Enumeration of cytotoxic CD8 T cells ex vivo during the response to Listeria monocytogenes infection

Abstract

Cytotoxicity is a key effector function of CD8 T cells. However, what proportion of antigen-specific CD8 T cells in vivo exert cytotoxic activity during a functional CD8 T-cell response to infection still remains unknown. We used the Lysispot assay to directly enumerate cytotoxic CD8 T cells from the spleen ex vivo during the immune response to infection with the intracellular bacterium Listeria monocytogenes. We demonstrate that not all antigen-responsive gamma interferon (IFN-gamma)-secreting T cells display cytotoxic activity. Most CD8 T cells detected at early time points of the response were cytotoxic. This percentage continuously declined during both the expansion and contraction phases to about 50% at the peak and to <10% of IFN-gamma-producing cells in the memory phase. As described for clonal expansion, this elaboration of a program of differentiation after an initial stimulus was not affected by antigen or CD4 help but, like proliferation, could be influenced by later reinfection. These data indicate that cytotoxic effector function during the response to infection is regulated independently from IFN-gamma secretion or expansion or contraction of the overall CD8 T-cell response.

FIG. 1.

Transduced P815-LacZ cells can reliably detect cytotoxic CD8 T cells. (A) Increasing numbers of P815-LacZ cells per well were added to a monolayer of 200,000 resting 2C cells at day 7 after in vitro stimulation, and the numbers of IFN-γ ELISPOTs (y axis) and Lysispots (x axis) per well are plotted. (B) Increasing numbers of P815-LacZ cells per well were incubated with a constant number of 2C cells, and the number of IFN-γ ELISPOTs per well was determined. sfu, spot-forming units.

FIG. 2.

CD8 T-cell response to L. monocytogenes infection as measured by IFN-γ ELISPOT and Lysispot assays. BALB/c mice were infected i.v. with L. monocytogenes and the LLO_91-99-specific CD8 T-cell responses in the spleen were analyzed by IFN-γ ELISPOT and Lysispot assays. (A and D) Total numbers of spots per spleen are shown; averages are depicted as black (IFN-γ) or gray (cytotoxicity) bars and data for individual mice as open circles. (B and E) Total numbers of cytotoxic cells per spleen (y axis) are plotted versus total numbers of IFN-γ-secreting cells (x axis). (C and F) Fractions of ex vivo cytotoxic T cells expressed as percentages of IFN-γ-secreting cells. Two separate experiments are shown.

FIG. 3.

Cytotoxicity of the CD8 T-cell population at the peak of the response is not directly influenced by the presence/absence of the pathogen. BALB/c mice were i.v. infected with L. monocytogenes and 36 h later treated with ampicillin (Amp) or left untreated. LLO_91-99-specific CD8 T-cell responses in the spleen were analyzed on day 8 after infection. Total numbers per spleen (A) and per million splenocytes (B) and fractions of cytotoxic cells expressed as percentages of IFN-γ-secreting cells (C) are shown. Averages are depicted as bars and data for individual mice as open circles. Similar results were obtained in two separate experiments.

FIG. 4.

Cytotoxicity of the CD8 T-cell population at the peak of the response is not directly influenced by the presence of CD4 T cells but can be enhanced by reinfection. BALB/c mice were i.v. infected with L. monocytogenes and then either injected with GK1.5 on days 3 and 4 after infection to deplete CD4 cells (A) or reinfected with 1 × 10⁶ Listeria cells on day 6 after infection (B) or were left untreated. LLO_91-99-specific CD8 T-cell responses in the spleen were analyzed on day 8 after infection. Fractions of cytotoxic cells are expressed as percentages of IFN-γ-secreting cells. Averages are depicted as bars and data for individual mice as open circles. Similar results were obtained in two separate experiments each.