Multiplex peptide-MHC tetramer staining using mass cytometry for deep analysis of the influenza-specific T-cell response in mice

Antigen-specific T cells play a crucial role for the host protective immunity against viruses and other diseases. The use of mass cytometry together with a combinatorial multiplex tetramer staining has successfully been applied for probing and characterization of multiple antigen-specific CD8⁺ T cells in human blood samples. The present study shows that this approach can also be used to rapidly assess the magnitude of influenza-specific CD8⁺ T cell epitope dominance across lymph nodes and lungs in a murine model of a highly pathological influenza infection. Moreover, we show feasibility of extending this approach to include concurrent identification of virus-specific CD4⁺ T cells. By using a double coding approach, we probed for five influenza-specific MHCI-peptide complexes as well as one influenza-specific MHCII-peptide complex in the presence of irrelevant control peptides and show that this approach is capable of tracking antigen-specific T cells across individual lymph nodes and lungs. The simultaneous staining with 26 surface maker molecules further facilitated an in-depth characterization of T cells reacting with influenza epitopes and revealed tissue specific phenotypic differences between CD4⁺ T cells targeting the same pathogenic epitope. In conclusion, this approach provides the possibility for a rapid and comprehensive analysis of antigen-specific CD8⁺ and CD4⁺ T cells in different disease settings that might be advantageous for subsequent vaccine formulation strategies.