Antigenic peptide binding to MHC class II molecules at increased peptide concentrations

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Major histocompatibility complex (MHC) class II molecules present peptides to T cells.
• Typically, MHC class II molecules bind only low percentages of antigenic peptides in vitro.
• Peptide binding efficiency varies with peptide sequence and size.

Purpose of the Study:

• To develop a method for achieving complete saturation of purified MHC class II molecules with antigenic peptides.
• To generate homogeneous MHC class II-peptide complexes for research and therapeutic applications.

Main Methods:

• Incubation of affinity-purified human HLA-DR2 with a myelin basic protein (MBP) analog peptide (MBP (83-102)Y83) at neutral pH.
• Use of a 100-fold molar excess of antigenic peptide.
• Kinetic analysis, competitive binding assays, and quantitative peptide binding measurements using biotinylated peptides.
• Analysis of purified complexes using reverse-phase HPLC, mass spectrometry, and 2D gel electrophoresis.

Main Results:

• Complete saturation (100-125% occupancy) of HLA-DR2 with MBP (83-102)Y83 peptide was achieved at 300-500-fold molar excess peptide concentrations.
• Specificity of binding was confirmed using a control peptide and competitive assays.
• Purified complexes contained a single peptide entity, free of endogenous polypeptides.

Conclusions:

• High peptide concentrations enable the generation of MHC class II-peptide complexes with defined composition.
• These saturated complexes may be valuable for antigen-specific immunotherapy of autoimmune diseases.
• Further understanding of MHC-peptide-TCR interactions can be facilitated by these defined complexes.