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

Abstract

Induction of memory CD8 T cells is important for controlling infections such as malaria HIV/AIDS, and for cancer immunotherapy. Accurate assessment of antigen (Ag)-specific CD8 T-cells is critical for vaccine optimization and defining correlates of protection. However, conditions for determining Ag-specific CD8 T-cell responses ex-vivo using 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 influences the magnitude of Ag-specific and bystander T cell responses. Indeed, following immunization with an attenuated whole sporozoite malaria vaccine in humans, significantly higher numbers of IFN-γ producing memory CD8 T-cells comprised of antigen specific and bystander responses were detected by increasing the duration of Ag-stimulation prior to addition of BFA. Mechanistic analyses of virus-specific CD8 T-cells in mice revealed that the increase in IFNg 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 (ex. 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.