Armitage R J, Sato T A, Macduff B M, Clifford K N, Alpert A R, Smith C A, Fanslow W C
Department of Immunology, Immunex Research and Development Corporation, Seattle, WA 98101.
Eur J Immunol. 1992 Aug;22(8):2071-6. doi: 10.1002/eji.1830220817.
We have identified the murine thymoma line EL4 as a source of biologically active CD40 ligand. Using a biotin-labeled soluble CD40.Fc fusion protein, consisting of the extracellular domain of human CD40 and the Fc region of human IgG1, EL4 cells were subjected to repeated flow cytometric cell sorting to select for cells with enhanced biotinylated CD40.Fc binding. After nine rounds of sorting, the number of CD40.Fc binding sites/cell had risen from 450 on the unsorted parental EL4 cells to 15,000 on EL40.9 cells (EL4 cells sorted with biotinylated CD40.Fc for nine rounds). Scatchard analysis of radiolabeled CD40.Fc binding revealed that the surface-expressed CD40 ligand on parental EL4 and EL40.9 cells bound its receptor with a single class of high-affinity sites (Kd = 0.5 nM). Supernatant (SN) from the sorted EL40.9 cells was found to contain human and murine B cell stimulatory activity which could be removed by preclearing with immobilized CD40.Fc, confirming the presence of soluble CD40 ligand in the preparations. EL40.9 supernatant enhanced soluble CD23 (sCD23) release and induced IgE secretion from interleukin 4-stimulated human B cells. In addition, EL40.9 SN contained proliferative activity for anti-IgM-activated murine B cells which could be removed by treatment with immobilized CD40.Fc. However, the same SN had no demonstrable activity on the proliferation of human B cells. The results presented here describe, for the first time, a source of membrane-bound and soluble CD40 ligand. The soluble form of this murine ligand has activity on murine and human B cells and induces some of the functional responses predicted for the ligand based on the action of stimulatory antibodies directed against the human CD40 surface molecule.
我们已确定小鼠胸腺瘤细胞系EL4是生物活性CD40配体的一个来源。使用由人CD40的胞外结构域和人IgG1的Fc区域组成的生物素标记的可溶性CD40.Fc融合蛋白，对EL4细胞进行反复的流式细胞术细胞分选，以选择与生物素化的CD40.Fc结合增强的细胞。经过九轮分选后，每个细胞的CD40.Fc结合位点数量从未分选的亲本EL4细胞上的450个增加到EL40.9细胞（用生物素化的CD40.Fc分选九轮的EL4细胞）上的15000个。对放射性标记的CD40.Fc结合进行Scatchard分析表明，亲本EL4和EL40.9细胞表面表达的CD40配体以单一类别的高亲和力位点（Kd = 0.5 nM）结合其受体。发现分选的EL40.9细胞的上清液（SN）含有人和小鼠B细胞刺激活性，该活性可通过用固定化的CD40.Fc预先清除而去除，这证实了制剂中存在可溶性CD40配体。EL40.9上清液增强了可溶性CD23（sCD23）的释放，并诱导白细胞介素4刺激的人B细胞分泌IgE。此外，EL40.9 SN含有抗IgM激活的小鼠B细胞的增殖活性，该活性可通过用固定化的CD40.Fc处理而去除。然而，相同的SN对人B细胞的增殖没有明显活性。此处呈现的结果首次描述了膜结合型和可溶性CD40配体的一个来源。这种小鼠配体的可溶性形式对小鼠和人B细胞具有活性，并诱导基于针对人CD40表面分子的刺激抗体的作用而预测的该配体的一些功能反应。
