American Society of Hematology

Fig. 5.

$Fig. 5. HCMV-infected DCs are refractory to produce IL-12 or TNF-α when stimulated with LPS or CD40L. / Cytokine production by mock-infected or HCMVTB40/E-infected DCs upon stimulation with LPS or CD40L was measured by FACS analysis. (A) A representative experiment showing IL-12 and TNF-α production by DCs upon stimulation with LPS. DCs were either mock infected (first panel) or were infected with HCMVTB40/E (MOI = 10) for 48 hours and then stimulated with LPS/IFN-γ/TNF-α for 22 hours in the presence of GolgiPlug for the last 19 hours. The Y axis represents the MFI of IL-12 or TNF-α and the X axis shows the MFI of the HCMV pp52-specific antibody. The numbers express the proportion of cytokine-producing cells. (B) The summary of 3 experiments, showing the mean and standard deviation of cytokine-producing mock-infected and HCMV-infected (Ag-positive or Ag-negative) DCs following LPS stimulation. Significant differences to the mock-infected control are indicated as *P < .05. (C) IL-12 and TNF-α production by DCs following stimulation with CD40L. Mock-infected DCs were either left unstimulated or were prestimulated with LPS/IFN-γ/TNF-α. One group of DCs was infected with HCMVTB40/E (MOI = 10 PFU/cell) for 48 hours. DCs were then stimulated with CD40L-expressing fibroblasts at DC/Fb ratio = 10:1 in the presence of IFN-γ (100 ng/mL) for 22 hours. GolgiPlug was added for the last 21 hours of the incubation. The results are expressed as percent of DCs positive for the studied cytokine. A representative of 2 similar experiments is shown. (D) HCMV induces up-regulation of nuclear NF-κB in DCs. Immunoblotting of nuclear NF-κB proteins is shown from DCs following HCMV infection. DCs on day 5 of culture in GM-CSF and IL-4 were either mock infected (-), infected with HCMV (MOI = 10) (+), or UV-inactivated virus was added (UV) for 24 hours. DCs were then either treated with LPS/IFN-γ/TNF-α (right panel, +LPS) for 24 hours or were left untreated (left panel, immature DCs). Nuclear extracts were analyzed for RelB expression by separation of the proteins on a 10% SDS-PAGE gel followed by electrotransfer to PVDF membranes and incubation with polyclonal antibodies against RelB and actin.$

HCMV-infected DCs are refractory to produce IL-12 or TNF-α when stimulated with LPS or CD40L.

Cytokine production by mock-infected or HCMV_TB40/E-infected DCs upon stimulation with LPS or CD40L was measured by FACS analysis. (A) A representative experiment showing IL-12 and TNF-α production by DCs upon stimulation with LPS. DCs were either mock infected (first panel) or were infected with HCMV_TB40/E (MOI = 10) for 48 hours and then stimulated with LPS/IFN-γ/TNF-α for 22 hours in the presence of GolgiPlug for the last 19 hours. The Y axis represents the MFI of IL-12 or TNF-α and the X axis shows the MFI of the HCMV pp52-specific antibody. The numbers express the proportion of cytokine-producing cells. (B) The summary of 3 experiments, showing the mean and standard deviation of cytokine-producing mock-infected and HCMV-infected (Ag-positive or Ag-negative) DCs following LPS stimulation. Significant differences to the mock-infected control are indicated as *P < .05. (C) IL-12 and TNF-α production by DCs following stimulation with CD40L. Mock-infected DCs were either left unstimulated or were prestimulated with LPS/IFN-γ/TNF-α. One group of DCs was infected with HCMV_TB40/E (MOI = 10 PFU/cell) for 48 hours. DCs were then stimulated with CD40L-expressing fibroblasts at DC/Fb ratio = 10:1 in the presence of IFN-γ (100 ng/mL) for 22 hours. GolgiPlug was added for the last 21 hours of the incubation. The results are expressed as percent of DCs positive for the studied cytokine. A representative of 2 similar experiments is shown. (D) HCMV induces up-regulation of nuclear NF-κB in DCs. Immunoblotting of nuclear NF-κB proteins is shown from DCs following HCMV infection. DCs on day 5 of culture in GM-CSF and IL-4 were either mock infected (-), infected with HCMV (MOI = 10) (+), or UV-inactivated virus was added (UV) for 24 hours. DCs were then either treated with LPS/IFN-γ/TNF-α (right panel, +LPS) for 24 hours or were left untreated (left panel, immature DCs). Nuclear extracts were analyzed for RelB expression by separation of the proteins on a 10% SDS-PAGE gel followed by electrotransfer to PVDF membranes and incubation with polyclonal antibodies against RelB and actin.

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