Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography

CD1 molecules are specialized in presenting lipids to T lymphocytes, but id entification and isolation of CD1-restricted lipid-specific T cells has bee n hampered by the lack of reliable and sensitive techniques. We here report the construction of CD1d-glycolipid tetramers from fully denatured human C D1d 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 lip ids is a rapid method for the generation of functional and specific CD1d te tramers, which unlike previously published protocols ensures isolation of C D1d tetramers loaded with a single lipid species. The use of human CD1d-alp ha -galactosylceramide tetramers for ex vivo staining of peripheral blood l ymphocytes and intrahepatic T cells from patients with viral liver cirrhosi s allowed for the first time simultaneous analysis of frequency and specifi city of natural killer T cells in human clinical samples, Application of th is protocol to other members of the CD1 family will provide powerful tools to investigate lipid-specific T cell immune responses in health and in dise ase.