Intracellular CD154 expression reflects antigen-specific CD8⁺ T cells but shows less sensitivity than intracellular cytokine and MHC tetramer staining

A recent report showed that analysis of CD154 expression in the presence of the secretion inhibitor Brefeldin A (Bref A) could be used to assess the entire repertoire of antigen-specific CD4⁺ T helper cells. However, the capacity of intracellular CD 154 expression to identify antigen-specific CD8⁺ T cells has yet to be investigated. In this study, we compared the ability of intracellular CD154 expression to assess antigen-specific CD8⁺ T cells with that of accepted standard assays, namely intracellular cytokine IFN-γ staining (ICS) and MHC class I tetramer staining. The detection of intracellular CD154 molecules in the presence of Bref A reflected the kinetic trend of antigen-specific CD8⁺ T cell number, but unfortunately showed less sensitivity than ICS and tetramer staining. However, ICS levels peaked and saturated 8 h after antigenic stimulation in the presence of Bref A and then declined, whereas intracellular CD154 expression peaked by 8 h and maintained the saturated level up to 24 h post-stimulation. Moreover, intracellular CD154 expression in antigen-specific CD8⁺ T cells developed in the absence of CD4⁺ T cells changed little, whereas the number of IFN-γ-producing CD8⁺ T cells decreased abruptly. These results suggest that intracellular CD154 could aid the assessment of antigen-specific CD8⁺ T cells, but does not have as much ability to identify heterogeneous CD4⁺ T helper cells. Therefore, the combined analytical techniques of ICS and tetramer staining together with intracellular CD154 assays may be able to provide useful information on the accurate phenotype and functionality of antigen-specific CD8⁺ T cells.