Georgia Stavrakis: Pre-conjugation of MHC Pentamers for simultaneous analysis of ag-specific T cells

Описание: Full title: 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.

Georgia Stavrakis is currently a PhD candidate at the Johns Hopkins Bloomberg School of Public Health studying in the department of Molecular Microbiology and Immunology. She received her undergraduate degree in Microbiology and Immunology from McGill University in Montreal, Qc.