Bloom B R, Jimenez L, Marcus P I
J Exp Med. 1970 Jul 1;132(1):16-30. doi: 10.1084/jem.132.1.16.
A general method is described for enumerating antigen-sensitive lymphocytes obtained from individuals having delayed hypersensitivity, in this case from highly tuberculin-sensitive guinea pigs. The method is based on the observation that resting lymphocytes are generally unable to support replication of RNA viruses, whereas antigen-"activated" lymphocytes can. Lymph node lymphocytes from individual animals were cultured in the presence or absence of tuberculin purified protein derivatives (PPD). After various periods of time, the cells were infected either with Newcastle disease virus or vesicular stomatitis virus, and plated in agar over a monolayer of cells susceptible to the virus. Wherever a lymphocyte yielded infectious virus, a discrete plaque in the monolayer could be seen. The increase in plaques of the antigen-stimulated cells over the background of the control sample was taken as the number of antigen-sensitive cells in the population. When lymphocytes from normal guinea pigs or from guinea pigs immunized to produce only circulating antibody to PPD were similarly tested, no increase in plaque-forming units (PFU) was observed. The average increase in PFU due to antigenic stimulation varied from 1 per 1000 lymphocytes at 24 hr to 16 per 1000 lymphocytes at 96 hr. By employing inhibitors of mitosis (colchicine, vinblastine, and thymidine) it was evident that the increase in PFU at least up to 48 hr was primarily due to initial antigen-reactive cells and not their progeny.
本文描述了一种用于计数从患有迟发型超敏反应的个体（在此例中为高度结核菌素敏感的豚鼠）获取的抗原敏感淋巴细胞的通用方法。该方法基于以下观察结果：静止淋巴细胞通常无法支持RNA病毒的复制，而抗原“激活”的淋巴细胞则可以。将来自个体动物的淋巴结淋巴细胞在有或没有结核菌素纯化蛋白衍生物（PPD）的情况下进行培养。在不同时间段后，用新城疫病毒或水泡性口炎病毒感染细胞，并将其接种在对该病毒敏感的单层细胞上的琼脂中。只要淋巴细胞产生感染性病毒，就可以在单层中看到一个离散的噬斑。抗原刺激细胞的噬斑相对于对照样品背景的增加被视为群体中抗原敏感细胞的数量。当对来自正常豚鼠或仅免疫产生针对PPD的循环抗体的豚鼠的淋巴细胞进行类似测试时，未观察到噬斑形成单位（PFU）的增加。由于抗原刺激导致的PFU平均增加量从24小时时每1000个淋巴细胞1个变化到96小时时每1000个淋巴细胞16个。通过使用有丝分裂抑制剂（秋水仙碱、长春碱和胸腺嘧啶核苷），很明显至少在48小时内PFU的增加主要是由于初始抗原反应性细胞而非其后代。
