Use of the CD107 mobilization assay reveals that cytotoxic T lymphocytes with novel MHC-Ib restriction are activated during Listeria monocytogenes infection

Abstract

Detection of cytotoxic activity by pathogen-specific T cells of unknown antigenic specificity is difficult due to the limitations of using infected cells, instead of peptide-pulsed cells, as targets. We report here that the recently described CD107 mobilization assay readily allowed for the ex vivo detection of cytotoxic T lymphocytes (CTL) with a novel MHC-Ib restriction that specifically recognized Listeria monocytogenes-infected macrophages. The CD107 mobilization assay is likely to be a useful tool for detection of CD8(+) T cells that recognize a wide variety of intracellular pathogens.

Fig. 1

OVA_257-264 specific T cells express CD107 on the cell surface following exposure to peptide-coated or Lm OVA-infected BMMϕ. The cytotoxic potential of line DM102 CD8⁺ T cells was assessed by CD107 mobilization assay. (A) C57BL/6 BMMϕ were incubated for 1 h in RP-10 media containing the indicated final concentration of synthetic OVA_257-264 peptide. (B) BMMϕ were infected with Lm OVA for 3 h at the MOI indicated. DM102 CD8⁺ T cells were added at an E:T ratio of 1:2. The percentage (A) or total number (B) of CD107a/b⁺ cells (gated on CD8⁺ cells) is shown.

Fig. 2

Listeria-infected cells can readily be used as targets in a CD107 mobilization assay, but not in a ⁵¹Cr release assay, for ex vivo detection of CTL activity in the spleen. C57BL/6 mice were infected with 2 × 10³ CFU of Lm OVA (immune) or given PBS (naïve) and splenocyte effectors were harvested 7 d later. The OVA_257-264 specific T cell line DM102 was used as a positive control. (A) BM12 macrophages were incubated with OVA_257-264 peptide (final conc. of 1 μM) for 1 h or infected for 3 h with Lm OVA (MOI = 0.5). CD8⁺ T cells were added at the given effector:target ratios (for splenocyte effectors, CD8⁺ T cells were assumed to be ~10% of the total population) and the % specific lysis was determined 4 h later. (B) C57BL/6 BMMϕ were infected (MOI=0.2) for 4 h with Lm OVA or incubated with OVA_257-264 peptide (final conc. of 1 μM) for 1 hr prior to the addition of either splenocyte effectors or DM102 T cells (E:T = 1:2). The % OVA-specific CD8⁺ T cells present in the spleen was determined by subtracting the number of CD107⁺ cells exposed to untreated BMMϕ (range = 0.5 − 1.5%) from the number of CD107⁺ cells exposed to either Lm-infected or peptide-coated BMMϕ. Average values +/- SD for groups of mice (n=3) from one of three separate experiments are shown. For the DM102 cell line, the mean +/- SD for pooled data obtained from all three experiments is given.

Fig. 3

Listeria-specific CTL with a novel MHC-Ib restriction were activated during L. monocytogenes infection. MHC-Ia deficient mice were either infected with 1 × 10³ CFU of Lm SDA1 (7 dpi) or given PBS (naïve). Splenocytes were harvested 7 d later, enriched for CD8⁺ T cells, and added (E:T= 1:2) to BMMϕ infected for 4 h with Lm SDA1 (MOI=0.1-1.0) for analysis in a CD107 mobilization assay. The % Lm-specific CD8⁺ T cells present in the spleen was determined by subtracting the number of CD107⁺ cells exposed to uninfected BMMϕ (typically ~1.0%) from the number of CD107⁺ cells exposed to Lm-infected BMMϕ. In some experiments, the infected BMMϕ were either pre-treated with anti-IL-12 + anti-IL-18 monoclonal Abs (final conc. 1 μg/ml) prior to the addition of splenocytes or separated from the CD8⁺-enriched immune splenocytes by incubation in a Transwell filter (0.4 μM pore size) insert. (A) Representative FACS plots for splenocytes from individual mice exposed to Listeria-infected macrophages. (B) Data for individual mice pooled from 6 separate experiments (n=2-3 per group for each exp.) is shown; mean values are indicated by thick horizontal lines. P values for the results of paired T test analyses are shown at the top of the graph.