cells fused with K562 cells induce a p210^BCR-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

【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 p210^BCR-ABL protein-specific CTL response in vitro after fusion with K562 cells which express p210^BCR-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 p210^BCR-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 p210^BCR-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 p210^BCR-ABL (b3a2) protein to DCs for processing and presentation，which may broaden the spectrum of possible DC-based clinical applications.

Key words： Dendritic cells； K562； Cell fusion； P210^BCR-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。

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 p210^BCR-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〕.

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.

The purpose of the present study was to investigate whether human DCs after fused with K562 cells expressing the p210^BCR-ABL protein，were capable of presenting antigen to induce a p210^BCR-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 p210^BCR-ABL protein-specific CTLs in vitro.

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℃ .

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^＋