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

   


Authors: Karadimitris, A.; Gadola, S.; Altamirano, M.; Brown, D.; Woolfson, A.; Klenerman, P.; Chen, J. L.; Koezuka, Y.; Roberts, I. A. G.; Price, D. A.; Dusheiko, G.; Milstein, C.; Fersht, A.; Luzzatto, L.; Cerundolo, V.
Article Title: Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography
Abstract: 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 β2-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-α-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.
Keywords: human cell; hepatitis c; liver cirrhosis; t lymphocyte; animals; mice; mice, knockout; cells, cultured; membrane protein; cell isolation; ligands; beta 2 microglobulin; protein folding; technique; oxidation-reduction; tetramer; staining and labeling; ceramides; galactosylceramide; glycolipid; glycolipids; cd1 antigen; antigens, cd1; protein denaturation; chromatography; humans; human; priority journal; article
Journal Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 98
Issue: 6
ISSN: 0027-8424
Publisher: National Academy of Sciences  
Date Published: 2001-03-13
Start Page: 3294
End Page: 3298
Language: English
DOI: 10.1073/pnas.051604498
PUBMED: 11248072
PROVIDER: scopus
PMCID: PMC30647
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-0035853062&partnerID=40&md5=89b264e8e358d935ef5b97a0c8500d34
Notes: Export Date: 21 May 2015 -- Source: Scopus
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MSK Authors
  1. Anastasios Karadimitris
    13 Karadimitris
  2. Lucio Luzzatto
    105 Luzzatto
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