Impact of bacteria on the phenotype, functions, and therapeutic activities of invariant NKT cells in mice
J. Clin. Invest. Sungjune Kim, et al. 118:2301 doi:10.1172/JCI33071 [Go to this article.]

Figure 4
Live L. monocytogenes infection induces hyporesponsiveness of iNKT cells to α-GalCer rechallenge ex vivo. (A) In vivo dynamics of the iNKT cell population in response to L. monocytogenes infection. Mice were injected with α-GalCer (5 μg/mouse, i.p.) or infected with L. monocytogenes and sacrificed at the indicated time points, and spleen and liver mononuclear cells were prepared and stained with anti–TCR-β–FITC, anti-NK1.1–PE, anti-B220–PerCP, and tetramer-allophycocyanin. Numbers indicate the percentage of TCR-β⁺tetramer⁺ cells among B220^– cells. Representative plots from 4–7 mice per group are shown. (B) Total spleen iNKT cell counts and percentage of liver iNKT cells at the indicated times, for a total of 4–7 mice per group, pooled from 2 separate experiments. *P < 0.05 compared with naive animals. (C) The in vitro α-GalCer recall response of mice at the indicated times after infection. Mice were infected with L. monocytogenes and sacrificed 3 days or 1, 2, or 4 weeks later, and splenocytes (2 × 10⁵ per well) were cultured with graded doses of α-GalCer. After 3 days, proliferation was assessed by [³H]thymidine incorporation, and culture supernatants were evaluated for IL-4 and IFN-γ levels by ELISA. Proliferation and cytokine results represent the mean ± SEM of 4–8 mice pooled from 2 separate experiments. *P < 0.05 compared with naive splenocytes cultured with the same dose of α-GalCer. (D) iNKT cell cytokine production. Spleen cells were prepared at the indicated times, and 2 × 10⁵ cells were cultured for 6 hours in plain medium or 100 ng/ml α-GalCer in the presence of GolgiPlug. Cells were then harvested and surface stained with tetramer-PE and anti-B220–PerCP, followed by intracellular staining with anti–IFN-γ–FITC and anti–IL-4–allophycocyanin. Data are shown for B220^–tetramer⁺ cells. Numbers indicate the percentage of cells within each quadrant. Results shown are representative of 2 independent experiments.