Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography.
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.
Nuffield Department of Medicine, Institute of Molecular Medicine, Oxford OX3 9DS, United Kingdom.
A
A
Karadimitris
S
S
Gadola
M
M
Altamirano
D
D
Brown
A
A
Woolfson
P
P
Klenerman
J L
JL
Chen
Y
Y
Koezuka
I A
IA
Roberts
D A
DA
Price
G
G
Dusheiko
C
C
Milstein
A
A
Fersht
L
L
Luzzatto
V
V
Cerundolo
D000818:Animals; D018949:Antigens, CD1 / Q000235:genetics / Q000378:metabolism*; D055610:Antigens, CD1d; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism*; D006526:Hepatitis C / Q000097:blood / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008103:Liver Cirrhosis / Q000097:blood / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010084:Oxidation-Reduction; D017510:Protein Folding; D013194:Staining and Labeling
C116527:AGL 563; D018949:Antigens, CD1; D055610:Antigens, CD1d; C526877:CD1D protein, human; D002518:Ceramides; D006017:Glycolipids; D008024:Ligands