HCMV infection of DCs induces no IL-12 and only a low level of TNF-α production.

Cytokine production by DCs following HCMV infection was measured by intracellular staining of cytokines in brefeldin A–treated, fixed, and permeabilized DCs by FACS analysis as described in “Patients, materials, and methods.” DCs were generated as described in the presence of GM-CSF and IL-4. On day 5 of the culture they were either mock infected or infected with HCMV_TB40/E (MOI = 10), or UV-irradiated HCMV was added (MOI = 10), or LPS was added in the presence of IFN-γ and TNF-α. After 2 hours the medium was removed, the cells were washed once, and fresh complete medium containing GM-CSF and brefeldin A, in the form of GolgiPlug, was added. The cells were incubated for 22 hours and immunofluorescent labeling of fixed and permeabilized cells with FITC-conjugated HCMV pp52-specific antibody and either PE-conjugated IL-12 specific antibody or PE-conjugated TNF-α–specific antibody was carried out as described. (A) A representative experiment showing the original FACS data of the IL-12 production, or (B) TNF-α production by DCs following mock infection (first panel); infection with HCMV (second panel); treatment with UV-inactivated HCMV (third panel); LPS treatment (fourth panel). The Y axis represents the mean fluorescence intensity (MFI) of IL-12 or TNF-α and the X axis shows the MFI of the HCMV pp52-specific antibody. The numbers represent the proportions of cytokine-producing DCs. In the HCMV-infected group the cytokine production by the HCMV Ag-negative and by the HCMV Ag-positive DCs was calculated separately. (C) The summary of IL-12 production and (D) TNF-α production by DCs following HCMV infection. Means and standard deviations were calculated from 4 experiments. Significant differences to the mock-infected control are indicated as follows: *P < .05, **P < .01.