人树突状细胞与K562细胞的融合以及体外诱导p210蛋白特异性CTL的研究
作者：范国昌，吴祖泽，王艳飞，邱兆华
单位：
关键词：树突状细胞；K562；细胞融合；P210蛋白；特异性CTL
癌症990601Human peripheral blood mononuclear cells (PBMCs)-derived dendritic
cells fused with K562 cells induce a p210BCR-ABL protein-specific
cytotoxic T cell response in vitro
FAN Guo-chang，WU Chu-tse*，WANG Yan-fei，et al.Department of Experimental Hematology，Beijing Institute of Radiation Medicine，Beijing 100850，P.R.China
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【Abstract】 Objective：To investigate whether human dendritic cells (DCs) derived from cultured human peripheral blood mononuclear cells (PBMCs) were capable of presenting antigen to induce a p210BCR-ABL protein-specific CTL response in vitro after fusion with K562 cells which express p210BCR-ABL protein.Methods：DCs were derived from human PBMCs in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). To show the successful fusion of DCs with K562 cells，we first used K562 cells expressing green fluorescence protein(GFP) in one group， while in the other group，DCs fused with normal K562 cells，and these fusion cells were cocultured with autologous T lymphocytes for two rounds.Results：After 5～7 days of incubation，these adherent cells appeared in clusters or dispersed，and the results of flow cytometric analysis showed the cell surface contained markers typical of dendritic cells，i.e.，they were positive for HLA-DR，HLA-A，B，C，CD1α ，CD80，CD86 and negative for CD14 (monocytes). The fusion cells demonstrated morphologically distinct cell aggregates. Moreover，most of these cells that expressed GFP， exhibited a DC morphology with veiled processes and dendrites. Furthermore，these fusion cells can induce a p210BCR-ABL protein-specific cytotoxic T cell response in vitro using the lactate dehydrogenase (LDH) release assay.Conclusions：The direct fusion of DCs with K562 cells could result in induction of an effective p210BCR-ABL (b3a2) protein-specific immunogen， either by confering sufficient DC function to K562 cells for activation of T cells or by facilitating the delivery of tumor p210BCR-ABL (b3a2) protein to DCs for processing and presentation，which may broaden the spectrum of possible DC-based clinical applications.
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Key words： Dendritic cells； K562； Cell fusion； P210BCR-ABL protein； Specific CTL
【摘要】目的：探讨人外周血单核细胞来源的树突状细胞(Dendritic cells，DC)与人白血病细胞K562融合后，能否诱导出p210蛋白特异性的CTL，为DC疫苗的临床应用提供理论基础。方法：外周血来源的贴附单核细胞，在人GM-CSF(1000U/ml)和IL-4(1000U/ml)作用下，培养5～7天后，与人白血病细胞K562进行融合，融合细胞再与自体的淋巴细胞共同孵育10～14天，采用乳酸脱氢酶释放试验分析其对p210蛋白阳性细胞的杀伤效果。为显示融合效果，对照试验组K562细胞用绿色荧光蛋白(GFP)进行标记。结果：贴附的单核细胞在GM-CSF和IL-4作用下，培养5天后，约有70%的DC产生，流式细胞仪分析表明，这些细胞为HLA-DR、HLA-A，B，C以及CD1α阳性，并高表达共刺激分子CD80和CD86。DC与GFP标记的K562细胞融合24h后，表达GFP蛋白的细胞中约有60%呈现出典型的DC形态特征。乳酸脱氢酶释放试验结果表明，融合细胞能激活淋巴细胞产生p210蛋白特异性的CTL。结论：树突状细胞与肿瘤细胞融合后，能诱导出肿瘤特异性的CTL，显示出DC疫苗抗肿瘤作用的广泛前景。
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中图分类号：R735.3 文献标识码：A 文章编号：1000-467X(1999)06-0617-07
The bcr-abl oncoprotein (a p210BCR-ABL protein)， the product of the fused bcr-abl gene in the Philadelphia (Ph.) chromosome is known to be chronic myelogenous leukemia (CML) specific and critical to leukemogenic process〔1～4〕， but if p210-positive cells are proved to be susceptible targets it will become critical to optimize any vaccination schedule for effective presentation of bcr-abl peptide to the immune system. The dendritic cells (DCs) have the capacity to migrate through tissues，can uptake，process and present antigen， and stimulate both a naive and memory T-cell response〔5〕.Recent studies have indicated that in respect of inducing an antitumor immune response DC is more effective than other approaches，such as viral vectors expressing tumors antigens，gene-modified tumor cells，naked DNA， or peptide emulsified in adjuvant〔6〕.DC-based vaccines are now under active investigation and many vehicles for the delivery of tumor antigen to DCs are being considered： viral vectors，naked and plasmid DNA，RNA，liposomes with nucleic acid or protein，and tumor lysates，apoptotic cells and peptides. Antigen has even been presented by fusion of DCs with whole tumor cells〔7～17〕.
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DC-tumor fusion cells have the potential to stimulate immunity against multiple tumor Ags and could induce synergistic protection through CD4+ and CD8+ T cell-mediated immune responses. Celluzzi et al〔16〕showed that short-term coculture of DCs and tumor cells， with or without prior fusion， resulted in a potent immunogen capable of inducing CTL-mediated protective antitumor immunity and regression of established tumors. Gong et al〔10〕also showed that the fusion cells could stimulate naive T cells in the primary mixed lymphocyte reaction and induce MC38 tumor-specific CTLs in vivo.
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The purpose of the present study was to investigate whether human DCs after fused with K562 cells expressing the p210BCR-ABL protein，were capable of presenting antigen to induce a p210BCR-ABL protein-specific CTL response in vitro. In one group，we first used K562 cells that expressed green fluorescence protein(GFP)，and after the fusion of DCs with K562 cells，the fusion cells not only expressed GFP ， but also exhibited a DC morphology with veiled processes and dendrites; while in the other group，after DCs fused with normal K562 cells，fusion cells were then cocultured with autologous T lyphocytes for two rounds， resulting in stimulating naive T cells and inducing a p210BCR-ABL protein-specific CTLs in vitro.
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1 MATERIALS AND METHODS
1.1 Cell lines and culture condition
Jurkat (HLA-A9，A25，B7，B41)，a T leukemia/lymphoma cell line，and K562，a human leukemia cell line，lacking MHC I and II and expressing the b3a2 translocation，were incubated in RPMI-1640 medium(Gibco，BRL，Grand Island，NY) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco，BRL)，25 mmol/L HEPES，2 mmol/L L-glutamine (Sigma，St. Louis，MO) and antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml)，in 5% CO2 atmosphere at 37℃ .
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1.2 Generation of DCs in vitro
DCs were prepared from PBMCs as previously described〔18〕.Briefly，PBMCs were isolated from leukapheresed blood of healthy donor (HLA-A25，B7) by density centrifugation on Ficoll-Hypaque grad(ents1.077g/ml)(Bio-tech Co.，Institute of Hematology，CAMS，Tianjing，China) for 20 min at 2000 rpm at room temperature. After four to five washes in PBS to remove platelets，cells were resuspended at 107/ml in RPMI-1640 medium and incubated for 1.5～2 h in 75-cm2 tissue culture flasks (Nunc， Denmark) (5% CO2，37℃ ). Then nonadherent cells were gently washed out with PBS and cryopreserved for use as T cell responder. The remaining adherent cells were further cultured (5% CO2， 37℃ ) in RPMI-1640 medium supplemented with 10% FCS， 1000 U/ml rhGM-CSF (Biotinge Biomedicine Co. Beijing，China) and 1000 U/ml rhIL-4 (Promega， Madison， WI). After 5～7 days，nonadherent cells were harvested. DCs generated in this way were 60% -80% pure based on morphology and the expression of a CD14-， CD1α+，CD80+，CD86+，MHC class I and II+ immunophenotype as assessed by flow cytometry.
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1.3 Flow cytometry
For immunophenotyping，DCs were washed and resuspended in 0.5 ml (1× 106 cells/ml) PBS supplemented with 0.2% FCS and 0.1% NaN3 and incubated (at 4℃ in dark place for 30 min) with 5 μ l of one of the following mAb：PE-conjugated anti-CD14，PE-conjugated anti-HLA-A，B，C (Immunotech，Hamburg，Germany)， PE-conjug-ated anti-CD1α (Coulter，Krefeld，Germany)，FITC-con-jugated anti-HLA-DR，FITC-conjugated anti-CD80，FITC-conjugatedanti-CD86 (Caltag Laboratories，Bur-lingame，CA).Surface expression was analyzed using a FCAScan flow cytometer (FACS440，Be-cton，Dickinson).
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1.4 Adenovirus-mediated transfer of GFP gene into K562 cells
K562 cells，incubated in freshly prepared RPMI -1640 medium supplemented with 10% FCS 24 h before infection，were washed twice with PBS and centrifuged.The recombinant virus Ad5RSVGFP (kindly provided by Dr. DAI Yifan from Baxter Inc.，USA) was added to 2× 106 resuspended cells in serum-free medium at a multiplicity of infections (MOI) of 400 p.f.u/cell，and incubated at 37℃，5% CO2 for 1.5～2 h in a small volume (< 500 μl). Culture medium was then added in order to obtain a cell concentration of 0.5× 106 /ml and incubation was continued for 48 h.Then they were used for cell fusion to evaluate the effect of such fusion.A part of K562 cells would be used for induction of CTLs in vitro.
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1.5 Fusion of DCs and K562 cells
On the 6th day of culture DCs were mixed with K562 cells at a ratio of 5∶1 for cell fusion by adding 40% polyethylene glycol(PEG)(Promega，WI，USA)，which had been warmed to 37℃ as described〔16〕.After PBS washing，fused cells were cultured overnight at 37℃ in RPMI-1640 (10% FCS) containing 1000 U/ml rhGM-CSF (Biotinge Biomedicine Co. Beijing，China) and 800 U/ml rhIL-4 (Promega，Madison，WI). Fluorescence and light microscopies were used to evalute the efficiency of cell fusion and the morphology of fused cells. These GFP labeled fused cells were not used to induce CTL generation.
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1.6 LipofectAMINE -mediated gene transfer to Jurkat cells
Plasmid EGFP， carrying a red-shifted humanized variant of the GFP gene，was driven by the CMV-IE promoter，and the plasmid p210BCR-ABL，containing bcr-abl (b3a2) fusion gene of 8.5 Kb，was driven by CMV promoter.For liposomal gene transfer，2×106 cells were washed once in serum-free RPMI-1640 without antibiotics and then resuspended in 0.8 ml culture medium and seeded in a six-well plate. Lipofection with lipofectAMINE (Gibco BRL，Madison，WI) was performed according to the manufacturer’s instructions. Briefly，increasing amounts of lipofect-AMINE (2～25 μ l) were diluted in 100 μ l RPMI-1640 and mixed with 4 μg of plasmid DNA，which was also diluted in 100 μ l RPMI-1640. After incubation for 30 min at room temperature to allow formation of DNA-liposome complexes，the lipid-DNA solution was added to the cells，mixed gently and incubated for 5 h at 37℃ , 5% CO2. Following incubation，1 ml of complete medium was added to each well. Gene activity was assayed on the 2nd day after transfection. Cell lysis and Western blots were performed as described〔19〕，a mouse MAb directed against c-abl was bought from Oncogene Science.
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1.7 CTL generation
After thawing， the cryopreserved non-adherent lymphocytes were stimulated with irradiated (30 Gy) fused cells at a stimulator-to-responder ratio of 1∶ 3. The mixed cells were cultured in RPMI 1640 (10% FCS) containing 10 ng/ml of IL-2 (Biotinge Biomedicine Co. Beijing， China). 0n day 7，the lymphocytes were restimulated with irradiated (30 Gy) fused cells (1∶5 stimulator-to-responder ratio)，and CTL assays were performed on day 5～6 postre-stimulation， in which DCs alone were used as control.
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1.8 T-cell cytotoxicity assay
T-cell cytotoxicity was measured in vitro using the lactate dehydrogenase (LDH) release assay according to the manual of reagent kit for CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega，WI，USA).Briefly，Jurkat cells (p210+) and Jurkat cells (pEGFP+ ) used as target cells were cocultured with effector T cells at various ratios for 6h in 96-well round-bottomed plates in RPMI-1640. Spontaneous release of effector and target cells was controlled by separate incubation of the respective cell populations.The percentage of cytotoxicity was calculated according to the following formula：cytotoxicity % = [(E-Se-St)]/[(Mt-St)]× 100. Where E stands for the LDH release by effector-target coculture; Se，the spontaneous release by effector cells alone; St，the spontaneous release by target cells alone; and Mt， the maximal release by target cells.
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2 RESULTS
2.1 Generation of dendritic cells in vitro
Freshly isolated PBMCs appeared as dispersed，spherical cells with a smooth surface by fluorescence microscopy (Fig 1). After 2～3 days of incubation，the cells appeared larger and aggregated into nonadherent grape-like clusters with short projections extending from the surface. Between days 5～7，in clusters or dispersed，nonadherent cells with large-cell bodies and long dendritic projections were the predominant population. In addition，there were minor populations of strongly adherening macrophages and loosely adherening mononuclear cells.Starting with 5× 108 PBMCs，we typically ended up with more than 3× 107 dendritic cell precursors; the purity of these cells was ～70%.Figure 2 shows the cell surface phenotype of DC precursors using flow cytometric analyses. The cells contained markers typical of dendritic cells〔18〕，i.e.，they were positive for HLA-DR，HLA-A，B，C，CD1α，CD80，CD86 and negative for CD14 (monocytes) (Fig 2).
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Fig 1 Appearance of human monocyte-derived DCs in culture：A)
adherent monocytes used for initiation of dendritic cells; B)
after 3 days，culture with GM-CSF and IL-4，monocytes clustered into aggregates typical of dendritic cell precursors; C)on day 6，DCs with obviously dendritic processes， and some untransformed cell clusters.
Fig 2 Flow cytometry evaluation of cell surface phenotype of DCs derived from human PBMCs on day 6 of incubation in the presence of GM-CSF and IL-4.
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2.2 Fusion of DCs and K562 cells
The direct fusion of DCs with tumor cells could result in an effective enhancement of tumor-specific immunogenicity，either by conferring sufficient DC function to tumor cells for activation of T cells or by facilitating the delivery of tumor Ags to DCs for processing and presentation.To observe the results of fusion of DCs and K562 cells，K562 cells were pre-transfected with AdRSVGFP at a MOI of 400 p.f.u/cell. We found that more than 90% of the cells were positive for GFP(Fig 3) 48 h after infection.Twenty-four hours after fusion of DCs and K562 cells (GFP+ )，the fusion cells were morphologically demonstrated distinct cell aggregates that could not be easily disrupted. Moreover， most of the fusion cells that expressed GFP， exhibited a DC morphology with veiled processes and dendrites (Fig 4).
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Fig 3 Expression of GFP in K562 cells. A) transmitted light micrograph
of K562 cells 48 h after infection with AdRSVGFP; B) same field as in A) under fluorescence microscope using a fluorescein filter set.
Fig 4 Twenty-four hours after fusion of DCs with K562 cells (GFP+)，the fusion cells demonstrated disti-nct cell aggregates，and expressed GFP，exihibited typical DC morphology (A，B，C).A) and B) are the same field under fluorescence microscope using a fluoscein filter set.
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2.3 Expression of p210BCR-ABL protein in p210BCR-ABL-transferred Jurkat cells
On the 2nd day after transfection with the plasmid p210BCR-ABL in Jurkat cells， Western blot analysis indicated that p210BCR-ABL could be expressed in the p210BCR-ABL-transferred Jurkat cells， and not expre-ssed in the pEGFP-transferred Jurkat cells (Fig 5).
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Fig 5 Western-blot analyses showing the expression of p210BCR-ABL.Whole-cell lysates were elestropho-retically separated on 0.1% SDS～7.5% PAGE and blotted onto nitrocellulose filters，and blots were hybridized with an antibody directed against c-abl. Lane 1 represents lysate from K562 cells served as positive control. Lane 2 contains the lysate of pEGFP-transferred Jurkat cells and Lane 3～5，the lysate of p210BCR-ABL-transferred Jurkat cells.
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HLA-matched，p210BCR-ABL-transferred-Jurkat cells or pEGFP-transferred Jurkat cells were used as targets in CTL cytotoxicity assays (Fig 6). K562 cells were also tested as targets to assess the extent of non-MHC-restricted cytotoxicity of the fusion cells against p210BCR-ABL -positive cells. After two rounds of restimulation with fusion cells (DCs/K562)，a killing rate of 23% was detected on p210BCR-ABL-trans ferred-Jurkat cells at an E：T ratio of 40：1，but very low cytotoxicity was shown towards pEGFP-transferred Jurkat cells.Very low levels of cytotoxicity were observed with K562 target cells. As expected，DCs alone stimulated T cells showed only little response. The results suggest that the cytotoxicity is directed at MHC-matched p210BCR-ABL-positive cells，and very low levels of non-MHC-restricted cytotoxicity.
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Fig6 CTLs were tested in cytotoxicity assays after two rounds of stimulation with
the autologous DCs fused with K562 cells, and the fusion cells can induce a p210BCR-ABL protein-specific CTL in vitro. The data points shown were calculated using the mean value of triplicate cultures.
3 DISCUSSION
Among hematologic malignancies，CML is being intensively evaluated as a possible candidate of DC-based immunotherapy.It is well known that CML is characterized by a specific translocation of the c-abl oncogene (9q34) to the bcr regionon chromosome 22 (22q11)〔2〕，resulting in generation of p210BCR-ABL protein which is an attractive target for induction of a MHC-restricted cytotoxic T-cell immunity againist tumors using antigen-presenting cells〔3，4〕，and now there is a growing list of different peptides identified from the p210BCR-ABL protein that bind to HLA alleles(HLA-A2.1，A3，A11，B8 and HLA-DR1 -4，-3，-11，15)〔20～24〕and elicit CTL in vitro，so it is possible that DCs can bind exogenous peptides to their MHC class molecules，and lead to induction of cytotoxic T cells. However，the use of DCs pulsed ex vivo with these synthetic bcr-abl peptides for effective cell therapy in clinical practice is likely to be hampered by a) the limited availability of patients with HLA genotype compatible for the utilized bcr-abl antigen peptide(s)，b) the occurrence of independent mechanisms facilitating tumor cells to escape in vivo such as loss of expression of bcr-abl antigen peptide(s) during tumor progression，and c) the short duration of the immune response thus requiring annoying boost vaccinations.In this regard，we have tried to take some other ways in transducing p210BCR-ABL gene into human monocyte-derived DCs to overcome the above-mentioned limitations，such as construction of recombinant adenovirus carrying p210BCR-ABL gene，however，the 8.5 kb of p210BCR-ABL gene is too large to generate recombinant adenovirus;and to directly transfer the plasmid p210BCR-ABL to DCs with cationic liposomes has also failed. Nevertheless，DCs fused with p210BCR-ABL protein-positive K562 cells may lead to cytoplasmic express of several different peptide fragments，among which the cell will ultimately select those fitting its own MHC molecules.In this way the fusion cells could induce a higher p210BCR-ABL protein-specific CTL frequency.Intriguingly，in CML〔25〕，DCs have been generated from Ph+ cells and these Ph+ DCs are capable of stimulating cytolytic T-cell responses against the parent leukemia cells，and our present work demonstrates that DCs fused with K562 cells can effectively induce a p210BCR-ABL protein-specific CTL response in vitro which may broaden the spectrum of possible DC-based clinical applications. On the other hand，DCs used in the present experiment were generated from human PBMCs having HLA-A25，B7 molecules， while the target cells were Jurkat cells having HLA-A9，A25，B7，B41 molecules. From these genetic backgrounds it is suggested that in the fusion protein (p210BCR-ABL protein) there should be a short polypeptide being able to bind HLA-A25 or B7 molecules. Whether it is true awaits confirmation in further studies.
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4 ACKNOWLEDGMENT
We thank Mr. Zhang Shuangxi (Beijing Institute of Basic Medical Sciences， Beijing， China) for technical assistance with flow cytometry and Dr. Kong Fanhua (Beijing Beitaiping Road Hospital，Beijing，China) for help in the blood typing， and discussions and Dr. Wang Fusheng and Prof. Li Yuanmin for review of the manuscript.
*通讯作者：Tel：86-10-66930008 Fax：86-10-68158311 E-mail：wuct@nic.bmi.ac.cn
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作者单位：军事医学科学院放射医学研究所三室(北京100850)
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收稿日期：1999-09-08
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单位：
关键词：树突状细胞；K562；细胞融合；P210蛋白；特异性CTL
癌症990601Human peripheral blood mononuclear cells (PBMCs)-derived dendritic
cells fused with K562 cells induce a p210BCR-ABL protein-specific
cytotoxic T cell response in vitro
FAN Guo-chang，WU Chu-tse*，WANG Yan-fei，et al.Department of Experimental Hematology，Beijing Institute of Radiation Medicine，Beijing 100850，P.R.China
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【Abstract】 Objective：To investigate whether human dendritic cells (DCs) derived from cultured human peripheral blood mononuclear cells (PBMCs) were capable of presenting antigen to induce a p210BCR-ABL protein-specific CTL response in vitro after fusion with K562 cells which express p210BCR-ABL protein.Methods：DCs were derived from human PBMCs in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). To show the successful fusion of DCs with K562 cells，we first used K562 cells expressing green fluorescence protein(GFP) in one group， while in the other group，DCs fused with normal K562 cells，and these fusion cells were cocultured with autologous T lymphocytes for two rounds.Results：After 5～7 days of incubation，these adherent cells appeared in clusters or dispersed，and the results of flow cytometric analysis showed the cell surface contained markers typical of dendritic cells，i.e.，they were positive for HLA-DR，HLA-A，B，C，CD1α ，CD80，CD86 and negative for CD14 (monocytes). The fusion cells demonstrated morphologically distinct cell aggregates. Moreover，most of these cells that expressed GFP， exhibited a DC morphology with veiled processes and dendrites. Furthermore，these fusion cells can induce a p210BCR-ABL protein-specific cytotoxic T cell response in vitro using the lactate dehydrogenase (LDH) release assay.Conclusions：The direct fusion of DCs with K562 cells could result in induction of an effective p210BCR-ABL (b3a2) protein-specific immunogen， either by confering sufficient DC function to K562 cells for activation of T cells or by facilitating the delivery of tumor p210BCR-ABL (b3a2) protein to DCs for processing and presentation，which may broaden the spectrum of possible DC-based clinical applications.
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Key words： Dendritic cells； K562； Cell fusion； P210BCR-ABL protein； Specific CTL
【摘要】目的：探讨人外周血单核细胞来源的树突状细胞(Dendritic cells，DC)与人白血病细胞K562融合后，能否诱导出p210蛋白特异性的CTL，为DC疫苗的临床应用提供理论基础。方法：外周血来源的贴附单核细胞，在人GM-CSF(1000U/ml)和IL-4(1000U/ml)作用下，培养5～7天后，与人白血病细胞K562进行融合，融合细胞再与自体的淋巴细胞共同孵育10～14天，采用乳酸脱氢酶释放试验分析其对p210蛋白阳性细胞的杀伤效果。为显示融合效果，对照试验组K562细胞用绿色荧光蛋白(GFP)进行标记。结果：贴附的单核细胞在GM-CSF和IL-4作用下，培养5天后，约有70%的DC产生，流式细胞仪分析表明，这些细胞为HLA-DR、HLA-A，B，C以及CD1α阳性，并高表达共刺激分子CD80和CD86。DC与GFP标记的K562细胞融合24h后，表达GFP蛋白的细胞中约有60%呈现出典型的DC形态特征。乳酸脱氢酶释放试验结果表明，融合细胞能激活淋巴细胞产生p210蛋白特异性的CTL。结论：树突状细胞与肿瘤细胞融合后，能诱导出肿瘤特异性的CTL，显示出DC疫苗抗肿瘤作用的广泛前景。
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中图分类号：R735.3 文献标识码：A 文章编号：1000-467X(1999)06-0617-07
The bcr-abl oncoprotein (a p210BCR-ABL protein)， the product of the fused bcr-abl gene in the Philadelphia (Ph.) chromosome is known to be chronic myelogenous leukemia (CML) specific and critical to leukemogenic process〔1～4〕， but if p210-positive cells are proved to be susceptible targets it will become critical to optimize any vaccination schedule for effective presentation of bcr-abl peptide to the immune system. The dendritic cells (DCs) have the capacity to migrate through tissues，can uptake，process and present antigen， and stimulate both a naive and memory T-cell response〔5〕.Recent studies have indicated that in respect of inducing an antitumor immune response DC is more effective than other approaches，such as viral vectors expressing tumors antigens，gene-modified tumor cells，naked DNA， or peptide emulsified in adjuvant〔6〕.DC-based vaccines are now under active investigation and many vehicles for the delivery of tumor antigen to DCs are being considered： viral vectors，naked and plasmid DNA，RNA，liposomes with nucleic acid or protein，and tumor lysates，apoptotic cells and peptides. Antigen has even been presented by fusion of DCs with whole tumor cells〔7～17〕.
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DC-tumor fusion cells have the potential to stimulate immunity against multiple tumor Ags and could induce synergistic protection through CD4+ and CD8+ T cell-mediated immune responses. Celluzzi et al〔16〕showed that short-term coculture of DCs and tumor cells， with or without prior fusion， resulted in a potent immunogen capable of inducing CTL-mediated protective antitumor immunity and regression of established tumors. Gong et al〔10〕also showed that the fusion cells could stimulate naive T cells in the primary mixed lymphocyte reaction and induce MC38 tumor-specific CTLs in vivo.
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The purpose of the present study was to investigate whether human DCs after fused with K562 cells expressing the p210BCR-ABL protein，were capable of presenting antigen to induce a p210BCR-ABL protein-specific CTL response in vitro. In one group，we first used K562 cells that expressed green fluorescence protein(GFP)，and after the fusion of DCs with K562 cells，the fusion cells not only expressed GFP ， but also exhibited a DC morphology with veiled processes and dendrites; while in the other group，after DCs fused with normal K562 cells，fusion cells were then cocultured with autologous T lyphocytes for two rounds， resulting in stimulating naive T cells and inducing a p210BCR-ABL protein-specific CTLs in vitro.
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1 MATERIALS AND METHODS
1.1 Cell lines and culture condition
Jurkat (HLA-A9，A25，B7，B41)，a T leukemia/lymphoma cell line，and K562，a human leukemia cell line，lacking MHC I and II and expressing the b3a2 translocation，were incubated in RPMI-1640 medium(Gibco，BRL，Grand Island，NY) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco，BRL)，25 mmol/L HEPES，2 mmol/L L-glutamine (Sigma，St. Louis，MO) and antibiotics (penicillin 100 U/ml and streptomycin 100 μg/ml)，in 5% CO2 atmosphere at 37℃ .
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1.2 Generation of DCs in vitro
DCs were prepared from PBMCs as previously described〔18〕.Briefly，PBMCs were isolated from leukapheresed blood of healthy donor (HLA-A25，B7) by density centrifugation on Ficoll-Hypaque grad(ents1.077g/ml)(Bio-tech Co.，Institute of Hematology，CAMS，Tianjing，China) for 20 min at 2000 rpm at room temperature. After four to five washes in PBS to remove platelets，cells were resuspended at 107/ml in RPMI-1640 medium and incubated for 1.5～2 h in 75-cm2 tissue culture flasks (Nunc， Denmark) (5% CO2，37℃ ). Then nonadherent cells were gently washed out with PBS and cryopreserved for use as T cell responder. The remaining adherent cells were further cultured (5% CO2， 37℃ ) in RPMI-1640 medium supplemented with 10% FCS， 1000 U/ml rhGM-CSF (Biotinge Biomedicine Co. Beijing，China) and 1000 U/ml rhIL-4 (Promega， Madison， WI). After 5～7 days，nonadherent cells were harvested. DCs generated in this way were 60% -80% pure based on morphology and the expression of a CD14-， CD1α+，CD80+，CD86+，MHC class I and II+ immunophenotype as assessed by flow cytometry.
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1.3 Flow cytometry
For immunophenotyping，DCs were washed and resuspended in 0.5 ml (1× 106 cells/ml) PBS supplemented with 0.2% FCS and 0.1% NaN3 and incubated (at 4℃ in dark place for 30 min) with 5 μ l of one of the following mAb：PE-conjugated anti-CD14，PE-conjugated anti-HLA-A，B，C (Immunotech，Hamburg，Germany)， PE-conjug-ated anti-CD1α (Coulter，Krefeld，Germany)，FITC-con-jugated anti-HLA-DR，FITC-conjugated anti-CD80，FITC-conjugatedanti-CD86 (Caltag Laboratories，Bur-lingame，CA).Surface expression was analyzed using a FCAScan flow cytometer (FACS440，Be-cton，Dickinson).
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1.4 Adenovirus-mediated transfer of GFP gene into K562 cells
K562 cells，incubated in freshly prepared RPMI -1640 medium supplemented with 10% FCS 24 h before infection，were washed twice with PBS and centrifuged.The recombinant virus Ad5RSVGFP (kindly provided by Dr. DAI Yifan from Baxter Inc.，USA) was added to 2× 106 resuspended cells in serum-free medium at a multiplicity of infections (MOI) of 400 p.f.u/cell，and incubated at 37℃，5% CO2 for 1.5～2 h in a small volume (< 500 μl). Culture medium was then added in order to obtain a cell concentration of 0.5× 106 /ml and incubation was continued for 48 h.Then they were used for cell fusion to evaluate the effect of such fusion.A part of K562 cells would be used for induction of CTLs in vitro.
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1.5 Fusion of DCs and K562 cells
On the 6th day of culture DCs were mixed with K562 cells at a ratio of 5∶1 for cell fusion by adding 40% polyethylene glycol(PEG)(Promega，WI，USA)，which had been warmed to 37℃ as described〔16〕.After PBS washing，fused cells were cultured overnight at 37℃ in RPMI-1640 (10% FCS) containing 1000 U/ml rhGM-CSF (Biotinge Biomedicine Co. Beijing，China) and 800 U/ml rhIL-4 (Promega，Madison，WI). Fluorescence and light microscopies were used to evalute the efficiency of cell fusion and the morphology of fused cells. These GFP labeled fused cells were not used to induce CTL generation.
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1.6 LipofectAMINE -mediated gene transfer to Jurkat cells
Plasmid EGFP， carrying a red-shifted humanized variant of the GFP gene，was driven by the CMV-IE promoter，and the plasmid p210BCR-ABL，containing bcr-abl (b3a2) fusion gene of 8.5 Kb，was driven by CMV promoter.For liposomal gene transfer，2×106 cells were washed once in serum-free RPMI-1640 without antibiotics and then resuspended in 0.8 ml culture medium and seeded in a six-well plate. Lipofection with lipofectAMINE (Gibco BRL，Madison，WI) was performed according to the manufacturer’s instructions. Briefly，increasing amounts of lipofect-AMINE (2～25 μ l) were diluted in 100 μ l RPMI-1640 and mixed with 4 μg of plasmid DNA，which was also diluted in 100 μ l RPMI-1640. After incubation for 30 min at room temperature to allow formation of DNA-liposome complexes，the lipid-DNA solution was added to the cells，mixed gently and incubated for 5 h at 37℃ , 5% CO2. Following incubation，1 ml of complete medium was added to each well. Gene activity was assayed on the 2nd day after transfection. Cell lysis and Western blots were performed as described〔19〕，a mouse MAb directed against c-abl was bought from Oncogene Science.
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1.7 CTL generation
After thawing， the cryopreserved non-adherent lymphocytes were stimulated with irradiated (30 Gy) fused cells at a stimulator-to-responder ratio of 1∶ 3. The mixed cells were cultured in RPMI 1640 (10% FCS) containing 10 ng/ml of IL-2 (Biotinge Biomedicine Co. Beijing， China). 0n day 7，the lymphocytes were restimulated with irradiated (30 Gy) fused cells (1∶5 stimulator-to-responder ratio)，and CTL assays were performed on day 5～6 postre-stimulation， in which DCs alone were used as control.
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1.8 T-cell cytotoxicity assay
T-cell cytotoxicity was measured in vitro using the lactate dehydrogenase (LDH) release assay according to the manual of reagent kit for CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega，WI，USA).Briefly，Jurkat cells (p210+) and Jurkat cells (pEGFP+ ) used as target cells were cocultured with effector T cells at various ratios for 6h in 96-well round-bottomed plates in RPMI-1640. Spontaneous release of effector and target cells was controlled by separate incubation of the respective cell populations.The percentage of cytotoxicity was calculated according to the following formula：cytotoxicity % = [(E-Se-St)]/[(Mt-St)]× 100. Where E stands for the LDH release by effector-target coculture; Se，the spontaneous release by effector cells alone; St，the spontaneous release by target cells alone; and Mt， the maximal release by target cells.
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2 RESULTS
2.1 Generation of dendritic cells in vitro
Freshly isolated PBMCs appeared as dispersed，spherical cells with a smooth surface by fluorescence microscopy (Fig 1). After 2～3 days of incubation，the cells appeared larger and aggregated into nonadherent grape-like clusters with short projections extending from the surface. Between days 5～7，in clusters or dispersed，nonadherent cells with large-cell bodies and long dendritic projections were the predominant population. In addition，there were minor populations of strongly adherening macrophages and loosely adherening mononuclear cells.Starting with 5× 108 PBMCs，we typically ended up with more than 3× 107 dendritic cell precursors; the purity of these cells was ～70%.Figure 2 shows the cell surface phenotype of DC precursors using flow cytometric analyses. The cells contained markers typical of dendritic cells〔18〕，i.e.，they were positive for HLA-DR，HLA-A，B，C，CD1α，CD80，CD86 and negative for CD14 (monocytes) (Fig 2).
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Fig 1 Appearance of human monocyte-derived DCs in culture：A)
adherent monocytes used for initiation of dendritic cells; B)
after 3 days，culture with GM-CSF and IL-4，monocytes clustered into aggregates typical of dendritic cell precursors; C)on day 6，DCs with obviously dendritic processes， and some untransformed cell clusters.
Fig 2 Flow cytometry evaluation of cell surface phenotype of DCs derived from human PBMCs on day 6 of incubation in the presence of GM-CSF and IL-4.
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2.2 Fusion of DCs and K562 cells
The direct fusion of DCs with tumor cells could result in an effective enhancement of tumor-specific immunogenicity，either by conferring sufficient DC function to tumor cells for activation of T cells or by facilitating the delivery of tumor Ags to DCs for processing and presentation.To observe the results of fusion of DCs and K562 cells，K562 cells were pre-transfected with AdRSVGFP at a MOI of 400 p.f.u/cell. We found that more than 90% of the cells were positive for GFP(Fig 3) 48 h after infection.Twenty-four hours after fusion of DCs and K562 cells (GFP+ )，the fusion cells were morphologically demonstrated distinct cell aggregates that could not be easily disrupted. Moreover， most of the fusion cells that expressed GFP， exhibited a DC morphology with veiled processes and dendrites (Fig 4).
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Fig 3 Expression of GFP in K562 cells. A) transmitted light micrograph
of K562 cells 48 h after infection with AdRSVGFP; B) same field as in A) under fluorescence microscope using a fluorescein filter set.
Fig 4 Twenty-four hours after fusion of DCs with K562 cells (GFP+)，the fusion cells demonstrated disti-nct cell aggregates，and expressed GFP，exihibited typical DC morphology (A，B，C).A) and B) are the same field under fluorescence microscope using a fluoscein filter set.
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2.3 Expression of p210BCR-ABL protein in p210BCR-ABL-transferred Jurkat cells
On the 2nd day after transfection with the plasmid p210BCR-ABL in Jurkat cells， Western blot analysis indicated that p210BCR-ABL could be expressed in the p210BCR-ABL-transferred Jurkat cells， and not expre-ssed in the pEGFP-transferred Jurkat cells (Fig 5).
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Fig 5 Western-blot analyses showing the expression of p210BCR-ABL.Whole-cell lysates were elestropho-retically separated on 0.1% SDS～7.5% PAGE and blotted onto nitrocellulose filters，and blots were hybridized with an antibody directed against c-abl. Lane 1 represents lysate from K562 cells served as positive control. Lane 2 contains the lysate of pEGFP-transferred Jurkat cells and Lane 3～5，the lysate of p210BCR-ABL-transferred Jurkat cells.
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HLA-matched，p210BCR-ABL-transferred-Jurkat cells or pEGFP-transferred Jurkat cells were used as targets in CTL cytotoxicity assays (Fig 6). K562 cells were also tested as targets to assess the extent of non-MHC-restricted cytotoxicity of the fusion cells against p210BCR-ABL -positive cells. After two rounds of restimulation with fusion cells (DCs/K562)，a killing rate of 23% was detected on p210BCR-ABL-trans ferred-Jurkat cells at an E：T ratio of 40：1，but very low cytotoxicity was shown towards pEGFP-transferred Jurkat cells.Very low levels of cytotoxicity were observed with K562 target cells. As expected，DCs alone stimulated T cells showed only little response. The results suggest that the cytotoxicity is directed at MHC-matched p210BCR-ABL-positive cells，and very low levels of non-MHC-restricted cytotoxicity.
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Fig6 CTLs were tested in cytotoxicity assays after two rounds of stimulation with
the autologous DCs fused with K562 cells, and the fusion cells can induce a p210BCR-ABL protein-specific CTL in vitro. The data points shown were calculated using the mean value of triplicate cultures.
3 DISCUSSION
Among hematologic malignancies，CML is being intensively evaluated as a possible candidate of DC-based immunotherapy.It is well known that CML is characterized by a specific translocation of the c-abl oncogene (9q34) to the bcr regionon chromosome 22 (22q11)〔2〕，resulting in generation of p210BCR-ABL protein which is an attractive target for induction of a MHC-restricted cytotoxic T-cell immunity againist tumors using antigen-presenting cells〔3，4〕，and now there is a growing list of different peptides identified from the p210BCR-ABL protein that bind to HLA alleles(HLA-A2.1，A3，A11，B8 and HLA-DR1 -4，-3，-11，15)〔20～24〕and elicit CTL in vitro，so it is possible that DCs can bind exogenous peptides to their MHC class molecules，and lead to induction of cytotoxic T cells. However，the use of DCs pulsed ex vivo with these synthetic bcr-abl peptides for effective cell therapy in clinical practice is likely to be hampered by a) the limited availability of patients with HLA genotype compatible for the utilized bcr-abl antigen peptide(s)，b) the occurrence of independent mechanisms facilitating tumor cells to escape in vivo such as loss of expression of bcr-abl antigen peptide(s) during tumor progression，and c) the short duration of the immune response thus requiring annoying boost vaccinations.In this regard，we have tried to take some other ways in transducing p210BCR-ABL gene into human monocyte-derived DCs to overcome the above-mentioned limitations，such as construction of recombinant adenovirus carrying p210BCR-ABL gene，however，the 8.5 kb of p210BCR-ABL gene is too large to generate recombinant adenovirus;and to directly transfer the plasmid p210BCR-ABL to DCs with cationic liposomes has also failed. Nevertheless，DCs fused with p210BCR-ABL protein-positive K562 cells may lead to cytoplasmic express of several different peptide fragments，among which the cell will ultimately select those fitting its own MHC molecules.In this way the fusion cells could induce a higher p210BCR-ABL protein-specific CTL frequency.Intriguingly，in CML〔25〕，DCs have been generated from Ph+ cells and these Ph+ DCs are capable of stimulating cytolytic T-cell responses against the parent leukemia cells，and our present work demonstrates that DCs fused with K562 cells can effectively induce a p210BCR-ABL protein-specific CTL response in vitro which may broaden the spectrum of possible DC-based clinical applications. On the other hand，DCs used in the present experiment were generated from human PBMCs having HLA-A25，B7 molecules， while the target cells were Jurkat cells having HLA-A9，A25，B7，B41 molecules. From these genetic backgrounds it is suggested that in the fusion protein (p210BCR-ABL protein) there should be a short polypeptide being able to bind HLA-A25 or B7 molecules. Whether it is true awaits confirmation in further studies.
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4 ACKNOWLEDGMENT
We thank Mr. Zhang Shuangxi (Beijing Institute of Basic Medical Sciences， Beijing， China) for technical assistance with flow cytometry and Dr. Kong Fanhua (Beijing Beitaiping Road Hospital，Beijing，China) for help in the blood typing， and discussions and Dr. Wang Fusheng and Prof. Li Yuanmin for review of the manuscript.
*通讯作者：Tel：86-10-66930008 Fax：86-10-68158311 E-mail：wuct@nic.bmi.ac.cn
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作者单位：军事医学科学院放射医学研究所三室(北京100850)
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收稿日期：1999-09-08
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