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. 2009 Aug 24;77(11):4953–4965. doi: 10.1128/IAI.00844-09

FIG. 1.

FIG. 1.

Downregulation of MHC class II in IFN-γ-activated macrophages by F. tularensis infection supernatants. (A) BMDM were infected with F. tularensis LVS at an MOI of 100:1 or treated with medium alone (mock) for 2 h. Cells were treated with gentamicin for 1 h, washed, and incubated overnight to generate supernatants. Fresh reporter BMDM (pretreated or not pretreated 100 U of IFN-γ/ml for 24 h) were exposed to cleared infection supernatants for 15 h and analyzed for cell surface protein expression by staining for flow cytometry. Exposure of reporter BMDM to LVS infection supernatants leads to reduced surface expression of MHC class II and CD86, while FcγR and CD11b expression remains high. A lack of propidium iodide (PI) uptake indicates no decrease in cell viability. A vertical line represents the mean fluorescence intensity of the “+IFN” control for each surface marker. (B) After exposure to LVS or SchuS4 infection supernatants, the BMDM were lysed, and the MHC class II levels were analyzed by Western blotting. Exposure of macrophages to LVS or SchuS4 supernatants leads to a loss of total MHC class II, suggesting degradation after internalization. Shown are representative results from one of three independent experiments. (C) IFN-γ-pretreated BMDM were cultured with 10 μM HEL and F. tularensis LVS infection supernatant for 15 h. The BMDM were fixed with 1% paraformaldehyde, washed, and cocultured with the HEL peptide-specific h4Ly50.5 T-cell hybridoma line for 15 h. IL-2 levels were determined by cytometric bead array. Treatment of macrophages with LVS infection supernatants leads to a >90% loss of antigen presentation and resultant T-cell activation. The results are representative of one of three independent experiments ± the standard error of the mean (SEM). ***, P ≤ 0.001 (statistical difference from mock supernatant treatment).

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