MAKELA O, NOSSAL G J
J Exp Med. 1962 Jan 1;115(1):231-44. doi: 10.1084/jem.115.1.231.
The DNA-synthesizing capacity of single antibody-forming cells was tested by a combination of micromanipulatory and autoradiographic techniques. Rats were immunized with S. adelaide flagellin, a protein antigen known to contain significant contamination with somatic (O) antigen. Single cells from secondarily immunized rats were tested for production of anti-H and anti-O antibodies by previously described and newer techniques. Positive antibody producers were transferred onto clean dry slides by micromanipulation, and autoradiographs were performed. When rats had received tritiated thymidine 1 hour before killing, labeling of antibody-forming cells was taken to imply that the cell was preparing for further mitotic division. It was found that on the 2nd and 3rd day of a secondary response, many of the antibody-producing cells in the nodes (chiefly plasmablasts) were incorporating tritiated thymidine. At the height of the cellular response, however, at 4 and 5 days, the majority of active antibody producers (chiefly mature plasma cells) were incapable of DNA synthesis. There appeared to be an inverse relationship between the antibody-forming and DNA-synthesizing capacities of the cell population under study; as more of the cells studied formed detectable antibody, fewer of them incorporated the DNA precursor. The age of plasma cells was also studied. Animals were killed at the height of the cellular immune response, having previously received an injection of tritiated thymidine 1 to 48 hours before killing; i.e., at 63 to 110 hours after their secondary stimulus. As the interval between isotope injection and killing increased, the proportion of antibody-forming cells showing labeling increased. With an interval of 30 hours, about half the antibody-forming cells were labeled and of 48 hours, over 95 per cent were labeled. This was taken as evidence that, few, if any, antibody-forming cells found at the height of a secondary response were more than 48 hours old. On the basis of these experiments and those reported in the accompanying paper, a simplified scheme showing the development of an antibody-forming clone in the secondary response was proposed.
采用显微操作技术和放射自显影技术相结合的方法，对单个抗体形成细胞的DNA合成能力进行了检测。用阿德莱德沙门氏菌鞭毛蛋白免疫大鼠，该蛋白抗原已知含有大量体细胞（O）抗原污染。通过先前描述的和更新的技术，对二次免疫大鼠的单个细胞进行抗H和抗O抗体产生的检测。通过显微操作将阳性抗体产生细胞转移到干净干燥的载玻片上，并进行放射自显影。当大鼠在处死前1小时接受了氚标记的胸腺嘧啶核苷时，抗体形成细胞的标记被认为意味着该细胞正在为进一步的有丝分裂做准备。结果发现，在二次反应的第2天和第3天，淋巴结中的许多抗体产生细胞（主要是成浆细胞）正在掺入氚标记的胸腺嘧啶核苷。然而，在细胞反应的高峰期，即第4天和第5天，大多数活跃的抗体产生细胞（主要是成熟浆细胞）无法进行DNA合成。在所研究的细胞群体中，抗体形成能力和DNA合成能力之间似乎存在反比关系；随着所研究的形成可检测抗体的细胞增多，掺入DNA前体的细胞就越少。还研究了浆细胞的年龄。在细胞免疫反应的高峰期处死动物，这些动物在处死前1至48小时（即二次刺激后63至110小时）接受了氚标记的胸腺嘧啶核苷注射。随着同位素注射和处死之间的间隔增加，显示标记的抗体形成细胞的比例增加。间隔30小时时，约一半的抗体形成细胞被标记；间隔48小时时，超过95%的细胞被标记。这被视为证据，表明在二次反应高峰期发现的抗体形成细胞中，很少（如果有的话）超过48小时。基于这些实验以及随附论文中报道的实验，提出了一个简化的方案，展示了二次反应中抗体形成克隆的发展过程。
