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

Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography
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 lipidspecific 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 ?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–?-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.
0027-8424
3294-3298
Karadimitris, Anastasios
ed4cf62f-ca4f-444c-8145-d58367707da1
Gadola, Stephan
ef2fa6cf-2ccc-4fea-a7a5-cc03a9d13ab1
Altamirano, Myriam
c2d4d730-0db0-4d97-bf8a-82920533d890
Brown, Dave
897ef8ae-e86d-4084-8558-ae60d845fa0a
Woolfson, Adrian
f892daf4-24d7-461e-bb96-2f287d4e8e08
Klenerman, Paul
aa4c00a6-1a4d-4a4f-ac21-2131996344a7
Chen, Ji-Li
4fcaef6a-e7d4-4c47-8fb9-1e0387a4a5c6
Koezuka, Yasuhiko
93634148-bcc0-4efe-8ae0-1726c3889f96
Roberts, Irene A.
6e025a6a-755f-4340-9a45-a99b0fde116b
Price, David A.
62c74c13-f63c-4449-8ab3-9d3a127a8fe9
Dusheiko, Geoff
e29a38e2-7d7d-4f7f-bc79-7f37745790d3
Milstein, Cesar
c08692ef-9f5c-4cf1-ba9c-d00378914e7b
Fersht, Alan
e4efd480-bd06-47fc-b793-6ca2158052bc
Luzzatto, Lucio
b42dd704-f8f8-4c0f-9ba3-13401c711211
Cerundolo, Vincenzo
813bcd4a-ca19-48a3-86e4-71d131e2065a
Karadimitris, Anastasios
ed4cf62f-ca4f-444c-8145-d58367707da1
Gadola, Stephan
ef2fa6cf-2ccc-4fea-a7a5-cc03a9d13ab1
Altamirano, Myriam
c2d4d730-0db0-4d97-bf8a-82920533d890
Brown, Dave
897ef8ae-e86d-4084-8558-ae60d845fa0a
Woolfson, Adrian
f892daf4-24d7-461e-bb96-2f287d4e8e08
Klenerman, Paul
aa4c00a6-1a4d-4a4f-ac21-2131996344a7
Chen, Ji-Li
4fcaef6a-e7d4-4c47-8fb9-1e0387a4a5c6
Koezuka, Yasuhiko
93634148-bcc0-4efe-8ae0-1726c3889f96
Roberts, Irene A.
6e025a6a-755f-4340-9a45-a99b0fde116b
Price, David A.
62c74c13-f63c-4449-8ab3-9d3a127a8fe9
Dusheiko, Geoff
e29a38e2-7d7d-4f7f-bc79-7f37745790d3
Milstein, Cesar
c08692ef-9f5c-4cf1-ba9c-d00378914e7b
Fersht, Alan
e4efd480-bd06-47fc-b793-6ca2158052bc
Luzzatto, Lucio
b42dd704-f8f8-4c0f-9ba3-13401c711211
Cerundolo, Vincenzo
813bcd4a-ca19-48a3-86e4-71d131e2065a

Karadimitris, Anastasios, Gadola, Stephan, Altamirano, Myriam, Brown, Dave, Woolfson, Adrian, Klenerman, Paul, Chen, Ji-Li, Koezuka, Yasuhiko, Roberts, Irene A., Price, David A., Dusheiko, Geoff, Milstein, Cesar, Fersht, Alan, Luzzatto, Lucio and Cerundolo, Vincenzo (2001) Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proceedings of the National Academy of Sciences of the United States of America, 98 (6), 3294-3298. (doi:10.1073/pnas.051604498). (PMID:11248072)

Record type: Article

Abstract

CD1 molecules are specialized in presenting lipids to T lymphocytes, but identification and isolation of CD1-restricted lipidspecific 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 ?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–?-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.

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More information

Published date: 13 March 2001
Organisations: Infection Inflammation & Immunity

Identifiers

Local EPrints ID: 165259
URI: http://eprints.soton.ac.uk/id/eprint/165259
ISSN: 0027-8424
PURE UUID: ec77b154-5013-4576-814b-854bcb700c63

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Date deposited: 08 Oct 2010 14:26
Last modified: 14 Mar 2024 02:10

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Contributors

Author: Anastasios Karadimitris
Author: Stephan Gadola
Author: Myriam Altamirano
Author: Dave Brown
Author: Adrian Woolfson
Author: Paul Klenerman
Author: Ji-Li Chen
Author: Yasuhiko Koezuka
Author: Irene A. Roberts
Author: David A. Price
Author: Geoff Dusheiko
Author: Cesar Milstein
Author: Alan Fersht
Author: Lucio Luzzatto
Author: Vincenzo Cerundolo

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