Amoroso K, Lipsky P E
Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas 75235.
J Immunol. 1990 Nov 15;145(10):3155-61.
Activation of T cells by mAb to the CD3 molecular complex induces the differentiation of many more Ig-secreting cells (ISC) from resting human B cells in bulk cultures than do other modes of polyclonal B cell activation. In the current experiments, a limiting dilution assay was used to demonstrate that this increase in ISC generation reflects an increased frequency of responding B cells. Highly purified B cells were cultured at densities of between 1000 cells and 0.5 cell per microwell with fresh, mitomycin C-treated T cells (T mito) or T cell clones stimulated by immobilized mAb to CD3. After 5 days in culture, the number of wells containing ISC was determined, and the frequency of responding B cells was calculated. The proportion of B cells responding to anti-CD3-stimulated T cells was very large (10.7 +/- 2.8%) and greatly surpassed that induced by other polyclonal activators. B cells cultured with anti-CD3-stimulated T cell clones responded better than did those cultured with T mito. The addition of exogenous IL-2 or IL-6 to cultures supported by activated T mito enhanced the frequency of responding B cells, whereas IL-4 did not increase the generation of ISC and inhibited the augmentation of B cell responses induced by IL-2. Supplementation of cultures with mitomycin C-treated B cells as accessory cells had less of an effect. The addition of both accessory cells and IL-2 markedly increased B cell responsiveness, with precursor frequencies of 60 to 80% noted. In some experiments, cultures were carried out for 7 to 14 days and supernatants were analyzed for IgM, IgG, and IgA secretion. B cells activated by anti-CD3-stimulated T cells produced all three Ig isotypes. When the classes of Ig produced by single B cells were examined, it was observed that the stimulation of individual B cell precursors led to the production of multiple Ig isotypes, suggesting that isotype switching occurs in these cultures. These results demonstrate that under optimum culture conditions, T cells stimulated with immobilized anti-CD3 can activate the majority of human peripheral blood B cells to produce Ig and induce isotype switching by many.
与其他多克隆B细胞激活模式相比，用抗CD3分子复合物的单克隆抗体（mAb）激活T细胞能在大量培养中诱导更多静息人B细胞分化为分泌免疫球蛋白的细胞（ISC）。在当前实验中，采用有限稀释分析法来证明ISC生成的增加反映了反应性B细胞频率的增加。将高度纯化的B细胞以每微孔1000个细胞至0.5个细胞的密度与新鲜的、经丝裂霉素C处理的T细胞（T mito）或由固定化抗CD3 mAb刺激的T细胞克隆一起培养。培养5天后，确定含有ISC的孔数，并计算反应性B细胞的频率。对抗CD3刺激的T细胞产生反应的B细胞比例非常大（10.7±2.8%），大大超过其他多克隆激活剂诱导的比例。与用T mito培养的B细胞相比，与抗CD3刺激的T细胞克隆一起培养的B细胞反应更好。向由活化的T mito支持的培养物中添加外源性白细胞介素-2（IL-2）或白细胞介素-6可提高反应性B细胞的频率，而IL-4不会增加ISC的生成，反而会抑制IL-2诱导的B细胞反应增强。用经丝裂霉素C处理的B细胞作为辅助细胞补充培养物的效果较小。同时添加辅助细胞和IL-2可显著提高B细胞反应性，前体频率为60%至80%。在一些实验中，培养7至14天，并分析上清液中的IgM、IgG和IgA分泌情况。由抗CD3刺激的T细胞激活的B细胞可产生所有三种Ig同种型。当检查单个B细胞产生的Ig类别时，观察到单个B细胞前体的刺激会导致产生多种Ig同种型，这表明在这些培养物中发生了同种型转换。这些结果表明，在最佳培养条件下，用固定化抗CD3刺激的T细胞可激活大多数人外周血B细胞产生Ig，并诱导许多细胞发生同种型转换。
