Verfaillie C, Miller W, Kay N, McGlave P
Department of Medicine, University of Minnesota, Minneapolis.
Blood. 1989 Aug 1;74(2):793-7.
We generated a homogeneous population of cells with cytotoxic activity termed "adherent lymphokine-activated killer" (ALAK) cells from the peripheral blood of nine patients in the chronic phase of Ph1 positive chronic myelogenous leukemia (CML). The selective enrichment of CML ALAK cells depended on their propensity to adhere to plastic and proliferate when cultured in the presence of recombinant interleukin-2 (rIL-2) for 14 days. Culture of peripheral blood mononuclear cells under these conditions resulted in growth of a uniform population of cells with morphologic characteristics of large granular lymphocytes. The NKH1+/CD3- phenotype associated with IL-2-stimulated natural killer (NK) cells was present on 79% +/- 9% of cells. Absence of colony formation in conditions promoting the growth of CFU-GEMM indicated that the CML ALAK population was not contaminated with viable hematopoietic progenitors. Cytogenetic analysis of the CML ALAK population revealed 119/120 Ph1 negative metaphases and l/120 Ph1 positive metaphase in six patients. Southern blot analysis of CML ALAK failed to demonstrate a bcr gene rearrangement in seven patients known to have a bcr gene rearrangement in myeloid cells. Comparison of ALAK populations derived from peripheral blood of CML patients and normals revealed similar cytotoxicity against the NK-sensitive K562 cell line (104 +/- 36 LU v 88 +/- 19 LU; P = NS) and the NK-resistant Raji cell line (93 +/- 26 LU v 98 +/- 28 LU; P = NS). The proliferative capacity of CML ALAK cells (101 +/- 33 fold expansion) exceeds the growth potential of the normal ALAK cells (22.3 +/- 3 fold expansion; P = .02). Direct comparison of equal numbers of CML ALAK cells and a CML LAK cell population produced by incubation of peripheral blood mononuclear cells in rIL-2 for 14 days without adherence revealed that the CML LAK population had significantly lower lytic activity against K562 and Raji cell lines. We are able to expand CML peripheral blood mononuclear cells to provide a population of ALAK cells with potent cytotoxic activity. The CML ALAK population is relatively homogeneous, not contaminated with viable stem cells, not derived from a malignant lineage, and more cytotoxic than equal numbers of CML LAK cells. Further studies are underway to determine if this ALAK population may be effective in autologous killing of chronic myelogenous leukemia stem cells.
我们从9例处于Ph1阳性慢性粒细胞白血病（CML）慢性期患者的外周血中，培养出了具有细胞毒性活性的均一细胞群体，称为“贴壁淋巴因子激活的杀伤细胞”（ALAK细胞）。CML-ALAK细胞的选择性富集取决于它们在重组白细胞介素-2（rIL-2）存在下培养14天时，贴附于塑料培养皿并增殖的倾向。在这些条件下培养外周血单个核细胞，可产生具有大颗粒淋巴细胞形态特征的均一细胞群体。与IL-2刺激的自然杀伤（NK）细胞相关的NKH1+/CD3-表型存在于79%±9%的细胞中。在促进CFU-GEMM生长的条件下未形成集落，表明CML-ALAK细胞群体未被存活的造血祖细胞污染。对CML-ALAK细胞群体的细胞遗传学分析显示，6例患者中有119/120个Ph1阴性中期相和1/120个Ph1阳性中期相。对已知在髓系细胞中有bcr基因重排的7例患者的CML-ALAK细胞进行Southern印迹分析，未发现bcr基因重排。比较来自CML患者和正常人外周血的ALAK细胞群体，发现它们对NK敏感的K562细胞系（104±36 LU对88±19 LU；P=无显著性差异）和NK抗性的Raji细胞系（93±26 LU对98±28 LU；P=无显著性差异）具有相似的细胞毒性。CML-ALAK细胞的增殖能力（101±33倍扩增）超过正常ALAK细胞的生长潜力（22.3±3倍扩增；P=0.02）。直接比较等量的CML-ALAK细胞和通过在外周血单个核细胞中加入rIL-2培养14天而不进行贴壁培养产生的CML-LAK细胞群体，发现CML-LAK细胞群体对K562和Raji细胞系的裂解活性显著较低。我们能够扩增CML外周血单个核细胞，以提供具有强大细胞毒性活性的ALAK细胞群体。CML-ALAK细胞群体相对均一，未被存活的干细胞污染，不是来源于恶性谱系，并且比等量的CML-LAK细胞更具细胞毒性。正在进行进一步研究，以确定该ALAK细胞群体是否可能对慢性粒细胞白血病干细胞的自体杀伤有效。
