Studying MHC Class II Peptide Loading and Editing In Vitro

Author:

Kim, AeRyon

Ishizuka, Isabel Emiko

Hartman, Isamu Z

Poluektov, Yuri

Narayan,Kedar

Sadegh-Nasseri, Scheherazade
Author Address

Department of Inflammation and Oncology, Amgen Research, Amgen Inc., South San Francisco, CA, USA., Department of Immunology, Genentech Inc., South San Francisco, CA, USA., Office for Technology Development, UT Southwestern Medical Center, Dallas, TX, USA., MBL International, A JSR Life Sciences Company, Des Plaines, IL, USA., Center for Molecular Microscopy, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA., Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA., Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. ssadegh@jhmi.edu., Department of Biology, Johns Hopkins University, Baltimore, MD, USA. ssadegh@jhmi.edu., Graduate Program in Immunology, Johns Hopkins University, Baltimore, MD, USA. ssadegh@jhmi.edu.,

Abstract:

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of MHC class II molecules upon binding of antigenic peptides and of the effect of DM on the interactions. Using purified soluble molecules, in this chapter we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on this interaction. A simple qualitative method, Gentle SDS-PAGE Assay assesses the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide-MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore Surface Plasmon Resonance (SPR). We also describe an intrinsic tryptophan fluorescence assay for studying transient interactions of DM and MHC class II molecules.

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