surface markers on day 13. A panel of mAbs recognizing the following antigens was used: PE-anti-CD4, FITC-anti-CD8, and PE-anti-CD56. Thus, the ratio of CD8/CD4 and CD8/CD56 was analysed with the flow cytometry.
Analysis of CTL for cytotoxicity assays: The primary kidney cancer cell line 786-0(HLA-A2 haplotype) was used as one G250-positive target. The HLA haplotypes of all donors were compatible with this cell line. Lung cancer cell line A549 was used as negative target. The cells were used for cytotoxicity assays by MTT method.
2. Result
Ad/G250 transfected DCs successful. Ad/G250-loading of DCs resulted in: (1)G250 experssion in DCs. (2). RT-PCR amplificated G250 mRNA in Ad/G250 transfecting DCs. (3) high CD80、CD83、HLA-DR and CD86 experssion in DCs. (4) high CD8: CD4 and CD8: CD56 T cell ratios. (5) strong, rapid G250-specific CTLs. In vitro the cytotoxicity to 786-0 of T lymphocytes activated by Ad-G250 transfecting DCs were greater than those of T lymphocytes in the protein G250 group and control group. The CTL stimulated by Ad-G250 group had much higher cytotoxicity to renal carcinoma cell line 786-0, as compared with A549.
3. Conclusion
These date suggest that Ad-loading of DCs may be useful for immunotherapeutic protocols against self-antigens and that the G250 antigen is a potentially appropriate target for cell-mediated immunotherapeutic treatment for the kidney cancer.
The third part: Immune response induced by DC transfected by G250 gene in vitro inhibit growth of implanted tumor and reject challenge of tumor cells in nudes.
1. Methods
1.1 Generation of monocyte-derived DCs from peripheral blood mononuclear cells Peripheral blood was derived from healthy donors. Ficoll gradient-purified PBMCs
Were inoculated into six well culture plates for 4～6 hours, and the adherent cells were selected following three gentle washes. Immediately after the removal of the nonadherent cells, the adherent cells were divided into two groups: one was gene transfer group; one was protein pulsed gruoup. The gene transfer group was infected with Ad/G250 virus. After 5 hours of incubation, the medium/virus solution was removed, and the fresh AIM-V medium was added. GM-CSF at a final concentration of 800IU/ml was included in the medium in two groups throughout the culture. Human IL-4 at 1000IU/ml was added at day 3. On day 5, the protein pulsed group was pulsed with G250 protein (750 mg/ml). At the same time, cytokines TNF-αat a final concentration of 200IU/ml was included in the medium in the three groups throughout the culture. The two groups were cultured for a further 3 days. On day 8, they were defined as mDC.
1.2 The two groups CTLs generated by the two groups DCs.
On day 8, the T lymphocytes from the same healthy donors were added to Ad/G250-loaded DCs and protein-pulsed DCs. (ratio of 20:1, responders: dendritics) in six-well culture plates. The cultures were supplemented with recombinant human GM-CSF(800U/ml), recombinant human IL-2(10U/ml), and IL-7(20ng/ml). After 5 days of coculture, the cells were collected for inhibiting growth of implanted tumor in nudes mice.
1.3 Animals and establishment of the xenograft RCC model in nude mice
Female BALB/c-nu/nu mice were purchased and were age-matched (4 weeks of age) at the beginning of each experiment. They were maintained in specific pathogen-free (SPF) facilities at our institute. For tumor implants, 786-0 cells (2 x 10~6) in 200μl of PBS were injected s. c. into the right side of back of female nude mice 4 weeks of age. Tumor dimensions were measured with digital calipers to obtain two diameters of the tumor formed. The tumor incidence and tumor size were calculated with the equations shown below. Tumor size (16 days after inoculation) was calculated using the formula as follows: Tumor size =a x b, where a is the longest diameter and b is the shortest.
1.4 Immune response induced by DC transfected by G250 gene and pulsed with G250 protein in vivo prevent growth of implanted tumor in nude mice
The experimental mice were assigned randomly to three groups (n=5): control group, CTL-DC-Ad/G250 group and CTL-DC-Pro/G250 group. Mice in the control were inoculated subcutaneously with normal saline (200ul/each) into the right side of back of athymic nude mice simultaneously. Nude mice in the CTL-DC-Ad/G250 groups were treated with CTL (2x10~6 cells/each)induced by DC-Ad/G250 subcutaneously. Nude mice in the CTL-DC-Pro/G250 group were treated with CTL (2x10~6 cells/each) induced by DC-Pro/G250 subcutaneously. 72 hours after inoculation, 786-0(2x10~6 cells/each) were inoculated subcutaneously into the other side of back of athymic nude mice in the three groups simultaneously. From then on, the growth of implant tumor was observed every three day for 30 days.
1.5 Immune response of CTL induced by DC transfected by G250 gene in vivo inhabit growth of implanted tumor nude mice
On 16 days after inoculation in 1.3, there were 12 nude mice growing the xenograft. On 21 days after inoculation, 10 nude mice, that xenograft average size had reached 8.6±1.2mm~2, were selected for next experiments, another 2 nude mice, that xenograft size growed largerly than others, and 3 not growing xenograft were rejected from experiments. The 10 growing xenograft mice were assigned randomly to two groups (n=5): Control group and CTL-DC-Ad/G250 group. Mice in the control were inoculated s. c. with normal saline (200ul/each) into the other side of back of nude mice. Mice in the CTL-DC-Ad/G250 groups were treated with CTL (2x10~6 cells/each) induced by DC-Ad/G250 subcutaneously into the other side of back of nude mice. From then on, the growth of implant tumor was observed every three day until that day when the mice have been inoculated 45 days.
2. Results
2.1 Happenning rate of xenograft tumor
On the 30th day, there were no xenograft tumor growth in CTL-DC-Ad/G250 group and CTL-DC-Pro/G250 group; but the control group had 4 mice growing the xenograft tumor. The tumor formation incidences of 786-0 cells in the control group (n=5) were 80%.
2.2 Inhibitory growth of implanted tumor nude mice
On the 45th day of inoculation, we finished the experiments about the xenograft in nude mice. The mean volume of tumor of control group was 143.37±11.6 mm~2, whereas that of CTL-DC-Ad/G250 was 42.55±5.62mm~2 (t=2.355, P=0.047＜0.05)
Evidently, the CTL induced by DC transfected by Ad/G250 could prevent the growth of xenograft tumor, compared with the control group.
3. Conclusions
The CTL induced by DCs transfected by Ad/G250 could not only preclude the growth of xenograft tumor in nude mice and prevent the mice against the further challenge of 786-0 tumor cells, but also inhibit the growth of implanted tumor. The result suggest, as a new concept anti-tumor vaccine, DC transfected by Ad/G250 or pulsed by G250 protein may play an important role in therapy and prevent against kidney cancer.
引文
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