Combining Activation-Induced Markers With PD-L1 Selectively Enhances Detection of Antigen-Specific T Cells in...

Detection and characterization of antigen-specific T cells are important for studying immune responses upon infection, vaccination, or autoreactivity. The activation-induced marker (AIM) assay is a robust technique to identify and characterize antigen-specific CD4 and CD8 T cells. However, there is variability in the AIM assay, particularly in the type and number of activation markers used. In this study, we set out to define which marker combinations are most suited to optimally detect antigen-specific CD4 and CD8 T cells and if certain marker combinations preferentially detect specific CD4 T helper subsets. A multiparameter flow cytometry panel, including six common activation markers: CD40L, CD137, CD69, OX40, CD25, and PD-L1, was used for detecting antigen-specific T cells following infection (SARS-CoV-2 and CMV) or vaccination (mRNA-1273 SARS-CoV-2). We demonstrate that combining multiple activation markers increases the detection frequency of antigen-specific CD4 T cells compared to commonly used dual marker combinations. In addition, marker combinations including PD-L1 detected a higher frequency of antigen-specific CD4 T cells in SARS-CoV-2 and CMV infected but not in SARS-CoV-2-vaccinated individuals. Certain dual marker combinations preferentially detected specific CD4 T helper subsets. The majority of antigen-specific CD8 T cells were captured by the dual combination of CD69 plus CD25. In conclusion, combining CD137, CD69, OX40, CD25, and PD-L1 in an AIM assay results in robust and optimal detection of both specific CD4 T helper subsets and CD8 T cells in different antigenic contexts.