Klingemann H G, Deal H, Reid D, Eaves C J
Terry Fox Laboratory, University of British Columbia, Vancouver, Canada.
Exp Hematol. 1993 Aug;21(9):1263-70.
Previous studies in animal models have suggested that bone marrow-derived interleukin-2 (IL-2) activated natural killer (A-NK*) cells can be used to eliminate residual leukemic cells both in vivo and in vitro. As part of initial studies to develop a clinical protocol to exploit this phenomenon in combination with culture purging of leukemic cells, we examined a number of variables that might affect either IL-2 stimulation of natural killer (NK) cell activity or maintenance of primitive hematopoietic cells in cultures suitable for manipulating human marrow autografts. Cytotoxicity of IL-2 A-NK cells was determined using K562 and Daudi target cells in a standard 4-hour 51Cr release assay. Primitive hematopoietic cells were quantitated using both direct clonogenic progenitor assays and the long-term culture-initiating cell (LTC-IC) assay. The latter measures a very primitive cell type that gives rise to clonogenic cells after > 5 weeks of culture on pre-established marrow feeder layers. Light-density (< 1.070 g/cm3) human marrow cells cultured at 10(6) cells/mL for 7 days at 37 degrees C in the presence of 1000 U of either natural or recombinant human IL-2 showed a marked generation of A-NK cell function that was not seen if IL-2 was not added. The cytotoxicity was the same whether the cells had been maintained in standard fetal calf serum (FCS)-supplemented medium or in LTC medium (which also contains horse serum and 10(-6) M hydrocortisone) or whether adherence of cells during the culture period was promoted or prevented. The level of NK activity in IL-2-stimulated cultures of marrow cells from patients with acute myeloid leukemia (AML) in remission was not significantly different from that obtained with normal marrow. The initial numbers of clonogenic cells in these samples were close to normal values (60%). For both normal and AML patient samples, these declined slightly during the 7-day culture period, regardless of whether IL-2 was present. Starting values for LTC-IC in patient marrow samples were markedly reduced to approximately 7% of normal values, but as in normal cultures, did not decrease further in culture with or without IL-2. These results indicate that activation of bone marrow-derived NK cells can be obtained in 7-day cultures containing IL-2 under conditions that preserve the most primitive hematopoietic cells currently detectable.
先前在动物模型中的研究表明，骨髓来源的白细胞介素-2（IL-2）激活的自然杀伤（A-NK*）细胞可用于在体内和体外消除残留的白血病细胞。作为制定一项临床方案以利用这一现象并结合白血病细胞培养清除的初步研究的一部分，我们研究了一些可能影响IL-2刺激自然杀伤（NK）细胞活性或在适合处理人类骨髓自体移植的培养物中维持原始造血细胞的变量。使用K562和Daudi靶细胞在标准的4小时51Cr释放试验中测定IL-2 A-NK细胞的细胞毒性。使用直接克隆形成祖细胞试验和长期培养起始细胞（LTC-IC）试验对原始造血细胞进行定量。后者测量的是一种非常原始的细胞类型，在预先建立的骨髓饲养层上培养超过5周后可产生克隆形成细胞。低密度（<1.070 g/cm3）的人骨髓细胞在37℃下以10(6) 个细胞/mL的密度培养7天，在存在1000 U天然或重组人IL-2的情况下，显示出A-NK细胞功能的显著产生，如果不添加IL-2则不会出现这种情况。无论细胞是在补充有标准胎牛血清（FCS）的培养基中、在LTC培养基（其中还含有马血清和10(-6) M氢化可的松）中维持，还是在培养期间促进或阻止细胞贴壁，细胞毒性都是相同的。急性髓系白血病（AML）缓解期患者骨髓细胞经IL-2刺激培养后的NK活性水平与正常骨髓获得的值无显著差异。这些样本中克隆形成细胞的初始数量接近正常值（60%）。对于正常和AML患者样本，无论是否存在IL-2，这些数量在7天培养期内都略有下降。患者骨髓样本中LTC-IC的起始值明显降低至正常值的约7%，但与正常培养一样，在有或没有IL-2的培养中都没有进一步下降。这些结果表明，在含有IL-2的7天培养中，在保留目前可检测到的最原始造血细胞的条件下，可以获得骨髓来源NK细胞的激活。
