Levine B L, Bernstein W B, Connors M, Craighead N, Lindsten T, Thompson C B, June C H
Henry M. Jackson Foundation for the Advancement of Military Medicine, U.S. Military HIV Research Program, Bethesda, MD 20889, USA.
J Immunol. 1997 Dec 15;159(12):5921-30.
In this report, conditions for prolonged in vitro proliferation of polyclonal adult CD4+ T cells via stimulation with immobilized anti-CD3 plus anti-CD28 have been established. CD4+ cells maintained exponential growth for more than 60 days during which a total 10(9)- to 10(11)-fold expansion occurred. Cell cultures exhibited cyclical changes in cell volume, indicating that, in terms of proliferative rate, cells do not have to rest before restimulation. Indeed, electronic cell size analysis was the most reliable method to determine when to restimulate with additional immobilized mAb. The initial approximately 10(5)-fold expansion was autocrine, occurring in the absence of exogenous cytokines or feeder cells. Addition of recombinant human IL-2 after the initial autocrine expansion resulted in continued exponential proliferation. Phorbol ester plus ionomycin also induced long-term growth when combined with anti-CD28 stimulation. Analysis of the T cell repertoire after prolonged expansion revealed a diverse repertoire as assessed by anti-TCR Vbeta Abs or a PCR-based assay. Cytokines produced were consistent with maintenance of both Th1 and Th2 phenotypes; however, the mode of CD3 and CD28 stimulation could influence the cytokine secretion pattern. When anti-CD3 and anti-CD28 were immobilized on the same surface, ELISAs on culture supernatants revealed a pattern consistent with Th1 secretion. Northern analysis revealed that cytokine gene expression remained inducible. Spontaneous growth or cell transformation was not observed in more than 100 experiments. Together, these observations may have implications for gene therapy and adoptive immunotherapy. Furthermore, these culture conditions establish a model to study the finite lifespan of mature T lymphocytes.
在本报告中，已建立了通过固定化抗CD3加抗CD28刺激使多克隆成年CD4 + T细胞在体外长期增殖的条件。CD4 +细胞维持指数生长超过60天，在此期间总共发生了10^9至10^11倍的扩增。细胞培养物表现出细胞体积的周期性变化，这表明就增殖率而言，细胞在再次刺激前无需休息。实际上，电子细胞大小分析是确定何时用额外的固定化单克隆抗体进行再次刺激的最可靠方法。最初约10^5倍的扩增是自分泌的，在没有外源性细胞因子或饲养细胞的情况下发生。在最初的自分泌扩增后添加重组人IL-2导致持续的指数增殖。佛波酯加离子霉素与抗CD28刺激联合使用时也可诱导长期生长。长期扩增后对T细胞库的分析显示，通过抗TCR Vβ抗体或基于PCR的检测评估，其具有多样性。产生的细胞因子与Th1和Th2表型的维持一致；然而，CD3和CD28刺激的方式可能影响细胞因子分泌模式。当抗CD3和抗CD28固定在同一表面上时，对培养上清液的ELISA显示出与Th1分泌一致的模式。Northern分析显示细胞因子基因表达仍可诱导。在超过100次实验中未观察到自发生长或细胞转化。总之，这些观察结果可能对基因治疗和过继性免疫治疗有影响。此外，这些培养条件建立了一个研究成熟T淋巴细胞有限寿命的模型。
