Human CD1d–glycolipid tetramers generated byin vitrooxidative refolding chromatography

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.

Severe and selective deficiency of interferon‐γ‐producing invariant natural killer T cells in patients with myelodysplastic syndromes

“…The number of Va24 + Vb11 + NKT cells in fresh human PB lymphocytes is highly variable and often close to the lower limit of detection by flow cytometry (mean 0AE02-0AE06% in most studies), either using invariant T-cell receptors (TCR) or binding to CD1d glycolipid tetramers (Karadimitris et al, 2001). In particular, low NKT cell frequency in humans complicates the measurement of fresh NKT cell function by flow cytometry.…”

Ultra‐sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells

“…Melan-Aspecific cells as shown here), both tissue compartmentalization and self tolerance effects will serve to reduce the frequencies of these populations in the blood, so that ultra-sensitive techniques such as that shown here might be necessary. Finally, the technique can be readily applied to the study of other lymphocyte subsets present at low frequencies as e.g., antigen-specific CD4 + T helper cells by using MHC class II tetramers [19] and NKT cells by using CD1d tetramers, as recently published [20].…”

Virus‐specific CD8+ T lymphocytes within the normal human liver

“…NKT cells have been shown to play an important role in the regulation of immunity to pathogens within this environment [23,24]. Previous studies on the V § 24/V g 11 subset of NKT cells analyzed patients during persistent HCV infection and showed a comparatively high (up to 0.37%) percentage of these cells within the liver [25,26]. The NKT cell subset was also more highly activated ( G 90%) in the HCV-infected livers as compared to an average of 50% seen in the 'normal' liver.…”