Jantscheff P, Spagnoli G, Zajac P, Rochlitz C F
Kantonspital Basel, Research Department, Molecular Cancer Research-Oncology, Switzerland.
Cancer Immunol Immunother. 2002 Sep;51(7):367-75. doi: 10.1007/s00262-002-0295-1. Epub 2002 Jun 25.
Somatic cell hybrids of HLA-A2(+) EBV-transformed B- or dendritic cells (DC) and allogeneic HLA-A2(-) melanoma cell line Me15 were obtained by in vitro electrofusion using an electroporator. Before fusion, melanoma cells were stably transfected with green fluorescent marker protein (GFP) and neomycin resistance gene (neo(+)). Stably growing hybrid antigen-presenting cells (HAPC) expressing HLA-DR and HLA-A2 (or HLA-A30/31), and melanoma-associated antigens (MART-1, gp100) were selected by a double strategy of immunomagnetic MACS and neomycin selection. Fusion efficiency ranged between 3% and 18% (mean: 8.0+/-4.7%) as defined by simultaneous GFP and HLA-A2 detection. Expression of melanoma-associated antigens (MART-1, gp100) in hybrid cells was determined by reverse transcription-polymerase chain reaction (RT-PCR). HLA-restricted antigen-specific presentation of melanoma antigens was demonstrated by killing of semi-allogenic HAPC by HLA-A2-restricted MART-1 or gp100-specific cytotoxic T lymphocyte (CTL) clones. HLA restriction and antigen specificity were confirmed by inhibition of specific cytotoxicity by anti-HLA antibodies and cold target inhibition. During long-term (42-70 days) neomycin selection of HAPC, a drastic loss of antigen-presenting cell (APC)-derived determinants (e.g. HLA-DR, HLA-A2) was observed which, however, could be "reversed" by repeated MACSorting (days 10, 21 and 49). Our method allows the generation of semi-allogenic HAPC that constitutively proliferate in vitro. This opens the possibility of establishing a number of tumor-APC hybrids expressing defined HLA haplotypes and tumor antigens, of investigating their specific properties (e.g. antigen processing), and testing their diagnostic or therapeutic potential.
使用电穿孔仪通过体外电融合获得了 HLA - A2(+) Epstein - Barr 病毒（EBV）转化的 B 细胞或树突状细胞（DC）与同种异体 HLA - A2(-)黑色素瘤细胞系 Me15 的体细胞杂种。在融合之前，黑色素瘤细胞用绿色荧光标记蛋白（GFP）和新霉素抗性基因（neo(+)）进行稳定转染。通过免疫磁珠 MACS 和新霉素选择的双重策略，筛选出稳定生长的表达 HLA - DR 和 HLA - A2（或 HLA - A30/31）以及黑色素瘤相关抗原（MART - 1、gp100）的杂种抗原呈递细胞（HAPC）。通过同时检测 GFP 和 HLA - A2 确定融合效率在 3%至 18%之间（平均：8.0±4.7%）。通过逆转录 - 聚合酶链反应（RT - PCR）测定杂种细胞中黑色素瘤相关抗原（MART - 1、gp100）的表达。通过 HLA - A2 限制性 MART - 1 或 gp100 特异性细胞毒性 T 淋巴细胞（CTL）克隆杀伤半同种异体 HAPC，证明了 HLA 限制性黑色素瘤抗原的特异性呈递。通过抗 HLA 抗体抑制特异性细胞毒性和冷靶抑制，证实了 HLA 限制性和抗原特异性。在对 HAPC 进行长期（42 - 70 天）新霉素选择期间，观察到抗原呈递细胞（APC）衍生决定簇（如 HLA - DR、HLA - A2）的急剧丧失，然而，通过重复的 MAC 分选（第 10、21 和 49 天）可以“逆转”这种情况。我们的方法允许产生在体外组成性增殖的半同种异体 HAPC。这为建立许多表达特定 HLA 单倍型和肿瘤抗原的肿瘤 - APC 杂种、研究它们的特定特性（如抗原加工）以及测试它们的诊断或治疗潜力开辟了可能性。
