Cambier J C, Monroe J G, Neale M J
J Exp Med. 1982 Dec 1;156(6):1635-49. doi: 10.1084/jem.156.6.1635.
In an effort to further elucidate the early cellular events in generation of antibody responses, we have determined the requirements for antigen-specific initiation of the G0 to G1 transition by isolated trinitrophenol (TNP) -binding B lymphocytes. TNP-binding cells were isolated from normal B6D2F1 splenocyte populations using hapten affinity fractionation on disulfide-bonded TNP-gelatin-coated plates. Populations prepared in this way are greater than or equal to 96% immunoglobulin positive and 70-95% antigen binding. Isolated cells were cultured for 48 h in the presence of a variety of TNP conjugates including TNP-Brucella abortus (Ba), TNP-Ficoll, TNP-sheep erythrocytes (SRBC), TNP-human gamma globulin (HGG), or TNP-ovalbumin (OVA) before being harvested and subjected to acridine orange cell cycle analysis. As many as 80% of cells were in cycle by 48 h in response to TNP-Ba, a thymus-independent (TI1 antigen. A smaller proportion (congruent to 40%) were in cycle in response to TNP-Ficoll, a TI2 antigen. Significant activation was not detected in cultures challenged with the thymus-dependent immunogens TNP-SRBC, TNP-HGG, and TNP-OVA. Addition of interleukin 1 (IL-1), IL-2, B cell growth factor, and/or T cell-replacing factor to cultures did not facilitate responses to these immunogens, suggesting a requirement for antigen-specific T cell help for entry into cell cycle induced by thymus dependent antigens. Activation by TNP-Ba was antigen specific and independent of accessory cells, occurring with equal efficiency in bulk and single-cell cultures. Activation by TNP-Ba was inhibitable by anti-Fab and anti-mu antibodies, but not by anti-delta antibodies. Results indicate that activation of TNP-binding cells to enter cell cycle by TNP-Ba is independent of accessory cells and requires interaction of antigen with cell surface IgM. Exposure to thymus-dependent TNP-immunogens plus nonspecific helper factors is insufficient to cause entry of TNP-binding cells into cycle.
为了进一步阐明抗体应答产生过程中的早期细胞事件，我们确定了分离的三硝基苯酚（TNP）结合B淋巴细胞从G0期向G1期转变的抗原特异性起始条件。利用在二硫键连接的TNP-明胶包被板上进行半抗原亲和分级分离，从正常B6D2F1脾细胞群体中分离出TNP结合细胞。以这种方式制备的细胞群体免疫球蛋白阳性率大于或等于96%，抗原结合率为70 - 95%。分离的细胞在多种TNP偶联物存在下培养48小时，这些偶联物包括TNP-流产布鲁氏菌（Ba）、TNP-菲可（Ficoll）、TNP-绵羊红细胞（SRBC）、TNP-人γ球蛋白（HGG）或TNP-卵清蛋白（OVA），然后收获细胞并进行吖啶橙细胞周期分析。在TNP-Ba（一种胸腺非依赖性（TI1）抗原）刺激下，多达80%的细胞在48小时内进入细胞周期。在TNP-菲可（一种TI2抗原）刺激下，较小比例（约40%）的细胞进入细胞周期。在用胸腺依赖性免疫原TNP-SRBC、TNP-HGG和TNP-OVA刺激的培养物中未检测到明显的激活。向培养物中添加白细胞介素1（IL-1）、IL-2、B细胞生长因子和/或T细胞替代因子并不能促进对这些免疫原的应答，这表明由胸腺依赖性抗原诱导进入细胞周期需要抗原特异性T细胞辅助。TNP-Ba的激活是抗原特异性的，且不依赖于辅助细胞，在大量细胞培养和单细胞培养中效率相同。TNP-Ba的激活可被抗Fab和抗μ抗体抑制，但不能被抗δ抗体抑制。结果表明，TNP-Ba激活TNP结合细胞进入细胞周期不依赖于辅助细胞，且需要抗原与细胞表面IgM相互作用。暴露于胸腺依赖性TNP免疫原加上非特异性辅助因子不足以使TNP结合细胞进入细胞周期。
