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Nat Commun. 2015 Feb 6;6:6199. doi: 10.1038/ncomms7199.

Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets.

Author information

1
Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.
2
Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Helmholtz-Centre for Infection Research and the Hannover Medical School, Feodor-Lynen Str. 7-9, 30625 Hannover, Germany.
3
1] Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany [2] Cluster of Excellence Frankfurt - Macromolecular Complexes, Goethe-University Frankfurt, Max-von-Laue Str. 9, 60438 Frankfurt/Main, Germany.

Abstract

Presentation of peptides on major histocompatibility complex class I (MHC I) is essential for the establishment and maintenance of self-tolerance, priming of antigen-specific CD8(+) T cells and the exertion of several T-cell effector functions. Cytosolic proteasomes continuously degrade proteins into peptides, which are actively transported across the endoplasmic reticulum (ER) membrane by the transporter associated with antigen processing (TAP). In the ER lumen antigenic peptides are loaded onto MHC I, which is displayed on the cell surface. Here we describe an innovative flow cytometric approach to monitor time-resolved ER compartmentalization of antigenic peptides. This assay allows the analysis of distinct primary human immune cell subsets at reporter peptide concentrations of 1 nM. Thus, this ultrasensitive method for the first time permits quantification of TAP activity under close to physiological conditions in scarce primary cell subsets such as antigen cross-presenting dendritic cells.

PMID:
25656091
PMCID:
PMC4347055
DOI:
10.1038/ncomms7199
[Indexed for MEDLINE]
Free PMC Article

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