Kurnick J T, Warrens A R, Moscicki R A, Leary C P
Clin Immunol Immunopathol. 1983 Jun;27(3):444-51. doi: 10.1016/0090-1229(83)90096-x.
The culture of human T lymphocytes in interleukin-2 (IL-2) containing growth factor medium results in a significant shift in the T-lymphocytes subsets isolated from such cultures at weekly intervals. If normal peripheral blood mononuclear cells are stimulated with phytohemagglutinin (PHA) or in a mixed lymphocyte reaction (MLR), the resulting T lymphoblasts can be propagated in growth factor medium. Staining of the cultured cells with monoclonal antibodies was evaluated by indirect immunofluorescence on a laser-activated flow cytometer (Ortho Spectrum III). The antibodies used were: OKT3 (mature T lymphocytes), OKT4 (helper/inducer T lymphocytes), OKT8 (cytotoxic/suppressor T lymphocytes, OKT10 (immature and "activated" lymphocytes), OKT11a (cells which rosette with sheep erythrocytes), and OKIa-I (HLA-DR constant region). Both PHA and MLR activation resulted in initial preservation of the OKT4+ subset predominance over OKT8+ T lymphocytes noted on normal circulating blood lymphocytes. However, during culture in T-cell growth factor medium, there was a progressive increase in the percentage of OKT8+ cells, and a concomitant decrease in OKT4+ lymphoblasts. The increase in OKT8+ cells in the MLR-stimulated cultures was paralleled by an increase in specific cell-mediated cytotoxicity against the stimulating lymphocyte population. In addition to the shift in T-lymphocyte subset, there was virtual 100% staining with OKT3 and OKT11a, indicating the T-cell nature of the proliferating cells. OKT10 which was present on a small subset of fresh blood lymphocytes appeared rapidly in stimulated cultures, and was retained on virtually all lymphoblasts of either OKT4+ or OKT8+ subset. OKIa-1 cells increased slowly in PHA-stimulated cultures. HLA-DR+ T cells were detected earlier in MLR cultures. The activation of T lymphocytes results in a significant increase in the number of molecules of OKT11a bound per cell, in concert with the increased avidity of T lymphoblasts for sheep erythrocytes. The significant change in the phenotype and function of lymphoblasts isolated from long-term cultures demonstrates the importance of monitoring cultures, and the potential hazards in equating a cultured cell population with a freshly isolated one.
在含白细胞介素 -2（IL -2）的生长因子培养基中培养人T淋巴细胞，会导致每周从这类培养物中分离出的T淋巴细胞亚群发生显著变化。如果用植物血凝素（PHA）刺激正常外周血单个核细胞或在混合淋巴细胞反应（MLR）中刺激，产生的T淋巴母细胞可在生长因子培养基中增殖。通过在激光激活流式细胞仪（Ortho Spectrum III）上进行间接免疫荧光评估，用单克隆抗体对培养细胞进行染色。所用抗体如下：OKT3（成熟T淋巴细胞）、OKT4（辅助/诱导性T淋巴细胞）、OKT8（细胞毒性/抑制性T淋巴细胞）、OKT10（未成熟和“活化”淋巴细胞）、OKT11a（与绵羊红细胞形成玫瑰花结的细胞）以及OKIa -I（HLA -DR恒定区）。PHA和MLR激活均导致最初OKT4 +亚群相对于正常循环血淋巴细胞中所观察到的OKT8 + T淋巴细胞保持优势。然而，在T细胞生长因子培养基中培养期间，OKT8 +细胞的百分比逐渐增加，同时OKT4 +淋巴母细胞减少。MLR刺激培养物中OKT8 +细胞的增加与针对刺激淋巴细胞群体的特异性细胞介导细胞毒性的增加平行。除了T淋巴细胞亚群的变化外，OKT3和OKT11a的染色率几乎达到100%，表明增殖细胞的T细胞性质。存在于一小部分新鲜血液淋巴细胞上的OKT10在刺激培养物中迅速出现，并几乎保留在OKT +细胞或OKT8 +亚群的所有淋巴母细胞上。在PHA刺激的培养物中OKIa -1细胞缓慢增加。在MLR培养物中更早检测到HLA -DR + T细胞。T淋巴细胞的激活导致每个细胞结合的OKT11a分子数量显著增加，这与T淋巴母细胞对绵羊红细胞亲和力的增加一致。从长期培养物中分离出的淋巴母细胞在表型和功能上的显著变化表明监测培养物的重要性，以及将培养细胞群体等同于新鲜分离细胞群体的潜在风险。
