Preparation of multimeric peptide-MHC with SpyCatcher modules for antigen-specific T cell detection

Abstract

Peptide-major histocompatibility complex (pMHC) multimers, most commonly used in streptavidin-based tetrameric form, are exceptional for detecting, isolating, and analyzing antigen-specific T cells. However, classical pMHC tetramer staining methods may not detect fully functional T-cells with lower-affinity T-cell receptors (TCRs), which are common in antitumor and autoimmune responses. This limitation has driven the innovation of new methods to improve pMHC binding through multivalent scaffolds, thereby increasing the avidity of weak TCR-pMHC interactions. In this study, we present a novel method for creating multimeric pMHC complexes using the SpyCatcher/SpyTag system. Our approach simplifies the multimer production process by eliminating biotinylation steps, instead employing SpyTag technology for efficient assembly. The method allows for the production of MHC molecules with SpyTags, which can be coupled to various SpyCatcher variants, enabling diverse oligomeric configurations. Our research demonstrates that these novel multimeric pMHC complexes significantly enhance the binding avidity for low-affinity antigen-specific TCRs compared to classical tetramers. This advancement presents a versatile platform for T cell response studies across diverse immunological contexts, offering advantages in specificity, stability, and scalability for both basic research and clinical applications.