Protein transfer of glycosyl-phosphatidylinositol (GPI)-modified murine B7-1 and B7-2 costimulators
Protein transfer of glycosyl-phosphatidylinositol (GPI)-modified murine B7-1 and B7-2 costimulators
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DOI：
10.1097/00002371-199909000-00002
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发表时间：
1999-09-01
影响因子：
3.9
通讯作者：
Tykocinski, ML
中科院分区：
文献类型：
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作者：
Brunschwig, EB;Fayen, JD;Tykocinski, ML
评估了使用蛋白质转移作为增强小鼠肿瘤细胞免疫原性的手段的可行性。小鼠共刺激物 B7-1 (CD80) 和 B7-2 (CD86) 的糖基磷脂酰肌醇 (GPI) 修饰变体，分别命名为 B7-1 GPI 和 B7-2 CPI，是从用编码这些嵌合蛋白的谷氨酰胺合成酶扩增/表达构建体转染的 CHO-K1 细胞中免疫亲和纯化的。这些蛋白质在去污剂耗尽的假胶束中纯化后，被外源性掺入几种不同的鼠肿瘤系（EL-4、SMUCC-1、BW5147.3、P815、Ag104A 和 EMT6）的膜中。通过免疫荧光和流式细胞术记录了 B7.GPI 蛋白成功的膜涂色，并通过证明重新掺入的蛋白对磷脂酰肌醇-磷脂酶 C 敏感、对糖基-磷脂酰肌醇-磷脂酶 D 具有抗性，并且难以用二肉豆蔻酰磷脂酰胆碱囊泡去除来验证膜整合。值得注意的是，B7-1.GPI 和 B7-2.GPI 可以： 一起共涂到 EL-4 细胞表面上，在两者之间没有观察到干扰。使用标准体外增殖测定表明，两种 B7.GPI 蛋白在膜重新掺入后保留了共刺激器功能。这些发现进一步验证了蛋白转移共刺激物.GPI的治疗潜力，并为其在动物肿瘤模型中的组合使用铺平了道路。
The feasibility of using protein transfer as a means for enhancing the immunogenicity of murine tumor cells was evaluated. Glycosyl-phosphatidylinositol (GPI)-modified variants of the murine costimulators B7-1 (CD80) and B7-2 (CD86), designated B7-1 GPI and B7-2 CPI, respectively, were immunoaffinity-purified from CHO-K1 cells transfected with glutamine synthetase amplification/expression constructs encoding each of these chimeric proteins. The proteins, once purified in detergent-depleted pseudomicelles, were exogenously incorporated into the membranes of several different murine tumor lines (EL-4, SMUCC-1, BW5147.3, P815, Ag104A, and EMT6). Successful membrane painting with the B7.GPI proteins was documented by immunofluorescence and flow cytometry, and membrane integration was verified by demonstrating that the reincorporated proteins were phosphatidylinositol-phospholipase C-sensitive, glycosyl-phosphatidylinositol-phospholipase D-resistant, and refractory to removal with dimyristylphosphatidylcholine vesicles. Significantly, B7-1.GPI and B7-2.GPI could be: together copainted onto EL-4 cell surfaces with no interference observed between the two. A standard in vitro proliferation assay was used to show that both of the B7.GPI proteins retained costimulator function after membrane reincorporation. These findings further validate the therapeutic potential of protein-transferred costimulator.GPIs and pave the way for their combinatorial use in animal tumor models.