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Ultrasensitive quantification of TAP-dependent antigen compartmentalization

February 2015. TAP, the transporter protein complex associated with antigen processing, plays a key role in the adaptive immune system. Following TAP-mediated transport of cytosolic peptides into the endoplasmic reticulum (ER), antigenic peptides are loaded on to the major histocompatibility complex class I (MHC I) for presentation on the cell surface. This process 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.

A team of scientists from the Frankfurt and Hannover have developed a new 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 as low as 1 nM. The method thus permits for the first time quantification of TAP activity under close to physiological conditions in scarce primary cell subsets such as antigen crosspresenting dendritic cells.

High-throughput screens with small compound libraries depend on reliable, quick and simultaneous detection of multiple parameters such as peptide translocation, inhibitor binding and lineage marker expression. The new FACS-based translocation assay fulfills these requirements and enables multiplex formats and very fast readout. These key advantages and in particular the minute number of cells needed for the new assay will make new studies in the field of antigen presentation possible.  The method also allows further investigations into the role of TAP-dependent peptide translocation during viral infection and tumour development.

The work has been published in the latest issue of the journal Nature Communications. More ...


Robert Tampé
Institute of Biochemistry
Goethe-University Frankfurt

Full reference:
Hanna Fischbach, Marius Döring, Daphne Nikles1, Elisa Lehnert, Christoph Baldauf, Ulrich Kalinke, Robert Tampé. 2015. Ultrasensitive quantification of TAP-dependent antigen compartmentalization in scarce primary immune cell subsets. Nature Communications, DOI: 10.1038/ncomms7199. More ...