Hirst W J, Buggins A, Darling D, Gäken J, Farzaneh F, Mufti G J
Departments of Haematological Medicine and Molecular, King's College School of Medicine and Dentistry, London, UK.
Gene Ther. 1997 Jul;4(7):691-9. doi: 10.1038/sj.gt.3300437.
Gene modification of malignant cells to express immune stimulators (cytokines and immune costimulators) has provided the basis for a novel form of immunotherapy. Using a MPSV-based retroviral vector with hygromycin resistance gene as a selectable marker, we have studied retrovirus-mediated gene transfer of an immune costimulator, B7.1, into primary human acute myeloid leukaemia (AML) cells and the subsequent induction of immune costimulatory function. AML blasts from 10 patients were transduced by co-culture for 48 h with or without haemopoietic growth factors (HGFs). In the absence of HGFs, transduction efficiency (TE), as judged by % B7.1 expressing cells, was low, varying from 0.3 to 8.2% (median 1.5%). Addition of HGFs increased the median TE 1.8-fold with stem cell factor alone and 2.6-fold with SCF, interleukin-3 and GM-CSF. Effects on cell cycling alone could not explain this difference, suggesting other factors such as virus binding and promoter activity, are also involved. CFU-AL assays indicated a higher transduction efficiency of clonogenic cells, which was not improved by growth factors. Limited duration of cell growth prevented significant expansion of transduced populations by culture in the presence of hygromycin. Although not increasing transduction efficiency, CD34 enrichment enhanced drug selection, by targeting cells with the greatest self-renewal capacity. Immunoselection of B7.1 expressing cells produced transduced populations with 30-60% expressing B7.1. In an allogeneic mixed leukaemic cell/T lymphocyte reaction (MLLR) transduced AML cells enriched by immunoselection were able to stimulate allogeneic T cells (CD4 and CD8 positive), which could be inhibited by a solubilised B7 receptor, CTLA4.Ig. Our results demonstrate that using a replication incompetent retroviral vector, it is possible to introduce the immune costimulator B7.1 into primary AML-blasts and by immunoselection, enrich the transduced cells, which may be used for subsequent administration as an autologous cellular vaccine.
对恶性细胞进行基因改造以表达免疫刺激因子（细胞因子和免疫共刺激分子）为一种新型免疫疗法奠定了基础。我们使用了一种基于MPSV的带有潮霉素抗性基因作为选择标记的逆转录病毒载体，研究了逆转录病毒介导的免疫共刺激分子B7.1基因向原代人急性髓性白血病（AML）细胞的转移以及随后免疫共刺激功能的诱导。将来自10名患者的AML原始细胞与有或无造血生长因子（HGFs）共同培养48小时进行转导。在没有HGFs的情况下，以表达B7.1的细胞百分比判断的转导效率（TE）较低，范围从0.3%到8.2%（中位数为1.5%）。添加HGFs后，单独使用干细胞因子时中位数TE增加了1.8倍，使用干细胞因子、白细胞介素-3和粒细胞巨噬细胞集落刺激因子时增加了2.6倍。仅对细胞周期的影响无法解释这种差异，提示还涉及其他因素，如病毒结合和启动子活性。集落形成单位-AML（CFU-AL）测定表明克隆形成细胞的转导效率更高，生长因子并未改善这一效率。细胞生长持续时间有限，阻止了在潮霉素存在下通过培养使转导群体显著扩增。虽然未提高转导效率，但CD34富集通过靶向具有最大自我更新能力的细胞增强了药物选择。对表达B7.1的细胞进行免疫选择产生了30%至60%表达B7.1的转导群体。在同种异体混合白血病细胞/T淋巴细胞反应（MLLR）中，通过免疫选择富集的转导AML细胞能够刺激同种异体T细胞（CD4和CD8阳性），这可被可溶性B7受体CTLA4.Ig抑制。我们的结果表明，使用无复制能力的逆转录病毒载体，有可能将免疫共刺激分子B7.1引入原代AML原始细胞，并通过免疫选择富集转导细胞，这些细胞可作为自体细胞疫苗用于后续给药。
