Bystander responses impact accurate detection of murine and human antigen-specific CD8+ T cells
Matthew D. Martin, … , Robert A. Seder, Vladimir P. Badovinac
Matthew D. Martin, … , Robert A. Seder, Vladimir P. Badovinac
Published September 3, 2019; First published June 20, 2019
Citation Information: J Clin Invest. 2019;129(9):3894-3908. https://doi.org/10.1172/JCI124443.
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Categories: Research Article Immunology

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 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.

Authors

Matthew D. Martin, Isaac J. Jensen, Andrew S. Ishizuka, Mitchell Lefebvre, Qiang Shan, Hai-Hui Xue, John T. Harty, Robert A. Seder, Vladimir P. Badovinac

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Figure 2

Delayed addition of BFA leads to bystander activation of CD8+ T cells.

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Delayed addition of BFA leads to bystander activation of CD8+ T cells. (...
(A) Experimental design. Mice received adoptive transfer (AT) of naive P14 cells and were infected with LCMV-Armstrong. Approximately 3 weeks after infection, splenocytes were harvested and ICS was conducted. (B) Representative dot plots of IFN-γ production following 8-hour incubation without peptide and with BFA present for the entire incubation (0+8) or the final hour (7+1). Plots on the left are gated lymphocytes, plots in the middle are gated CD8+ T cells (Thy1.1– = endogenous CD8+ T cells, Thy1.1+ = P14 cells), and plots on the right are gated P14 cells. Numbers inside plots indicate the percentage of cells producing IFN-γ out of all gated cells. (C) Representative dot plots of IFN-γ production following 8-hour incubation with GP33 peptide. (D) Representative dot plots of IFN-γ production following 8-hour incubation with NP396 peptide. (E) Left: Summary graphs of the percentage of endogenous CD8+ T cells producing IFN-γ following stimulation with GP33 peptide out of all CD8+ T cells with BFA present for the entire incubation (0+8) or the final hour (7+1). Right: Percentage of endogenous CD8+ T cells producing IFN-γ when BFA was added for the final hour of incubation normalized to the percentage when BFA was present for the entire incubation (dotted line, 100%). Representative data from more than 3 independent experiments. n = 4. Dots indicate individual mice. Solid red lines indicate the mean. **P < 0.01 as determined by paired Student’s t test.