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Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets.
Fischbach, Hanna Döring, Marius Nikles, Daphne Lehnert, Elisa Baldauf, Christoph Kalinke, Ulrich Tampé, Robert
Fischbach, Hanna
Döring, Marius
Nikles, Daphne
Lehnert, Elisa
Baldauf, Christoph
Kalinke, Ulrich
Tampé, Robert
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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.
Citation
Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets. 2015, 6:6199 Nat Commun
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PubMed ID
PubMed Central ID
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Article
Language
en
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Series/Report no.
ISSN
2041-1723