Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography
Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography
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DOI：
10.1073/pnas.051604498
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发表时间：
2001-03-13
影响因子：
11.1
通讯作者：
Cerundolo, V
中科院分区：
文献类型：
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作者：
Karadimitris, A;Gadola, S;Cerundolo, V
CD1 分子专门向 T 淋巴细胞呈递脂质，但由于缺乏可靠和灵敏的技术，CD1 限制性脂质特异性 T 细胞的鉴定和分离受到阻碍。我们在这里报告了通过使用氧化重折叠色谱技术从完全变性的人 CD1d 分子构建 CD1d-糖脂四聚体。我们证明，在合成脂质存在的情况下，分子伴侣和折叠酶辅助的变性 CD1d 分子和 β (2)-微球蛋白的重折叠是一种生成功能性和特异性 CD1d 四聚体的快速方法，与之前发布的方案不同，它确保了加载单一脂质种类的 CD1d 四聚体的分离。使用人 CD1d-α-半乳糖神经酰胺四聚体对病毒性肝硬化患者的外周血淋巴细胞和肝内 T 细胞进行离体染色，首次能够同时分析人类临床样本中自然杀伤 T 细胞的频率和特异性，将该方案应用于 CD1 家族的其他成员将为研究脂质特异性 T 细胞提供强大的工具 健康和疾病中的免疫反应。
CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipid-specific T cells has been hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human CD1d molecules by using the technique of oxidative refolding chromatography. We demonstrate that chaperone- and foldase-assisted refolding of denatured CD1d molecules and beta (2)-microglobulin in the presence of synthetic lipids is a rapid method for the generation of functional and specific CD1d tetramers, which unlike previously published protocols ensures isolation of CD1d tetramers loaded with a single lipid species. The use of human CD1d-alpha -galactosylceramide tetramers for ex vivo staining of peripheral blood lymphocytes and intrahepatic T cells from patients with viral liver cirrhosis allowed for the first time simultaneous analysis of frequency and specificity of natural killer T cells in human clinical samples, Application of this protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in disease.