A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC50 determination

• Published in: Journal of immunological methods Vol. 406; pp. 21 - 33
• Main Authors: Yin, Liusong, Stern, Lawrence J.
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01-04-2014
• Subjects: Binding competition; DM-susceptibility; Fluorescence polarization; IC50; MHCII–peptide complex; Off-rate; Fluorescence polarization; MHCII–peptide complex; Off-rate; DM; FP; Binding competition; koff,DM and koff; MHCII; IC50; DM-susceptibility; IC50,DM; kdis,DM and kdis; kass,DM and kass
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2014.02.008

HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC50 (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC50 value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC50 is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.
•ΔIC50 is a fast, reliable and high-throughput measure of MHCII DM-susceptibility.•ΔIC50 correlates with DM-susceptibility measured conventionally by off-rate.•ΔIC50 accounts for the effect of DM in both peptide association and dissociation.