Bystander responses impact accurate detection of murine and human antigen-specific CD8 T cells

Induction of memory CD8(+) T cells is important for controlling infections such as malaria and HIV/AIDS and for cancer immunotherapy. Accurate assessment of antigen-specific (Ag-specific) CD8(+) T cells is critical for vaccine optimization and for defining correlates of protection. However, conditions for determining Ag-specific CD8(+) T cell responses ex vivo using intracellular cytokine staining (ICS) may be variable, especially in humans with complex antigens. Here, we used an attenuated whole parasite malaria vaccine model in humans and various experimental infections in mice to show that the duration of antigenic stimulation and timing of brefeldin A (BFA) addition influence the magnitude of Ag-specific and bystander T cell responses. Indeed, after immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-γ–producing memory CD8(+) T cells comprising Ag-specific and bystander responses were detected when the duration of Ag stimulation prior to addition of BFA was increased. Mechanistic analyses of virus-specific CD8(+) T cells in mice revealed that the increase in IFN-γ–producing CD8(+) T cells was due to bystander activation of Ag-experienced memory CD8(+) T cells, and correlated with the proportion of Ag-experienced CD8(+) T cells in the stimulated populations. Incubation with anti-cytokine antibodies (e.g., IL-12) improved accuracy in detecting bona fide memory CD8(+) T cell responses, suggesting this as the mechanism for the bystander activation. These data have important implications for accurate assessment of immune responses generated by vaccines intended to elicit protective memory CD8(+) T cells.