Optimizing pre-conjugation of biotinylated MHC class I Pentamers to streptavidin fluorophores for simultaneous analysis of different antigen-specific T cells

To study virus specific CD8+ T cell responses recognizing different epitopes in the same infection, we sought to develop a method to maximize flexibility for simultaneous staining of multiple MHC class I epitopes. We optimized streptavidin-PE and -APC as fluorophores for MHC I pentamer staining, allowing us to change the fluorophore tag on pentamers in combinations defined by the epitopes specific for a given sample. We conjugated streptavidin -PE and -APC to biotinylated MHC I pentamers derived from CMV, EBV and HCV epitopes to optimize the assay. This allowed us to develop a novel strategy to pre-conjugate pentamers following careful titration of the amount of streptavidin-fluor to biotinylated pentamer and the volume of the conjugated fluor-pentamer used to stain for antigen specific T cells. By evaluating multiple unique viral epitopes in an infection, we can explore CD8+ T cell phenotypes in contexts such as reinfections, with sequence evolution, and viral clearance.