Alloreactive monoclonal antibodies select Kd molecules with different peptide profiles

Area of Science:

• Immunology
• Molecular Biology
• Structural Biology

Background:

• Class I major histocompatibility complex (MHC) molecules present peptides to T cells, playing a crucial role in immune responses.
• Understanding the antigenic structure of MHC class I molecules is vital for dissecting immune recognition and developing targeted therapies.

Purpose of the Study:

• To investigate the impact of site-directed mutations on H-2Kd molecule antigenicity.
• To explore the role of bound peptides in shaping the serologic epitopes of MHC class I molecules.

Main Methods:

• Generated and analyzed 25 site-directed mutants of the H-2Kd molecule.
• Assessed the binding of five H-2Kd-reactive monoclonal antibodies (mAbs) to these mutants.
• Eluted and analyzed peptides bound to H-2Kd molecules precipitated by different mAbs.
• Utilized peptide variants to identify specific peptide residues contributing to epitopes.

Main Results:

• Single amino acid substitutions on alpha-helices generally enhanced mAb binding, particularly with charged or polar residues.
• Mutations at positions 58 and 166 abolished binding for one specific mAb, identifying key residues for that epitope.
• Most mutations diminishing mAb binding occurred within the peptide-binding groove, suggesting peptide influence.
• Peptide repertoire analysis revealed distinct profiles for different alloreactive mAbs, with one mAb showing a unique profile.
• Identification of a specific peptide residue influencing an alloreactive mAb's recognition.

Conclusions:

• Amino acid substitutions on alpha-helices can modulate H-2Kd molecule antigenicity and antibody binding.
• Peptide binding significantly contributes to the serologic epitopes of MHC class I molecules.
• Distinct peptides bound to MHC molecules can alter their recognized serologic determinants, influencing immune recognition.