Borrebaeck C A, Möller S A
J Immunol. 1986 May 15;136(10):3710-5.
The antigen-specific activation of murine nonimmunized B lymphocytes subsequently used in hybridization experiments has been investigated by using phylogenetically conserved antigens or autologous immunogens. This in vitro immunization was supported by B cell growth and differentiation factors derived from phorbol myristate acetate-stimulated EL-4 thymoma cells and mixed lymphocyte cultures (MLC). A filter immuno-plaque assay was used to evaluate the effect of different activation procedures on the number of antigen-specific plaque-forming cells (PFC). We first determined the requirement for MLC-derived lymphokines in the in vitro immunization. An optimal number of antigen-specific PFC was obtained when using 33 to 50% of the supernatant from a 48-hr MLC to support the activation. B cell growth and differentiation factors derived from EL-4 cultures were then tested for their abilities to potentiate the number of PFC by using both unseparated spleen cells and highly purified Ig-positive B cells as target cells. The combination of lymphokines found in supernatants from 25% EL-4 thymoma culture and 33% MLC yielded the highest number of PFC when used to support an in vitro immunization. This optimal factor preparation was used to determine the kinetics (4 to 7 days) and the dose response (0.01 to 10 micrograms antigen/ml) of antigen-specific B cell activation before using the immunized splenocytes as parental cells in cell fusion experiments. Mouse albumin and hemoglobin, actin (25 micrograms/ml), RNA polymerase II (5 micrograms/ml), as well as syngeneic mouse serum were used to immunize BALB/c spleen cells in vitro. We obtained antigen-specific PFC by using all of the different immunogens, including syngeneic mouse serum, and the in vitro immunized cells were then used in hybridization experiments. The specific efficiencies of each fusion that made use of cells immunized with mouse albumin, hemoglobin, syngeneic mouse serum, actin, or RNA polymerase II were 12, 31, 33, 52, and 22%, respectively, which illustrated the apparent lack of immune tolerance found when the immunization was performed in culture.
通过使用系统发育保守抗原或自体免疫原，对随后用于杂交实验的未免疫小鼠B淋巴细胞的抗原特异性激活进行了研究。来自佛波酯肉豆蔻酸酯刺激的EL-4胸腺瘤细胞和混合淋巴细胞培养物（MLC）的B细胞生长和分化因子支持了这种体外免疫。使用滤膜免疫斑试验来评估不同激活程序对抗原特异性斑形成细胞（PFC）数量的影响。我们首先确定了体外免疫中对MLC衍生的淋巴因子的需求。当使用来自48小时MLC的33%至50%的上清液来支持激活时，可获得最佳数量的抗原特异性PFC。然后，通过使用未分离的脾细胞和高度纯化的Ig阳性B细胞作为靶细胞，测试了来自EL-4培养物的B细胞生长和分化因子增强PFC数量的能力。当用于支持体外免疫时，来自25% EL-4胸腺瘤培养物和33% MLC的上清液中发现的淋巴因子组合产生了最高数量的PFC。在将免疫的脾细胞用作细胞融合实验的亲本细胞之前，使用这种最佳因子制剂来确定抗原特异性B细胞激活的动力学（4至7天）和剂量反应（0.01至10微克抗原/毫升）。使用小鼠白蛋白、血红蛋白、肌动蛋白（25微克/毫升）、RNA聚合酶II（5微克/毫升）以及同基因小鼠血清在体外免疫BALB/c脾细胞。我们使用所有不同的免疫原，包括同基因小鼠血清，获得了抗原特异性PFC，然后将体外免疫的细胞用于杂交实验。利用用小鼠白蛋白、血红蛋白、同基因小鼠血清、肌动蛋白或RNA聚合酶II免疫的细胞进行的每次融合的特异性效率分别为12%、31%、33%、52%和22%，这说明了在培养中进行免疫时明显缺乏免疫耐受性。
