Comparative analysis of genetically modified dendritic cells and tumor cells as therapeutic cancer vaccines

We have directly compared the efficacy of two immunotherapeutic strategies for the treatment of cancer: "vaccination" of tumor-bearing mice with genetically modified dendritic cells (DCs), and vaccination with genetically modified tumor cells. Using several different preexisting tumor models that make use of B16F10 melanoma cells expressing a target tumor antigen (human melanoma-associated gene [MAGE]-1), we found that vaccination with bone marrow-derived DCs engineered to express MAGE-1 via adenoviral-mediated gene transfer led to a dramatic decrease in the number of metastases in a lung metastasis model, and led to prolonged survival and some long-term cures in a subcutaneous preexisting tumor model. In contrast, vaccination with granulocyte/macrophage colony-stimulating factor (GM-CSF)-transduced tumor cells, previously shown to induce potent antitumor immunity in standard tumor challenge assays, led to a decreased therapeutic effect in the metastasis model and no effect in the subcutaneous tumor model. Further engineering of DCs to express either GM-CSF, tumor necrosis factor alpha, or CD40 ligand via retroviral-mediated gene transfer, led to a significantly increased therapeutic effect in the subcutaneous tumor model. The immunological mechanism, as shown for GM-CSF-transduced DCs, involves MAGE-1-specific CD4(+) and CD8(+) T cells. Expression of GM-CSF by DCs led to enhanced cytotoxic T lymphocyte activity, potentially mediated by increased numbers of DCs in draining lymph nodes. Our results suggest that clinical studies involving the vaccination with genetically modified DCs may be warranted.     

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.     

RNA transfected dendritic cells as cancer vaccines

Immunization with dendritic cells loaded with tumor antigens could represent a powerful method of inducing antitumor immunity. Studies from several laboratories have shown that immunization with dendritic cells pulsed with specific antigens prime cytotoxic T-cells and engender tumor immunity. This review will focus on the use of dendritic cells transfected with RNA as cancer vaccines, with emphasis on the potential advantages of using RNA. The majority of cancer patients who lack an identified tumor antigen and/or cannot provide sufficient tumor tissue for antigen preparation will be excluded from treatment with cancer vaccines based on using either specific tumor antigens or mixtures of tumor-derived antigens in the form of peptides or proteins isolated from tumor cells. Vaccination with the mRNA content of tumor cells would extend the scope of vaccination to this group of patients as well because RNA can be amplified from very few cancer cells.     

Developing DNA vaccines that call to dendritic cells

DNA vaccination is a novel immunization strategy that has great potential for the development of vaccines and immune therapeutics. This strategy has been highly effective in mice, while less immunogenic in nonhuman primates and humans. Enhancing DNA vaccine potency remains a challenge. It is likely that APCs, and especially DCs, play a paramount role in the presentation of vaccine antigen to the immune system. A new study reports the synergistic recruitment, expansion, and activation of DCs in vivo in a mouse model through covaccination with plasmids encoding macrophage inflammatory protein-1alpha (MIP-1alpha), fms-like tyrosine kinase 3 ligand (Flt3L), and the DNA vaccine. Such cooperative strategies delivering vaccine in a single, simple platform result in improved cellular immunity in vivo, including enhanced tetramer responses and IFN-gamma secretion by antigen-specific cells.     

The use of dendritic cells for cancer vaccination

Dendritic cells (DC) are the most potent antigen presenting cells (APC) and the only ones capable of presenting novel antigens to na?ve T-cells. Large numbers of DC can be generated in vitro in the presence of appropriate cytokine cocktails using either adherent peripheral blood mononuclear cells (PBMC) or CD34+ precursors. More than 20 preclinical studies have demonstrated the effectiveness of antigen-loaded DC to mediate antitumor immune responses. Three clinical trials have been reported to date that show DC as a promising tool for the immunotherapy of cancer. However, completion and analysis of randomized trials to establish the appropriate antigen(s), adjuvant(s), dose, route and schedule will be crucial. Future DC-based therapies will include genetic modification of DC, the use of CD34+ precursors, direct delivery of DC to tumors, and application of tumor lysates or apoptotic cells as sources of additional, as yet undefined, antigens.     

基因修饰骨髓间充质干细胞修复关节软骨损伤

背景：随着生物技术的发展,通过转基因技术修饰细胞,从而获得长期稳定表达的生物活性因子以治疗关节软骨损伤逐渐引起重视。目的：就基因修饰的骨髓间充质干细胞在修复关节软骨损伤中的应用作一综述。方法：由第一作者检索1990至2011年PubMed数据库（http：／／www．ncbi．nlm．nih.gov／PubMed）有关基因修饰骨髓间充质干细胞修复关节软骨损伤的文献,英文检索词为“cartilage,genetherapy,mesenchymalstemcells,tissueengineering,bioactivefactor,vector”。共纳入15篇文献归纳总结。结果与结论：骨髓间充质干细胞已被广泛应用于修复关节软骨损伤。通过转基因技术将特定外源基因导入骨髓间充质干细胞,联合细胞治疗和基因治疗可达到更好的治疗效果,在关节软骨损伤的治疗中有广阔的应用前景。     

Antitumor reactivity of lymph node cells primed in vivo with dendritic cell-based vaccines.

Tumor lysate-pulsed dendritic cells were used to generate nodal effector T cells in the murine MCA 205 tumor model. Dendritic cells were derived from bone marrow and cultured in granulocyte-macrophage colony-stimulating factor/interleukin 4 before pulsation with tumor lysate. Multiple subcutaneous administrations of tumor lysate-pulsed dendritic cells (TP-DCs) resulted in an approximately eightfold hypertrophy of the vaccine draining nodes, with an increased influx of dendritic (CD11c+/CD80+) cells and B (B220+) cells. The vaccine-primed lymph node (VPLN) cells were secondarily activated with anti-CD3/interleukin 2 and exhibited specific interferon-gamma release to tumor antigen. The adoptive transfer of TP-DC VPLN cells resulted in regression of established 3-day pulmonary metastases. The antitumor reactivity of TP-DC VPLN cells was comparable to anti-CD3/interleukin 2 activated tumor-draining lymph node cells. However, the admixture of keyhole limpet hemocyanin (KLH) with tumor lysate during pulsation of dendritic cells significantly enhanced the induction of tumor-reactive VPLN cells. Tumor lysate-pulsed dendritic cells can be used as a strategy to generate effector T cells for adoptive immunotherapy.     

Comparative analysis of antigen loading strategies of dendritic cells for tumor immunotherapy

Dendritic cells (DCs) loaded with antigens can effectively stimulate host immune responses to syngeneic tumors, but there is considerable controversy as to which forms of antigen-loading are most immunogenic. Here, the authors compared immunotherapeutic reactivities of DCs loaded with a variety of antigen preparations. Because DC maturation stages affect their capacities of antigen processing and presentation, two DC populations were used for the current analysis: in vivo Flt-3 ligand-induced mature DCs and in vitro bone marrow-derived DCs, which were less mature. To facilitate a direct comparison, the LacZ gene-transduced B16 melanoma model system was used, where beta-galactosidase served as the surrogate tumor-rejection antigen. DC loading strategies included pulsing with the beta-galactosidase protein, H-2K restricted peptide, tumor cell lysate, and irradiated tumor cells and fusion of DCs with tumor cells. Our results demonstrated that electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. For the treatment of 3-day established pulmonary tumor nodules, a single intranodal vaccination plus IL-12 resulted in a significant reduction of metastatic nodules, while other DC preparations were only marginally effective. Immunotherapy mediated by the fusion cells was tumor antigen-specific. Consistent with their therapeutic activity, fusion hybrids were the most potent stimulators to induce specific IFN-gamma secretion from immune T cells. Furthermore, fusion cells also stimulated a small amount of IL-10 production from immune T cells. However, this IL-10 secretion was also induced by other DC preparations and did not correlate with in vivo therapeutic reactivity.     

致敏树突状细胞对射频消融治疗大鼠实体瘤疗效的影响

目的通过观察射频消融(RFA)治疗大鼠Walker256实体瘤后局部应用树突状细胞(DC)的疗效,探讨其临床应用的可能性。方法经Walker256细胞反复冻融获得肿瘤抗原并致敏大鼠骨髓细胞衍化的DC,大鼠腋窝皮下接种Walker256细胞获得实体瘤模型。30只SD大鼠分成三组,每组10只:对照组1仅行RFA治疗;对照组2行RFA 未致敏DC治疗;实验组行RFA 冻融抗原致敏DC治疗。超声评价治疗前后各组肿瘤体积变化,记录大鼠的荷瘤生存期,各组间比较。结果实验组瘤体平均体积在治疗后第一天与对照组无显著差异,但在治疗后第2、3天测得瘤体体积显著小于其他两组对照组(P<0.05)。实验组生存期显著长于对照组(P<0.05),对照组间瘤体体积及荷瘤生存期的差异无统计学意义(P>0.05)。结论在RFA治疗大鼠Walker256实体瘤后应用致敏DC可有效地延缓肿瘤生长速度,延长大鼠荷瘤生存期,有可能为临床治疗肿瘤提供一新途径。     

Clinical applications of dendritic cell vaccines

Dendritic cells play a central role in the presentation of antigen to na?ve T-cells and the induction of primary immune responses. Preclinical studies have established that dendritic cells loaded with antigens ex vivo induce potent antitumor and antiviral immune responses in vitro and in vivo. This has lead to a proliferation of clinical trials testing their effectiveness in humans, particularly with advanced malignancies. The few reported studies suggest that clinically relevant immune responses may be induced against some types of malignancies. Many questions regarding the best type of dendritic cell, degree of maturity, choice of antigen, route and schedule of administration, targeting to lymphoid tissue and use of additional adjuvants will need to be answered in preclinical and clinical studies as the field of dendritic cell-based immunotherapy progresses.     

高强度聚焦超声制备肿瘤抗原致敏树突状细胞

目的:探讨用高强度聚焦超声辐照肿瘤细胞制备肿瘤抗原致敏树突状细胞的可行性,为高强度聚焦超声开辟新的应用领域。方法:实验于2004-01/2005-04在泰安市中心医院中心实验室完成。①实验材料:BALB/C清洁级小鼠20只。CT-26肿瘤细胞为BALB/C小鼠来源的结肠癌细胞,由解放军第二军医大学免疫学研究所惠赠。②实验方法:应用白细胞介素4 粒细胞-巨噬细胞集落刺激因子联合诱导培养小鼠骨髓细胞。设立4组:高强度聚焦超声辐照组将CT-26细胞置于含10%小牛血清的RPMI1640培养液中,进入对数生长期后调整细胞浓度为5×109L-1,采用1000W/cm2×30s剂量辐照CT-26细胞,离心,取上清过滤除菌。冻融组将CT-26细胞调整浓度至5×109L-1,-80℃冷冻,35℃水浴复温,循环4次,裂解离心,取上清过滤除菌。单纯CT-26细胞组将CT-26细胞调整浓度至5×109L-1,细胞不经其他任何处理。空白对照组将树突状细胞与抗原按1∶10接种,培养4～6h。从小鼠脾脏制备脾细胞,分离纯化T细胞,调整浓度至2×109L-1,T细胞与上述4组负载的树突状细胞按20∶1接种,培养基中加入白细胞介素2,共孵育48h。③实验评估:对扩增培养的树突状细胞首先进行形态学观察,再采用流式细胞仪分析细胞表型。锥虫蓝染色观察高强度聚焦超声辐照后CT-26细胞拒染率和形态学变化。ELISA法检测各组致敏树突状细胞诱导T细胞分泌白细胞介素12及干扰素-γ的含量。结果:①骨髓源性树突状细胞形态及细胞表面表型分析:体外培养小鼠骨髓细胞6～8d,细胞表面出现较多毛刺样突起,拉长,为典型的树突状细胞特征。流式细胞仪检测树突状细胞表面分子CD80、CD86、H-2Kd及I-Ad呈高表达。②高强度聚焦超声辐照后细胞拒染率和形态学的变化:超声辐照剂量为1000W/cm2×30s时,无细胞存活,完全失去细胞形态,全部被撕裂成碎片。③致敏树突状细胞诱导T细胞分泌细胞因子情况:与空白对照组比较,高强度聚焦超声辐照组、冻融组、单纯CT-26细胞组分泌的白细胞介素12及干扰素-γ含量均明显升高(P均<0.05);且高强度聚焦超声辐照组、冻融组升高幅度大于单纯CT-26细胞组,但此两组比较差异无显著性意义(P>0.05)。结论:高强度聚焦超声能使肿瘤细胞灭活、破碎,其制备的肿瘤抗原可体外致敏树突状细胞,并使树突状细胞分泌白细胞介素12,诱导T细胞分泌干扰素-γ。     

Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro

Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.     

Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines

DCs are critical for priming adaptive immune responses to foreign antigens. However, the utility of harnessing these cells in vivo to optimize the immunogenicity of vaccines has not been fully explored. Here we investigate a novel vaccine approach that involves delivering synergistic signals that both recruit and expand DC populations at the site of antigen production. Intramuscular injection of an unadjuvanted HIV-1 envelope (env) DNA vaccine recruited few DCs to the injection site and elicited low-frequency, env-specific immune responses in mice. Coadministration of plasmids encoding the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) and the DC-specific growth factor fms-like tyrosine kinase 3 ligand with the DNA vaccine resulted in the recruitment, expansion, and activation of large numbers of DCs at the site of inoculation. Consistent with these findings, coadministration of these plasmid cytokines also markedly augmented DNA vaccine---elicited cellular and humoral immune responses and increased protective efficacy against challenge with recombinant vaccinia virus. These data suggest that the availability of mature DCs at the site of inoculation is a critical rate-limiting factor for DNA vaccine immunogenicity. Synergistic recruitment and expansion of DCs in vivo may prove a practical strategy for overcoming this limitation and potentiating immune responses to vaccines as well as other immunotherapeutic strategies.     

Update on tumor vaccines

Summary Vaccination against tumor has always been an attractive idea for the treatment of patients bearing tumor. By harnessing the host's own immune response the attack on tumor cells would act on a continuing basis, with emerging tumor cells stimulating their own destruction. However, the approach has been hampered by our poor understanding of the nature of tumor antigens and of the pathways by which immune cells might operate against tumor growth. Recent developments in molecular biology and immunology are remedying this deficiency and bringing vaccination to the forefront of new approaches to treatment of a range of tumors. Results obtained in B-cell tumors, where the idiotypic immunoglobulin at the cell surface provides a well-defined tumor antigen, are already indicating exciting possibilities as well as delineating problems. There is considerable clinical evidence that patients have some intrinsic ability to control tumor growth and that certain tumors remain dormant for long periods. Attempts to understand and perhaps stimulate the mechanisms involved are being made through the use of biological modifiers and by manipulating potential effector cells in vitro. Ideally this approach, which may include non-specific and specific elements, could be combined with specific vaccination in order to combat the apparent ability of many tumor cells to evade host defences.     

Anchoring cytokines to tumor cells for the preparation of anticancer vaccines without gene transfection in mice

The authors have investigated a new way of combining cytokines with tumor cells to prepare anticancer vaccines. This method may offer an alternative to gene therapy approaches. It consists in anchoring recombinant cytokines to the cell membrane. Attachment is mediated by the transmembrane domain of diphtheria toxin (T) genetically fused to the cytokine and is triggered by an acid pH pulse. The authors found that the fusion protein T-hIL-2 anchored to the surface of tumor cells retained its IL-2 activity while remaining exposed for several days. Interestingly, vaccination of mice with these modified tumor cells induced a protective antitumor immunity mediated by tumor-specific cytotoxic T lymphocytes. This procedure presents several advantages as compared with the conventional approaches based on the transfection of tumor cells with cytokine genes. It does not require the culture of tumor cells from the patients and the selection of transfected clones, it eliminates the safety problems connected with viral vectors, and it allows the control of the amount of cytokines delivered with the vaccine.     

Current methods for loading dendritic cells with tumor antigen for the induction of antitumor immunity

The immunotherapy of cancer is predicated on the belief that it is possible to generate a clinically meaningful antitumor response that provides patient benefit, such as improvement in the time to progression or survival. Indeed, immunotherapeutics with dendritic cells (DC) as antigen-presenting delivery vehicles for cell-based vaccines have already improved patient outcome against a wide range of tumor types (1-9). This approach stimulates the patient's own antitumor immunity through the induction or enhancement of T-cell immunity. It is generally believed that the activity of cytotoxic T lymphocytes (CTL), the cells directly responsible for killing the tumor cells in vivo, are directed by DC. Therefore, the goal of many current designs for DC-based vaccines is to induce strong tumor-specific CTL responses in patients with cancer. In practice, most studies for DC-based cancer vaccine development have focused on the development of methods that can effectively deliver exogenous tumor antigens to DC for cross-priming of CD8+ T cells through the endogenous MHC class I processing and presentation pathway (10). To date, many methods have been developed or evaluated for the delivery of defined and undefined tumor antigens to DC. This review provides a brief summary on these methods, the techniques used in these methods, as well as the advantages and disadvantages of each method.     

In vitro production of dendritic cells from human blood monocytes for therapeutic use

Dendritic cells (DC) are professional antigen-presenting cells that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now developed. For this purpose, their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. In the present review, we give our experience of such a procedure: it includes collection of mononuclear cells by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + human serum (autologous patient's serum or AB serum) or in a serum-free medium (AIM V). The characteristics of monocyte-derived DC grown in these various conditions varied mainly regarding their phenotype and their morphology in confocal microscopy, whereas no significant differences were found in their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The DC were also cryopreserved in bags (either by putting the bags directly in a -80 degrees C mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degrees C/min) in a solution containing 10% dimethyl sulfoxide (Me(2)SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after freezing and thawing were not statistically different (around 70%). The immunophenotype of DC, as well as the T lymphocyte-stimulating capacity, were not modified by the freezing--thawing procedure. The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.     

转染A549细胞总RNA的树突状细胞疫苗抗肿瘤效应的体外研究

目的探讨人肺腺癌细胞株A549总RNA转染的人树突状细胞(DCs)在体外诱导特异性抗肿瘤免疫反应的能力。方法利用密度梯度离心法从血细胞分离机新鲜采集的外周富集血中分离出单个核细胞(PBMCs),贴壁获得单核细胞,加入重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素-4(rhIL-4)进一步诱导为树突状细胞(DCs),同时利用提取的A549细胞总RNA转染DCs来制备疫苗,并与T细胞混合培养诱导扩增细胞毒性T淋巴细胞(CTLs);实验分为转染A549细胞总RNA的DC组、未转染DC组和转染空脂质体DC组,分别以流式细胞术(FCM)鉴定DCs表型、酶联免疫吸附法(ELISA)检测IL-12、IFN-γ分泌水平、3H-TdR掺入检测T细胞增殖能力及乳酸脱氢酶(LDH)释放法测定杀伤肿瘤活性并比较各组差异。结果1)转染总RNA组DCs表面CD40、CD80、CD83、HLA-DR的表达较未转染组DCs和转染空脂质体组DCs均有升高,CD40在3组的表达率分别为(69.01±7.67)%、(19.28±3.51)%和(39.62±2.72)%;CD80在3组的表达率分别为:(74.39±6.46)%、(15.48±3.03)%和(25.70±2.92)%;CD83在3组的表达率分别为:(81.79±4.61)%、(13.29±2.34)%和(31.42±1.97)%;HLA-DR在3组的表达率分别为:(76.53±5.13)%、(49.23±4.05)%和(29.94±3.53)%,差异有统计学意义(P<0.05);2)转染总RNA组DCs的IL-12和IFN-γ分泌水平较未转染组及转染空脂质体组明显升高(P<0.05),3组IL-12分别为(543.24±68.33)、(77.11±27.65)和(167.78±31.84)pg/ml;3组IFN-γ分别为:(122.95±32.84)、(41.06±10.97)和(56.08±15.96)pg/ml;3)3H-TdR掺入试验所得的cpm,3组分别为(7437±720)、(3 807±476)和(4 543±719),阳性对照组为:16 739±91,差异有统计学意义(P<0.05);4)LDH释放法可见转染A549总RNA的DC组的特异性杀瘤活性(58.54±8.27)%,明显高于未转染组(36.03±4.03)%和转染空脂质体组(33.47±8.21)%,差异具有统计学意义(P<0.05)。结论人肺腺癌细胞A549总RNA转染的DC疫苗体外诱导的特异性抗肿瘤免疫能力显著增强,有望为肺腺癌的治疗提供新的手段。