Figure 5. T cell signaling modulation is mediated via a secreted factor from macrophages.

(A) Mice were treated i.v. with 0.5 μg LPS (light grey), 5 μg LPS (dark grey), or PBS as a control (white). After 6 hours, splenocytes were harvested and stimulated with IFNα (400 U/mL), IFNγ (40 ng/mL), IL-6 (40 ng/mL) or were left unstimulated. The mean (± SD) in fold change of pStat1 MFI post-stimulation was determined from B cells and CD4 T cells from two mice per group and is representative of at least two separate experiments. (B) BMDCs were cultured overnight without or with LPS (1 μg/mL), stimulated with either IFNα, IFNγ, IL-6, IL-10, or GM-CSF for 15 minutes, and then fixed and analyzed. The gating for MHCII+ cells is shown in the upper left contour plots. Also shown are contour plots of CD11c compared to pStat1, pStat3, and pStat5. (C) BMDMs and primary T cells were cultured with LPS (10 μg/mL) or PAM3CysSK4 (10 μg/mL) for 6 hours, then stimulated 15 minutes with either IFNα, IFNγ, or IL-6 and assayed by flow cytometry for pStat1 levels. (D) The media from BMDMs treated with LPS or PAM3CysSK4 was collected and applied to T cells for 3 hours. T cells were then stimulated with IFNα, IFNγ, or IL-6 and assayed by flow cytometry for pStat1 levels. (E) BMDMs were infected ex vivo with wild-type Listeria or LLO- Listeria and then treated with gentamycin. Three mice each were intravenously injected with PBS control, BMDMs, Listeria-infected BMDMs, LLO- Listeria-infected BMDMs, or Listeria-cleared media to control for media exposure to bacteria. Six hours later, splenocytes were harvested and stimulated with IFNα, IFNγ or IL-6 and then assayed by flow cytometry for pStat1 levels. An asterisk signifies p < 0.05 compared to PBS control. Data are representative of at least two separate experiments.