BMX-A and BMX-S: Accessible cell-free methods to estimate peptide-MHC-I affinity and stability

• Published in: Molecular immunology Vol. 161; pp. 1 - 10
• Main Authors: Witney, Matthew J., Tscharke, David C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.09.2023
• Subjects: Affinity; Bead-based; bioactive properties; cell division; DimerX; dissociation; flow cytometry; major histocompatibility complex; MHC; peptidases; peptides; RMA/S; Stability; surface plasmon resonance; T-lymphocytes; Bead-based; Affinity; MHC; Stability; RMA/S; DimerX
• ISSN: 0161-5890; 1872-9142; 1872-9142
• DOI: 10.1016/j.molimm.2023.07.008

The affinity and stability of peptide binding to Major Histocompatibility Complex Class I (MHC-I) molecules are fundamental parameters that underpin the specificity and magnitude of CD8+ T cell responses. These parameters can be estimated in some cases by computational tools, but experimental validation remains valuable, especially for stability. Methods to measure peptide binding can be broadly categorised into either cell-based assays using TAP-deficient cell lines such as RMA/S, or cell-free strategies, such as peptide competition-binding assays and surface plasmon resonance. Cell-based assays are subject to confounding biological activity, including peptide trimming by peptidases and dilution of peptide-loaded MHC-I on the surface of cells through cell division. Current cell-free methods require in-house production and purification of MHC-I. In this study, we present the development of new cell-free assays to estimate the relative affinity and dissociation kinetics of peptide binding to MHC-I. These assays, which we have called BMX-A (relative affinity) and BMX-S (kinetic stability), are reliable, scalable and accessible, in that they use off-the-shelf commercial reagents and standard flow cytometry techniques. •Peptide:MHC-I affinity and stability are key parameters of CD8+ T cell responses.•We present a new bead-based platform (BMX) to measure p:MHC-I binding.•BMX uses a commercially-available tetramer-like reagent.•BMX-A and BMX-S are accessible methods to measure p:MHC-I affinity and stability.•The BMX platform is a suitable replacement for cell-based p:MHC-I binding assays.