肝癌相关肿瘤抗原AFP新的HLA—A2／A24限制性CTL表位的预测及分析

目的：预测肝癌肿瘤抗原AFP新的HLA—A2／A24限制性CTL表位,为肝癌CTL表位肽的筛选及鉴定提供依据。方法：选择与肝癌密切相关的肿瘤抗原AFP为研究目标,采用NetCTL1．2Server远程预测系统进行HLA—A2／A24限制性CTL表位预测。结果：共预测出与肝癌相关的肿瘤相关抗原AFP的8个HLA—A2限制性CTL表位,13个HLA—A24限制性CTL表位。结论：应用NetCTL1．2Server法预测CTL表位是一种高效准确的预测方法。所预测的肿瘤抗原AFP的HLA—A2／24限制性CTL表位经实验筛选、鉴定后,可为肝癌治疗性疫苗的制备选择合适的CTL表位提供依据。     

Dendritic cells fused with allogeneic breast cancer cell line induce tumor antigen-specific CTL responses against autologous breast cancer cells

Dendritic cell (DC)/tumor cell fusion vaccine has been revealed as a promising tool for the antitumor immunotherapy. Previous research has shown that fusion hybrids of human DCs and autologous tumor cells can induce cytotoxic T lymphocyte (CTL) responses against autologous tumor cells in animal models and human clinical trials. However, a major restriction factor for the clinical use is the difficulty for preparation of sufficient amount of autologous tumor cells especially for the patients with metastasis cancer whose primary tumor lesion is not clear or has been resected. In this study, allogeneic breast cancer cell line MCF-7 cells were electrofused to autologous DCs from patient with breast cancer as a strategy to deliver shared breast cancer antigens to DCs. Fusion cells generated by autologous DCs and allogeneic MCF-7 were able to induce autologous T lymphocytes proliferation, high levels of IFN-gamma production and CTL responses. CTLs induced by DCs/allogeneic MCF-7 fusion cells were able to kill autologous breast cancer cells in an antigen specific and HLA restriction manner. Our study may provide the experiment basis for the use of allogeneic breast cancer cell line in the DC/tumor cell fusion cell vaccination strategy.     

Specific killing of P53 mutated tumor cell lines by a cross-reactive human HLA-A2-restricted P53-specific CTL line

p53 is upregulated in the majority of spontaneous tumors and the HLA class I molecule HLA-A2 is expressed by approximately 50% of the caucasians. Potentially, these facts make HLA-A2-binding p53 peptides for CTL-inducing immunotherapy applicable to a broad range of cancer patients. In our study, we investigated the CTL-inducing capacity of autologous monocyte-derived dendritic cells (DC) maturated by exposure to CD40L and pulsed with a pool of 4 wild-type, HLA-A2-binding p53 peptides, and the p53-specific CD8(+) CTL lines established from healthy HLA-A2-positive donors were characterized. Reactivity to p53(65-73) and p53(187-197) peptides was obtained in the T-cell lines. Interestingly, cold target inhibition experiments demonstrated that the simultaneous recognition of the 2 peptides was the result of cross-reactivity, which was confirmed by killing experiments at the clonal CTL level. Furthermore, 4 HLA-A2(+) p53-mutated tumor cell lines were lysed by the CTL line, indicating that these peptides are endogenously processed and presented on HLA-A2 molecule. Thus, monocyte-derived DC pulsed with a pool of peptides are able to induce CTL reactivity to wild-type p53 peptides presented by several cancer cell lines. In addition, the recognition of 2 different p53 peptides by the same CTL clone suggests a promiscuous peptide recognition by the TCR involved. Taken together, these in vitro results suggest that vaccination with autologous DC pulsed with multiple p53 epitopes may induce an effective tumor-specific CTL response in vivo with the potential to eradicate p53-upregulated spontaneously occurring tumors.     

Dendritic cell vaccination with MAGE peptide is a novel therapeutic approach for gastrointestinal carcinomas.

The MAGE gene is selectively expressed in cancer tissues such as melanoma or gastrointestinal carcinomas, whereas no expression is observed in normal tissues except testis. There are several reports of successful induction of HLA class I-restricted antitumor CTLs using MAGE peptides, and some clinical trials with these immunogenic peptides were reported as effective for some patients with malignant melanoma. However, there are no similar studies in gastrointestinal carcinomas, which are important neoplasms. Autologous dendritic cells (DCs) were generated ex vivo and were pulsed with MAGE-3 peptide, depending on the patient's HLA haplotype (HLA-A2 or A24). Patients were immunized with DC pulsed with MAGE-3 peptide every 3 weeks at four times. Twelve patients with advanced gastrointestinal carcinoma (six stomach, three esophagus, and three colon) were treated, and no toxic side effects were observed. Peptide-specific CTL responses after vaccination were observed in four of eight patients. Improvement in performance status was recognized in four patients. Tumor markers decreased in seven patients. In addition, minor tumor regressions evidenced by imaging studies were seen in three patients. These results suggested that DC vaccination with MAGE-3 peptide is a safe and promising approach in the treatment of gastrointestinal carcinomas.     

腺病毒转染黑色素瘤抗原基因3的树突状细胞疫苗抗胃癌的免疫效应

目的 研究趋化因子巨噬细胞炎症蛋白1α(MIP-1α)动员的树突状细胞(DC),经黑色素瘤抗原基因3(MAGE-3)腺病毒转染后对胃癌细胞的免疫效应.方法 615小鼠尾静脉注射MIP-1α,分选得到B220-CD11c+细胞,加入细胞因子连续培养,检测其细胞表面标志和混合淋巴细胞反应(MLR).收集培养后的B220-CD11c+细胞,加入编码MAGE-3的重组腺病毒进行转染,制备表达肿瘤抗原的DC疫苗,以荷载小鼠前胃癌(MFC)全肿瘤细胞抗原制备的DC疫苗作为阳性对照.采用二苯基溴化四氮唑蓝(MTT)法,榆测活化的T淋巴细胞在体外对MFC细胞的杀伤作用.采用酶联免疫吸附试验(ELISA),检测γ干扰素(INF-γ)的分泌情况.DC疫苗皮下注射MFC荷瘤小鼠,观察小鼠瘤体生长情况和存活时间.结果 MIP-1α注射后,外周血中B220-CD11c+细胞明显升高.新鲜分离的B220-CD11c+细胞在体外经过细胞因子诱导分化后具有典型的DC表面标志,并在MLR中具有极强的刺激T淋巴细胞增殖的能力.腺病毒转染MAGE-3的DC激活的T淋巴细胞表现出对MFC细胞的特异性杀伤作用,产牛高水平的INF-γ[(1460.00±16.82)ps/ml].实验组荷瘤小鼠接受DC疫苗治疗后,肿瘤牛长缓慢,观察至MFC细胞接种后的第27天,其肿瘤体积仅为(3.46±1.12)cm3;实验组小鼠的肿瘤体积与对照组之间的差异有统计学意义(P<0.01).实验组小鼠存活时间明显延长,与对照组之间的差异有统计学意义(P<0.01).结论 注射趋化因子MIP-1α可快速动员并诱导分化为成熟DC.肿瘤抗原基因MAGE-3经腺病毒转染制备的DC疫苗,在体外可以诱导出针对胃癌细胞的特异性杀伤作用,在体内对MFC荷瘤小鼠有明显的免疫治疗作用.     

Antitumor Activity of Lentivirus-mediated Interleukin -12 Gene Modified Dendritic Cells in Human Lung Cancer in Vitro

Objectives: Dendritic cell (DC)-based tumor immunotherapy needs an immunogenic tumor associatedantigen (TAA) and an effective approach for its presentation to lymphocytes. In this study we explored whethertransduction of DCs with lentiviruses (LVs) expressing the human interleukin-12 gene could stimulate antigenspecificcytotoxic T cells (CTLs) against human lung cancer cells in vitro. Methods: Peripheral blood monocytederivedDCs were transduced with a lentiviral vector encoding human IL-12 gene (LV-12). The anticipated targetof the human IL-12 gene was detected by RT-PCR. The concentration of IL-12 in the culture supernatant of DCswas measured by ELISA.Transduction efficiencies and CD83 phenotypes of DCs were assessed by flow cytometry.DCs were pulsed with tumor antigen of lung cancer cells (DC+Ag) and transduced with LV-12 (DC-LV-12+Ag).Stimulation of T lymphocyte proliferation by DCs and activation of cytotoxic T-lymphocytes (CTL) stimulatedby LV-12 transduced DCs pulsed with tumor antigen against A549 lung cancer cells were assessed with methylthiazolyltetrazolium (MTT). Results: A recombinant lentivirus expressing the IL-12 gene was successfullyconstructed. DC transduced with LV-12 produced higher levels of IL-12 and expressed higher levels of CD83than non-transduced. The DC modified by interleukin -12 gene and pulsed with tumor antigen demonstratedgood stimulation of lymphocyte proliferation, induction of antigen-specific cytotoxic T lymphocytes and antitumoreffects. Conclusions: Dendritic cells transduced with a lentivirus-mediated interleukin-12 gene have anenhanced ability to kill lung cancer cells through promoting T lymphocyte proliferation and cytotoxicity.     

Transduction of dendritic cells with recombinant adenovirus encoding HCA661 activates autologous cytotoxic T lymphocytes to target hepatoma cells

Transduction of recombinant adenovirus into dendritic cells (DCs) is a promising new tool for cancer vaccine development. Here, we report that an adenovirus vector carrying hepatocellular carcinoma (HCC) antigen HCA661 and infected into DCs generates T-cell immunity against hepatoma cells. HCA661 is a novel cancer/testis (CT) antigen screened by SEREX from sera of an HCC patient. We constructed a recombinant adenovirus expressing the full-length cDNA of HCA661 gene and then transduced immature DCs, which had been generated with GM-CSF and IL-4 from peripheral blood mononuclear cell of HLA-A2(+) healthy donors. The resulting adenovirus-transduced DCs differentiated in the presence of monocyte-conditioned medium and poly [I] : poly [C], expressing the surface markers of mature DCs, including CD83, CD80, CD86 and HLA-DR. After maturation, the transduced DCs transcribed HCA661 mRNA and were able to prime the na?ve T cells to become cytotoxic T lymphocytes (CTLs). Intracellular flow cytometry and enzyme-linked immunospot assay showed that these CTLs were able to target a hepatoma cell line, HepG2, which is HLA-A2 and HCA661 positive. In summary, we found that this recombinant adenovirus can help to induce DC maturation and these mature DCs can activate T cells to target hepatoma cells. Therefore, this recombinant adenovirus may have potential for use in liver cancer immunotherapy.     

含甲胎蛋白肽段的重组腺病毒转染树突状细胞诱导细胞毒性T细胞对肝癌细胞的体外杀伤效应

目的 研究人外周血单核细胞来源的树突状细胞(DC)转染含甲胎蛋白(AFP,137-145)片段的重组腺相关病毒后所诱导的特异性T细胞对肝癌细胞株HepG2和SMMC-7721的体外杀伤作用.方法 抽取健康志愿者外周血,分离单核细胞,体外培养,使用含AFP片断的重组腺相关病毒转染未成熟DC,诱导特异性T细胞.检测体外培养的DC和细胞毒性T细胞(CTL)活性,应用四甲基偶氮唑蓝(MTT)法检测CTL对HepG2和SMMC-7721细胞的杀伤作用.结果 转染或未转染的体外培养的成熟DC高表达CD40、CD86和IL12,成熟DC诱导的CTL高表达IFNγ;修饰成熟后的DC后体外能诱导特异性CTL,该CTL在休外对肝癌细胞株HepG2和SMMC-7721均有杀伤作用.结论 重组腺相关病毒转染DC,不明显改变DC表型和刺激淋巴细胞增殖和分化功能,可诱导自体CTL增殖,含AFP(137～145)片断的腺相关病毒转染DC诱导自体CTL对肝癌细胞株HepG2和SMMC-7721细胞有明显杀伤作用,DC疫苗可以作为肝癌患者免疫治疗的有效补充.     

A combination of chemoimmunotherapies can efficiently break self-tolerance and induce antitumor immunity in a tolerogenic murine tumor model

Her-2/neu is a well-characterized tumor-associated antigen overexpressed in human carcinomas such as breast cancer. Because Her-2/neu is a self-antigen with poor immunogenicity due to immunologic tolerance, active immunotherapy targeting Her-2/neu should incorporate methods to overcome immunologic tolerance to self-proteins. In this study, we developed a tolerogenic tumor model in mice using mouse Her-2/neu as self-antigen and investigated whether genetic vaccination with DNA plasmid and/or adenoviral vector expressing the extracellular and transmembrane domain of syngeneic mouse Her-2/neu or xenogenic human Her-2/neu could induce mouse Her-2/neu-specific CTL responses. Interestingly, adenoviral vectors expressing xenogenic human Her-2/neu (AdhHM) proved capable of breaking immune tolerance and of thereby inducing self-reactive CTL and antibodies, but not to the degree required to induce therapeutic antitumor immunity. In attempting to generate therapeutic antitumor immunity against established tumors, we adopted several approaches. Treatment with agonistic anti-glucocorticoid-induced TNFR family-related receptor (GITR) antibody plus AdhHM immunization significantly increased self-reactive CTL responses, and alpha-galactosylceramide (alphaGalCer)-loaded dendritic cells (DC) transduced with AdhHM were shown to break self-tolerance in a tolerogenic murine tumor model. Furthermore, gemcitabine treatment together with either AdhHM plus agonistic anti-GITR antibody administration or alphaGalCer-loaded DC transduced with AdhHM showed potent therapeutic antitumor immunity and perfect protection against preexisting tumors. Gemcitabine treatment attenuated the tumor-suppressive environment by eliminating CD11b(+)/Gr-1(+) myeloid-derived suppressor cells. When combined with immunotherapies, gemcitabine offers a promising strategy for the Ag-specific treatment of human cancer.     

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.     

肝细胞癌p53蛋白、增殖细胞核抗原与临床关系的研究

目的　探讨在肝细胞癌中突变型 p5 3蛋白与增殖细胞核抗原 (PCNA)的相互关系及其意义。 方法　应用免疫组化ABC法检测肝细胞癌和肝硬化中 p5 3蛋白及增殖细胞核抗原。 结果　 37例肝细胞癌 (HCC)中 p5 3阳性 19例 ,33例肝硬化标本中p5 3阳性 2例 ,两者有显著差异 (P <0 .0 1) ;HCC中PCNA阳性细胞均数显著高于肝硬化组 (P <0 .0 1) ;p5 3阳性者PCNA阳性细胞数高于p5 3阴性者 (P <0 .0 5 )。p5 3与PCNA两者表达存在显著正相关关系 (r=0 .5 936 2 ,P <0 .0 5 ) ,p5 3表达与年龄、性别、组织分化无关 ,PCNA表达分别与组织分化及血AFP成负相关及正相关。结论　p5 3基因突变在由肝硬化转变为癌过程中是频发事件 ,其表达异常与HCC发生密切相关。PCNA与 p5 3的正相关提示p5 3突变型主要存在于异常增生细胞中 ,且PCNA可反映癌细胞分化及增殖程度 ,可为肝硬变与肝癌关系的探讨及临床诊断、治疗、预后提供参考资料。     

Generation of antigen-presenting cells using cultured dendritic cells and amplified autologous tumor mRNA

Novel antigen-presenting cells (APCs) were generated using cultured dendritic cells (DCs) and amplified tumor mRNA, and the potential of tumor antigen-reactive T cell induction by the tumor RNA-introduced DCs (DC/tumor RNA) was analyzed in a patient with melanoma antigen-encoding gene (MAGE3)-positive malignant melanoma of the esophagus. DCs were generated from an adherent fraction of peripheral blood mononuclear cells in the presence of granulocyte macrophage colony-stimulating factor and interleukin-4. Tumor mRNA was purified from tumor tissue, amplified in vitro using a T7 RNA polymerase system, and then introduced into DCs by electroporation (150 V/150 microF or 100 V/200 microF). The gene introduction efficiency was 44-55% as measured by enhanced green fluorescent protein reporter gene expression, and the viability of RNA-introduced DCs was approximately 80%. DC/tumor RNA could induce tumor antigen-reactive cytotoxic T lymphocytes (CTLs) in an mRNA-specific manner, but had no effect on the self-antigen-reactive T cells. DC/tumor RNA could induce the polyspecific antigen-reactive CTL responses mediated by both human leukocyte antigen class I and class II molecules, whereas MAGE3 peptide-pulsed DCs induced only the monospecific MAGE3-reactive CTL responses mediated by human leukocyte antigen class I molecules, showing the superiority of the DC/tumor RNA over the DC/peptide. It is suggested that the use of DC/tumor RNA as antigen-presenting cells may be more effective, convenient and practical for the DC-based anti-cancer immunotherapy.     

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor-associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte-derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA-derived CEA and MUC1 antigens and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4(+) and CD8(+) T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN-gamma. More importantly, coculture of fusion cells with patient-derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen-specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1-positive and HLA partially matched target cells. Antigen-specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA- and MUC1-specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen-specific CTL responses. Our report demonstrates the simultaneous induction of CRCA-specific CTL responses restricted by HLA-A2 and -A24.     

Enhanced HER-2/neu-specific antitumor immunity by cotransduction of mouse dendritic cells with two genes encoding HER-2/neu and alpha tumor necrosis factor

The present study uses an in vivo murine tumor model expressing the human HER-2/neu antigen to evaluate the potential vaccine using dendritic cells (DCs) infected with adenovirus AdVHER-2. We first investigated whether infected DCs (DC(HER-2)) engineered to express HER-2/neu could induce HER-2/neu-specific immune responses. Our data showed that (i) AdVHER2-infected DC(HER-2) expressed HER-2/neu by Western blot and flow cytometric analysis, and (ii) vaccination of mice with DC(HER-2) induced HER-2/neu-specific cytotoxic T-lymphocyte (CTL) responses, but protected only 25% of vaccinated mice from challenge of 3 x 10(5) MCA26/HER-2 tumor cells. Further, to enhance the efficacy of DC(HER-2) vaccine, we coinfected DCs with both AdVHER-2 and AdVTNF-alpha. The infected DCs (DC(HER-2/TNF-alpha)) displayed the expression of both HER-2/neu and TNF-alpha by flow cytometric and ELISA analysis. We next investigated whether DC(HER-2/TNF-alpha) could induce stronger HER-2/neu-specific immune responses. We found that DC(HER-2/TNF-alpha) displayed up-regulation of immunologically important CD40, CD86, and ICAM-I molecules compared with DC(HER-2), indicating that the former ones are more mature forms of DCs. Vaccination of DC(HER-2/TNF-alpha) induced stronger allogeneic T-cell proliferation and 36% enhanced HER-2/neu-specific T-cell responses in vitro than DC(HER-2) cells. More importantly, it stimulated the significant anti-HER-2/neu immunity in vivo, which protected 8/8 mice from challenge of 3 x 10(5) MCA26/HER-2 tumor cells. Therefore, DCs genetically engineered to express both the tumor antigen and cytokines such as TNF-alpha as an immunoadjuvant are likely to represent a new direction in DC vaccine of cancer.     

Fusion vaccine therapy by IL-2-gene-transduced dendritic cells and tumor cells

We evaluated the usefulness of fusion vaccine prepared from IL-2-gene-transduced splenic dendritic cells (DCs) and fibrosarcoma tumor cells (QRsP) in treating of lung metastasis. The IL-2 or LacZ gene was transferred into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumor cells were fused with IL-2 gene transduced DCs (fusion/IL-2) or LacZ gene transduced DCs (fusion/LacZ) by polyethyleneglycol. These fusion cells expressed major histocompatibility complex (MHC) class I and MHC class II, CD86, CD11c and CD8alpha. IFN-gamma and cytotoxic T lymphocyte (CTL) activity of splenic lymphocytes in mice vaccinated with fusion cells increased significantly as compared with those of DC or tumor cells vaccinated mice. CTL levels in fusion/IL-2-vaccinated mice were higher than that in fusion/LacZ-vaccinated mice. The number of lung metastasis in the fusion/IL-2 or fusion/LacZ-vaccineatd mice was significantly lower than that in mice vaccinated with DCs, tumor or PBS. The introduction of the IL-2 gene into fusion cells produced more potent therapeutic effects. Our results suggest that the fusion cells prepared from IL-2 gene transduced spleen derived DCs and tumor cells have the ability to induce therapeutic effect against lung metastasis.     

Identification of alpha-fetoprotein-derived peptides recognized by cytotoxic T lymphocytes in HLA-A24+ patients with hepatocellular carcinoma

Alpha-fetoprotein (AFP) has been proposed as a potential target forT-cell-based immunotherapy for hepatocellular carcinoma (HCC), but the number of its epitopes that have been identified is limited and the status of AFP-specific immunological responses in HCC patients has not been well-characterized. To address the issue, we examined the possibility of inducing AFP-specific cytotoxic T cells (CTLs) using novel HLA-A*2402-restricted T-cell epitopes (HLA, human leukocyte antigen) derived from AFP and then analyzed the relationship between its frequency of occurrence and clinical features associated with patients having HCC. Five AFP-derived peptides containing HLA-A*2402 binding motifs and showing high binding affinity to HLA-A*2402 induced CTLs to produce IFN-gamma and kill an AFP-producing hepatoma cell line. The frequency of AFP-specific CTLs was 30-190 per 1 x 10(6) peripheral blood mononuclear cells, which was the same as that of other immunogenic cancer associated antigen-derived epitopes. Analyses of the relationships between AFP-specific CTL responses and clinical features of patients with HCC revealed that AFP epitopes were more frequently recognized by CTLs in patients with advanced HCC correlating to tumor factors or the stage of TNM classification. The analyses of CTL responses before and after HCC treatments showed that the treatments changed the frequency of AFP-specific CTLs. In conclusion, we identified five HLA-A*2402-restricted T-cell epitopes derived from AFP. The newly identified AFP epitopes could be a valuable component of HCC immunotherapy and for analyzing host immune responses to HCC.     

Advances in dendritic cell-based vaccine of cancer

Dendritic cells (DCs) are potent antigen presenting cells that exist in virtually every tissue, and from which they capture antigens and migrate to secondary lymphoid organs where they activate na?ve T cells. Although DCs are normally present in extremely small numbers in the circulation, recent advances in DC biology have allowed the development of methods to generate large numbers of these cells in vitro. Because of their immunoregulatory capacity, vaccination with tumor antigen-presenting DCs has been proposed as a treatment modality for cancer. In animal models, vaccination with DCs pulsed with tumor peptides, lysates, or RNA or loaded with apoptotic/necrotic tumor cells could induce significant antitumor CTL responses and antitumor immunity. However, the results from early clinical trails pointed to a need for additional improvement of DC-based vaccines before they could be considered as practical alternatives to the existing cancer treatment strategies. In this regard, subsequent studies have shown that DCs that express transgenes encoding tumor antigens are more potent primers of antitumor immunity both in vitro and in vivo than DCs simply pulsed with tumor peptides. Furthermore, DCs that have been engineered to express certain cytokines or chemokines can display a substantially improved maturation status, capacity to migrate to secondary lymphoid organs in vivo, and abilities to stimulate tumor-specific T cell responses and induce tumor immunity in vivo. In this review we also discuss a number of factors that are important considerations in designing DC vaccine strategies, including (i) the type and concentrations of tumor peptides used for pulsing DCs; (ii) the timing and intervals for DC vaccination/boostable data on DC vaccination portends bright prospects for this approach to tumor immune therapy, either alone or in conjunction with other therapies.     

A HLA-A2 restricted human CTL line recognizes a novel tumor cell expressed p53 epitope

A p53 peptide-specific CTL line was generated through stimulation with autologous monocyte-derived dendritic cells (DC) pulsed with wild-type HLA-A2 binding p53 derived peptides. A p53 peptide-specific CD8(+) CTL line was established from a healthy HLA-A2 positive donor. The CTL line was characterized with respect to specificity, affinity and killing of cell lines derived from p53 mutated spontaneous tumors. The CTL line demonstrated lysis of p53(139-147) pulsed target cells and cold target inhibition experiments as well as antibody blocking confirmed that the killing was epitope-specific, HLA-A2 restricted and dependent on CD8-binding. Interestingly, the affinity of the CTL line was only in the micromole per liter range and target cells pulsed with less than 0.01 microM peptide were not recognized. Furthermore, 3 HLA-A2(+) p53 mutated tumor cell lines were efficiently lysed by the CTL line, indicating that this novel p53 peptide epitope is endogenously processed and presented by the HLA-A2 molecules of the tumor cells. In conclusion, CTL reactivity towards a wild-type p53 peptide was revealed through induction with DC pulsed with a pool of HLA-A2 binding p53 peptides. In addition, the CTL line, which expressed relatively low affinity for the HLA-A2/peptide complex, was able to kill 3 different HLA-A2(+) p53 mutated tumor cell lines. The present and our previous observations expand the number of p53-derived peptides suitable for vaccination protocols for cancer patients with p53 positive tumors.     

Maturation of dendritic cells is a prerequisite for inducing immune responses in advanced melanoma patients.

PURPOSE: We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo. EXPERIMENTAL DESIGN: Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients). RESULTS: All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and >40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells. CONCLUSIONS: We conclude that mature DC are superior to immature DC in the induction of immunological responses in melanoma patients, which may translate into improved clinical results.