Karadimitris, A and Gadola, S and Altamirano, M and Brown, D and Woolfson, A and Klenerman, P and Chen, JL and Koezuka, Y and Roberts, IAG and Price, DA and Dusheiko, G and Milstein, C and Fersht, A and Luzzatto, L and Cerundolo, V (2001) Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. P NATL ACAD SCI USA , 98 (6) 3294 - 3298.
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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.
|Title:||Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography|
|Keywords:||KILLER T-CELLS, V(ALPHA)14 NKT CELLS, ALPHA-GALACTOSYLCERAMIDE, CUTTING EDGE, CD1D TETRAMERS, LYMPHOCYTES, ACTIVATION, RECOGNITION, GENES, GLYCOSYLCERAMIDES|
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