热休克凋亡食管癌细胞负载自体树突状细胞制备及其表型特征的分析

目的:建立自体热休克凋亡食管癌细胞抗原制备、树突状细胞(DC)体外诱导以及抗原负载方法,为DC肿瘤疫苗的临床应用提供技术基础.方法:采用酶消化法从手术切除的食管癌新鲜组织获得单细胞悬液,热休克后用桦脂酸诱导其凋亡制备成细胞抗原;采用人工肝素抗凝采集外周静脉血,分离单个核细胞(PBMC),贴壁法获得单核细胞,经GM-CSF与IL-4体外诱导成未成熟树突状细胞(imDC);负载细胞抗原后制备成DC肿瘤疫苗,并对DC疫苗的形态、数量及表型特征进行分析.结果:肿瘤细胞抗原每克组织得率(9.89±2.46)×106,平均凋亡率(67.60±3.23)%.imDC活率>95%,imDC表型分析为CD11c+HLA-DR+、CD11c+CD80+、CD11c+CD83+和CD11c+CD86+,表达率分别为(88.30±3.59)%、(1.25±0.55)%、(6.13±1.79)%和(72.90±5.01)%.DC疫苗活率>95%,DC疫苗表型为CD11c+HLA-DR+CD11c+CD80+、CD11c+CD83+和CD11c+CD86+,表达率分别为(89.60±3.91)%、(34.20±3.85)%、(64.90±8.05)%和(86.10±6.22)%.负载自体抗原DC诱导CTL杀伤自体肿瘤细胞的杀伤率为(65.90±6.25)%,明显高于杀伤食管癌肿瘤细胞株的杀伤率[(32.56±2.68)%],P<0.01.结论:本方法稳定、安全及可靠,可制备出在体外诱导特异性CTL的DC肿瘤疫苗.     

肺癌gp96-多肽复合物/DC疫苗体外诱导CTL反应的实验研究

目的 研究热休克蛋白gp96－多肽复合物负载树突状细胞（dendritic cell,DC）后，能否诱导出gp96－多肽复合物特异性的细胞毒性T细胞（cytotoxic T lymphocyte,CTL）反应。方法 从一例肺癌患者肿瘤组织中提取gp96-多肽复合物和自体肿瘤细胞溶解物，分别负载从该患者骨髓血中培养的DC。以不同形式的抗原／DC疫苗分别刺由患者外周血中分离的淋巴细胞。采用ELISA法检测淋巴细胞所释放的IFN－γ量作为CTL反应的指标；以Cr^51释放实验分析致敏后的淋巴细胞对不同靶细胞的裂解和杀伤作用。结果 所有肿瘤抗原致敏淋巴细胞后均可以诱导产生CTL反应，其中以gp96－多肽复合物／DC疫苗诱导释放的IFN－γ量最高。肿瘤抗原致敏淋巴细胞后对原代培养的肿瘤细胞的杀伤作用高于PG细胞和K562细胞。结论 自体肿瘤组织中提取的gp96－多肽复合物能诱导出肿瘤特异性CTL反应，而负载DC后能激发起更强的CTL。     

负载Walker-256肿瘤细胞抗原的树突状细胞疫苗的制备

目的 制备负载大鼠Walker-256肿瘤细胞抗原的树突状细胞(DC)疫苗,为进一步研究打下基础.方法 从大鼠骨髓细胞中利用粒细胞巨噬细胞集落刺激因子(GM-CSF)和白细胞介导素4(IL-4)在体外定向诱导分化出DC,以液氮冻融法制备肿瘤抗原刺激DC制备肿瘤疫苗.ELISA检测培养液中IL-12的浓度.结果 得到大量具备典型光镜和电镜形态特征的DC,表达大鼠DC特异性标志OX62.负载抗原前后DC产生IL-12的水平存在明显差异(P＜0.05).结论 负载Walker-256肿瘤抗原的树突状细胞疫苗制备成功,具有促进Th1极化的潜能.     

树突状细胞肿瘤疫苗抗肿瘤研究进展

目的:探讨不同树突状细胞(DC)肿瘤疫苗的抗肿瘤机制及应用价值.方法:应用NCBI的PubMed文献数据库系统,以“树突状细胞、疫苗、肿瘤”为关键词检索2001-01-01-2011-12-31文献1 589篇,纳入标准:1)DC抗肿瘤的机制;2)肿瘤抗原肽负载的DC肿瘤疫苗;3)肿瘤全细胞抗原负载的DC肿瘤疫苗;4)肿瘤细胞来源的基因修饰的DC肿瘤疫苗;5)DC-CIK联合修饰疫苗.根据纳入标准符合分析的共30篇文献纳入分析.结果:DC是目前已知人体内功能最强抗原提呈作用的专职抗原呈递细胞,与肿瘤发展有着密切的关系,以DC为基础制备的不同类型肿瘤疫苗提高了抗肿瘤免疫反应,但存在不同的缺点.结论:DC肿瘤疫苗展示了良好的应用前景,对临床免疫治疗将有着积极的作用,但仍有待于进一步深入研究.     

树突状细胞在肿瘤免疫中的研究进展

树突状细胞(DC)是最强的抗原提呈细胞(APC),APC捕获抗原提呈给T、B淋巴细胞,从而引发一系列的免疫应答。肿瘤患者体内由于肿瘤的调变可以降低DC提呈功能,使淋巴细胞杀伤肿瘤的效率减弱,目前DC体外可以大量扩增,故各种DC疫苗和DC细胞治疗应运而生,通过抗原负载的DC提高抗原提呈能力,激活细胞毒T细胞(CTL)的杀伤能力,治疗肿瘤,但抗原负载的方法很多．哪种抗原负载提高提呈功能的效率最高,每一种肿瘤的抗原有其特异性。这些都是DC疫苗广泛开展的障碍,而且DC疫苗在免疫系统低下的老年人群由于淋巴细胞的免疫应答功能下降,可能会免疫失败,这些都值得我们进一步思考应对策略。     

树突状细胞疫苗肿瘤抗原负载方式的临床转化研究进展

树突状细胞（dendritic cell, DC）是体内功能最强的抗原提呈细胞,能将肿瘤抗原提呈给T细胞,是抗肿瘤免疫的启动者。DC疫苗的肿瘤免疫治疗虽已取得丰硕成果,但在其临床转化过程中还有很多问题需要解决,选择何种抗原负载方式则是其中之一。目前临床试验中常用的有肿瘤细胞裂解物负载DC、重组肿瘤相关抗原负载DC及携带肿瘤相关抗原信息的mRNA转染DC三种方法。本文对此三种方法在临床试验中的应用做一综述,为树突状细胞疫苗的临床应用提供参考。     

树突状细胞的肿瘤抗原负载

树突状细胞（DC）是体内功能最强的专职抗原提呈细胞，在诱导特异性抗肿瘤免疫反应中起着至关重要的作用。通过体外负载肿瘤抗原致敏DC可以有效地增强其抗原提呈能力，所制备的肿瘤抗原DC疫苗回输体内后可促进机体产生特异性细胞毒T淋巴细胞（CTL）和其他抗肿瘤免疫应答机制，有效地杀伤肿瘤细胞。目前已发展了多种针对DC负载肿瘤抗原的方法，有些方法已被应用到了人体肿瘤治疗。     

RNA转染树突状细胞肿瘤疫苗的研究进展

树突状细胞（DC）是体内功能最强的专职抗原递呈细胞,能激发并维持机体的初始免疫反应。通过不同方式将肿瘤抗原信息负载DC制备肿瘤疫苗是肿瘤免疫治疗的一个重要方面。而通过RNA转染DC制备核酸肿瘤疫苗是最近发展起来的新型制备疫苗的方法。现对RNA—DC疫苗的研究进展作一综述。     

树突状细胞疫苗治疗脑胶质瘤的研究进展

大量实验研究证明树突状细胞(dendritic cell，DC))疫苗是治疗脑胶质瘤的一种很有前景的技术。常用的DC负载肿瘤抗原的方法有肿瘤抗原肽致敏DC、肿瘤细胞提取物致敏DC和DC／肿瘤融合等，本文综述这几方面的研究进展。     

RNA转染树突状细胞肿瘤疫苗的研究进展

树突状细胞(DC)是体内功能最强的专职抗原递呈细胞,能激发并维持机体的初始免疫反应。通过不同方式将肿瘤抗原信息负载DC制备肿瘤疫苗是肿瘤免疫治疗的一个重要方面。而通过RNA转染DC制备核酸肿瘤疫苗是最近发展起来的新型制备疫苗的方法。现对RNA-DC 疫苗的研究进展作一综述。     

术中失血来源的树突状细胞用于肝癌治疗的体外实验研究

目的 探讨从肝癌患者术中失血来源的单个核细胞中培养树突状细胞(DC)的可行性,为个体化的DC介导免疫治疗提供新的细胞来源.方法 采集9例原发性肝细胞癌患者术中失血及8例脐血,分离其单个核细胞,其中贴壁的单个核细胞经重组人粒细胞巨噬细胞集落刺激因子(RHGM-CSF),重组人白细胞介素4(rhIL-4)诱导和负载癌细胞抗原,制成不同的DC瘤苗,悬浮的单个核细胞经细胞因子处理成为细胞因子诱导的杀伤细胞(CIK).采用二苯基溴化四氮唑蓝(MTT)法测定DC激活同源CIK的相对增殖率和CIK对肝癌细胞的杀伤效果.结果 肝癌患者术中失血来源的单个核细胞在体外能够诱导分化为具有典型形态和表型的DC.术中失血来源的DC表面标志物表达水平低于脐血来源的DC,但二者均能有效地激活CIK,并增强其对肝癌细胞的杀伤活性.负载患者自身癌细胞抗原的术中失血IX:和脐血DC,其激活的CIK增殖率分别为(388.9±137.3)%和(315.1±44.5)%,对肿瘤细胞的杀伤率分别为(87.1±8.0)%和(90.0±5.1)%;而负载SMMC-7721抗原的术中失血DC和脐血DC,其激活的CIK增殖率分别为(239.9±48.7)%和(226.3±32.3)%,对肿瘤细胞的杀伤率分别为(76.4±7.9)%和(81.1 ±4.3)%.在激活CIK和增强对肝癌细胞杀伤能力方面,相同抗原负载的两种Dc差异无统计学意义,但负载自身抗原优于负载SMMC-7721抗原.结论 肝癌患者术中失血来源的DC可有效激活CIK,并增强其对肝癌细胞的杀伤效应,为临床研究和应用DC瘤苗提供了一个新的来源.     

IL-27基因修饰树突状细胞活化的特异性CTL对人食管癌裸鼠移植瘤生长增殖的影响

研究IL-27基因转染树突状细胞（DC）体内诱导免疫杀伤食管癌细胞的效能及其机制。方法:基因转染的方法建立表达IL-27基因的树突状细胞（DC/IL-27）;构建人食管癌细胞裸鼠移植瘤模型,皮下注射食管癌细胞抗原致敏、IL-27基因修饰DC（DC/IL-27-Ag）活化的特异性CTL后观察荷瘤裸鼠抑瘤率;TUNEL法检测荷瘤裸鼠肿瘤细胞的原位凋亡;流式细胞术检测移植瘤细胞的细胞周期、凋亡率。结果:RT-PCR显示DC/IL-27细胞中有IL-27 p28和EBI3亚基基因表达提示转染成功;免疫接种DC/IL-27-Ag活化的CTL组的抑瘤率为58.28%,明显高于其他组,差异有统计学意义（P<0.01）。TUNEL法检测显示,DC/IL-27-Ag活化的CTL组凋亡率明显高于其他组（P<0.01）,流式细胞术（FCM）显示肿瘤组织内细胞增殖指数为（23.92±1.60）%,显著低于其他组,差异有统计学意义（P<0.01）;细胞凋亡率为（32.78±0.83）%,显著高于其他组,差异有统计学意义（P<0.01）。结论:DC/IL-27-Ag可活化特异性CTL,在裸鼠体内产生了抑制肿瘤生长的作用,显著抑制食管癌细胞增殖、促进其凋亡,为DC应用于食管癌的免疫治疗提供了理论和动物实验依据。      

T cell responses in colorectal cancer patients: evidence for class II HLA-restricted recognition of shared tumor-associated antigens

Few cases of anti-colon cancer specific T lymphocytes have been described so far. Moreover, the majority of these effectors were generated in vitro by stimulating PBMC from patients or healthy donors with peptides that were derived from proteins expressed and/or secreted by colon cancer tissue such as CEA, Mucin or Her-2/neu. The aim of our study was to evaluate the immunogenicity of colorectal carcinomas in an autologous setting. We exploited the antigen processing and presentation capacity of dendritic cells (DC) to establish an in vitro autologous system that can bypass the need of obtaining cultured tumor cells. DC were generated from the adherent monocyte fraction of PBMC taken from stage II/III colorectal cancer patients. A single cell suspension was prepared by mechanical and enzymatic disruption of the surgical specimens immediately after resection. DC were loaded with autologous tumor lysate, obtained by repeated freezing and thawing, before being used as stimulators for autologous PBL. HLA-class II restricted T cells that recognize the autologous tumor could be generated in a proportion of patients. The fine specificity of the anti-tumor T cells indicates that differentiation as well as tumor restricted antigens are expressed in colon cancer and that these antigens can evoke a class II HLA-restricted response in an autologous setting. Altogether these findings may open a new perspective for a DC based vaccination of colon cancer patients.     

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4⁺ and CD8⁺ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4⁺CD25⁺Foxp3⁺ T_reg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.     

自体宫颈癌-树突细胞疫苗激活的CTL杀伤效应

背景与目的:树突细胞(dendriticcells,DC)是目前已知的功能最强的抗原递呈细胞(antigen-presentingcell,APC),它可以在体内、外向T淋巴细胞递呈抗原,并诱发细胞毒T淋巴细胞(cytotoxicTlymphocyte,CTL)反应。本研究旨在探讨负载自体宫颈癌抗原的DC体外激发的CTL对自体宫颈癌细胞的杀伤效应。方法:先冻融宫颈癌细胞制备抗原,然后以GM-CSF、IL-4诱导自体外周血单个核细胞(peripheralbloodmononuclearcell,PBMC)获得DC并负载抗原,刺激自体T淋巴细胞制备宫颈癌抗原特异性CTL,观察CTL对宫颈癌细胞的杀伤活性。结果:负载自体宫颈癌抗原DC诱导的特异性CTL对自体宫颈癌细胞的体外杀伤率高达79.32%～89.27%,显著高于淋巴因子激活的杀伤细胞(lymphokine-activatedkillingcells,LAK)的杀伤率(t≥2.89,P<0.05);且对宫颈癌HeLa细胞株具有一定杀伤效应(40.35%～58.09%),但低于自体癌细胞组(t≥2.97,P<0.05);特异性CTL对HepG2、MCF7、A549、MGC803细胞无明显杀伤效应。结论:自体宫颈癌-树突细胞疫苗体外诱导的CTL具有高效而特异的抗自体宫颈癌细胞免疫活性,可望成为宫颈癌生物治疗的一个有力手段。     

Dendritic cell-mediated immunization in macroglobulinemia

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) specialized to initiate immunity and are critical targets for all vaccines. New methods to generate large quantities of these cells in the laboratory and load them with tumor antigens allows novel approaches targeting these APCs in the clinic. Injection of antigen loaded DCs can boost tumor-specific immunity in patients. Studies in myeloma suggest that the use of autologous tumor as a source of antigen may be the preferred approach. Boosting antitumor immunity may be a useful strategy to prevent tumor progression in patients with asymptomatic macroglobulinemia.     

Influence of drug-induced apoptotic death on processing and presentation of tumor antigens by dendritic cells

Here we have studied the effects of apoptotic cell death induced by chemotherapic agents on tumor phagocytosis by dendritic cells (DC) and presentation of the relevant antigen to T lymphocytes. Annexin-V-FITC (Ann-V) and propidium iodide (PI) staining was used to assess early apoptotic (Ann-V(+)/PI(-)) vs. late apoptotic/secondary necrotic (Ann-V(+)/PI(+)) death after a 24 hr observation of untreated and drug-treated gastric carcinoma cells. After treatments, the HLA-A*0201(+) tumor cell line KATO III was exposed for 24 hr to allogeneic, HLA-related GM-CSF, IL-4-driven immature (i) DC. Tumor-loaded iDC were tested for IL-12 release in an ELISA assay, incubated with the DC-maturating factor TNF-alpha and used as stimulators for autologous T lymphocytes. Generation of antitumor T response against KATO cells was evaluated in an anti-MHC class I MAb-blocked Interferon-gamma ELISPOT assay. After treatment with Cis-platin (cis), all dying cells were in early apoptosis, whereas secondary necrosis was the prevalent death pattern observed after epirubicin (epi) and doxorubicin (doxo). Doxo and epi increased tumor expression of heat shock protein (hsp) 70 and uptake of tumor cell components by DC, whereas cis treatment had no effect on hsp70 and was associated with poor tumor uptake by DC. Significant upmodulation of IL-12 was observed by DC that had taken up the doxo- and epi-treated tumors (p< 0.005 and p< 0.01, respectively). Increased IFN-gamma release was also observed after stimulation of T lymphocytes with DC loaded with doxo- and epi-treated (p< 0.02 and p< 0.005, respectively) but not with cis-treated DC. These data show that the products of early apoptosis cannot efficiently cross-activate MHC class I-restricted anti-tumor lymphocytes even in the presence of DC maturating factors, whereas secondary necrosis is associated with robust T cell response.     

Apoptotic tumor cells are superior to tumor cell lysate, and tumor cell RNA in induction of autologous T cell response in B-CLL.

B cell chronic lymphocytic leukemia (B-CLL) is a chronic leukemia manifested by increased numbers of B cells in circulation. The slow, smouldering nature of the disease in a significant proportion of the cases makes it an ideal target for immunotherapy. Dendritic cell (DC)-based immunotherapy is emerging as an exciting modality with significant clinical potential. In this study, three strategies for delivering antigens to DC, namely apoptotic bodies (Apo-DC), tumor lysates, and tumor RNA were studied in an autologous setting. In all six CLL patients, Apo-DC induced higher HLA-restricted, T cell responses than DC pulsed with tumor lysate or RNA. Real-time PCR confirmed higher expression of genes for IL-2 and IFN-gamma in T cells stimulated with Apo-DC. Concurrently, no IL-10 and low IL-4 responses indicated that the immune response was primarily of the Th1 type. Enzyme-linked immunospot assay revealed high IFN-gamma secretion by T cells when Apo-DC was used to stimulate autologous T cells in all patients. Our data suggest that cellular vaccines with DC loaded with apoptotic bodies may be a suitable approach for immunotherapy of B-CLL.     

Induction and clonal expansion of tumor-specific cytotoxic T lymphocytes from renal cell carcinoma patients after stimulation with autologous dendritic cells loaded with tumor cells

Melanoma and renal cell carcinoma (RCC) are considered to be the most immunogenic tumors in humans. To generate conditions to induce primary T-cell responses against RCC and to allow further expansion of tumor-specific cytotoxic T lymphocytes (CTL) for adoptive transfer, peripheral blood mononuclear cells from RCC patients were stimulated with primary autologous tumor cells or monocyte-derived dendritic cells (DC) loaded with either tumor lysate (TU-LY) or apoptotic tumor cells (TU-AP). Whereas repetitive stimulation (4x) with tumor cells alone induced a predominant population of CD3(-) natural killer cells, 4 weeks of stimulation with tumor-loaded DC favored induction and expansion of CD4+ T cells (>80%). However, 2 weekly stimulation cycles with tumor-loaded DC followed by restimulation with autologous irradiated tumor cells alone were optimal for induction of tumor-specific CTL responses in vitro. Using these culture conditions a marked increase of CD4+ T cells was observed during the first 2 weeks of stimulation with tumor-loaded DC. Subsequent restimulation with autologous tumor cells alone gave rise to 500-fold expansion of CD8+ T cells. These CD8+ T cells were shown to exhibit strong major histocompatibility complex class I-restricted cytotoxic activity against the autologous tumor. Comparison of TU-LY and TU-AP as a source of tumor antigen for loading DC did not show any difference in stimulating tumor-specific CTL. Length pattern analysis of the complementary determining region 3 (CDR3) of the T-cell receptor Vbeta chain revealed expansion of oligoclonal CTL populations with outgrowth of 1 or 2 clones after prolonged stimulation with autologous tumor cells. Our study demonstrated an efficient method for generating tumor-specific CTL in vitro that may be used to identify tumor cell antigens or that can be expanded for adoptive T-cell transfer in tumor immunotherapy.     

Induction of carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro using autologous dendritic cells loaded with CEA peptide or CEA RNA in patients with metastatic malignancies expressing CEA.

The application of dendritic cells (DC) to the active immunotherapy of cancer currently relies on the generation of potent DC capable of presenting tumor antigens such as carcinoembryonic antigen (CEA). It is unknown whether the T cells of patients with advanced malignancies can be reliably stimulated against tumor antigens by their autologous DC. In this study, starting with the peripheral blood mononuclear cells (PBMC) of patients with metastatic malignancies expressing CEA, autologous DCs were generated in vitro in serum-free media supplemented with GM-CSF and IL-4. The DCs from HLA A2 positive patients were loaded with the CEA peptide CAP-1 and the DCs from HLA A2 negative patients were depleted of bystander lymphocytes and loaded with mRNA encoding CEA. The DC preparations were tested to determine their phenotype and were used to stimulate autologous PBMC twice, separated by 10-14 days. The stimulated cells were then tested for their ability to lyse CEA-expressing target cells. We successfully generated an adequate number of DC for a clinical trial from all patients. The harvested DC preparations contained 49% DC and 87% DC if depleted of bystander lymphocytes. Phenotypic analysis showed the typical pattern of CD11c+ CD40+ CD86+ HLA-DR+ CD80(low) CD83(low) CD14(low). All preparations but one were able to stimulate CEA-specific cytotoxic T-lymphocyte (CTL) activity, suggesting that the majority of patients are not anergic to CEA and possess functional DC. The CTL activity was similar for the CEA peptide and CEA RNA-loaded DC.