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

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.

Variations in HLA-B cell surface expression, half-life and extracellular antigen receptivity

“…Based on these experiments, monocytes were found to have more intracellular HLA class I relative to lymphocytes populations ( Figure 4A ). Previous studies have described higher expression of TAP1 and higher activity of TAP complexes in monocytes relative to lymphocytes ( Fischbach et al, 2015 ). Additionally, monocytes generally also have more tapasin relative to lymphocytes ( Figure 4—figure supplement 1 ).…”

Optical control of the antigen translocation by synthetic photo-conditional viral inhibitors

“…4). After translocation, the NST-F peptide is specifically N-core glycosylated in the ER lumen based on the glycosylation targeting sequence (NST) 24,25. Before illumination, TAP-dependent peptide translocation was blocked by pc-ICP47 2–34 to a level similar to unmodified ICP47 2–34 (>85% inhibition; Fig.…”

Type I IFN and not TNF, is Essential for Cyclic Di-nucleotide-elicited CTL by a Cytosolic Cross-presentation Pathway

“…4 e), providing evidence on TAP transport contribution. To further validate these results, we performed a functional TAP assay ( Fischbach et al, 2015 ). The obtained results confirmed the importance of TAP activity for CDA-mediated cross-presentation.…”

“…Performed according to Fischbach et al (2015) , briefly, human PBMC were isolated from buffy coats of healthy donors using a Ficoll (Biocoll, Biochrom AG) gradient. PBMC were seeded in 24-well plates in a density of 1 × 10 6 cells/well and the corresponding wells were incubated for 24 h with CDA to a final concentration of 5 μg/ml.…”