The onset of CD8+-T-cell contraction is influenced by the peak of Listeria monocytogenes infection and antigen display

Abstract

The CD8+-T-cell response to infection with Listeria monocytogenes consists of expansion, contraction, and memory phases. The transition between expansion and contraction is reported to occur on different days postinfection with virulent (8 to 9 days) and attenuated (DeltaactA) (7 days) L. monocytogenes strains. We hypothesized that differences in the infectious courses, and therefore antigen (Ag) display, determine the precise time of the expansion/contraction transition in response to these infections. To test this, we infected BALB/c mice with 0.1 50% lethal dose of DeltaactA or virulent L. monocytogenes and measured bacterial numbers, Ag display, and Ag-specific CD8+-T-cell responses on various days after infection. We found that bacterial numbers and Ag display peaked between 12 and 36 h and between 36 and 60 h after infection with DeltaactA and virulent L. monocytogenes strains, respectively. Infection with DeltaactA L. monocytogenes resulted in a sharp peak in the Ag-specific CD8+-T-cell response on day 7, while infection with virulent L. monocytogenes yielded a prolonged peak with equivalent numbers of Ag-specific CD8+ T cells on days 6, 7, and 8 after infection. Truncating virulent infection with antibiotics on day 1 or 2 after infection resulted in a shift in the expansion/contraction transition from day 8 to day 7 after infection. However, antibiotic treatment beginning on day 3, after the peak of virulent L. monocytogenes infection and Ag display, had no effect upon the magnitude or timing of the CD8+-T-cell response. These results demonstrate a direct relationship between the course of infection and Ag display and that the timing of these events is important in shaping the T-cell response to infection.

FIG. 1.

The CD8⁺-T-cell response expansion/contraction transition is prolonged in virulent L. monocytogenes infection. BALB/c mice were infected with 2.6 × 10⁶ ΔactA L. monocytogenes organisms (Att.) or 1.2 × 10³ 10403s L. monocytogenes organisms (Vir.). (A) The total numbers of LLO_91-99- plus p60_217-225-specific CD8⁺ splenocytes were measured on the indicated days (d) after infection using ICS for IFN-γ. The first days of significant Ag-specific CD8⁺-T-cell decrease compared to day 7 postinfection are marked with asterisks. *, P ≤ 0.04; **, P ≤ 0.02. (B) Representative CD8⁺ gated FACS plots from representative mice 7 days after infection. The upper and lower numbers are the percentages of CD8⁺ cells that were IFN-γ positive in peptide-incubated or non-peptide-incubated splenocytes, respectively. (C) The total number of Ag-specific CD8⁺ T cells per spleen was normalized to the maximum response to each L. monocytogenes strain. (D) Portions of spleens from infected mice were plated on the indicated days after infection. LOD, limit of detection; the numbers in parentheses are the fractions of mice with measurable infection. Days with significant difference between Att. and Vir. infections: *, P ≤ 0.01; **, P ≤ 0.001. The data shown are means ± standard deviations of three mice per group and are representative of four or more experiments.

FIG. 2.

Direct ex vivo Ag presentation after ΔactA and virulent L. monocytogenes infection. (A) The DEAD assay was performed with responder Ag-specific CD8⁺-T-cell lines incubated with naïve splenocytes and increasing concentrations of cognate peptide. BALB/c mice were infected with 9.2 × 10⁶ ΔactA L. monocytogenes organisms (Att.) or 8.8 × 10³ 10403s L. monocytogenes organisms (Vir.). (B) Representative FACS plots from DEAD assays performed on infected or naïve splenocytes (Naï.) with a LLO91-99-specific T-cell line on the indicated days (d) after infection. The T-cell line was CFSE positive, and the numbers represent the percentages of CFSE-positive cells that were IFN-γ positive. (C) Composite DEAD assay results of two experiments using L. monocytogenes-specific T-cell lines and a negative-control, β-Gal-specific CD8⁺-T-cell line. The data shown are means ± standard errors of the means for six mice per group.

FIG. 3.

In vivo “functional” Ag presentation after ΔactA and virulent L. monocytogenes infection. L9.6 cells (1 × 10⁶) were transferred into BALB/c Thy1.1 mice on the indicated days (d) after infection or no infection with 1.1 × 10⁶ ΔactA (Att.) or 1.4 × 10³ 10403s (Vir.) organisms. Spleens were harvested 48 h after transfer, and the Thy1.2⁺ L9.6 cells were analyzed for CFSE dilution. (A) Representative histograms of L9.6 cells harvested from individual mice from uninfected (Un.) or infected mice. The numbers are the percentages of L9.6 cells that divided. (B) Average percentages of L9.6 cells that divided. The data shown are means ± standard deviations for three mice per group and are representative of two experiments.

FIG. 4.

Three days of infection are sufficient to shape the Ag-specific CD8⁺-T-cell response. BALB/c mice were infected with 1.6 × 10³ 10403s organisms (Vir.), and groups of mice were given ampicillin on different days (d) after infection. (A) Spleens from infected mice were plated 1 day after the initiation of Amp treatment, and CFU/spleen were determined. (B) Total numbers of LLO_91-99- and p60_217-225-specific CD8⁺ T cells per spleen on the indicated days after infection, determined by ICS for IFN-γ. The data shown are means ± standard deviations for three mice per group and are representative of two experiments.