Figure 10. Differences in the number of lung dendritic cells or the cytokines IL-15, IL-18, and TGFβ cannot account for the negative regulation of effector cells in coinfected animals.

A. Lung dendritic cells and macrophages were quantified in virus infected or coinfected animals on day 8 post influenza virus infection. DC and macrophage subsets were identified as follows. Live cells were gated on CD11c. Macrophages were identified based on forward/side scatter profile and CD11b staining, with recruited inflammatory macrophages defined as high expressers of CD11b, interstitial macrophages as intermediate expressers, and alveolar macrophages as low to absent expressers. For DC subsets, plasmacytoid DC were identified by intermediate levels of MHC class II and B220 positivity. Airway DC were defined by CD103 and MHC Class II positivity. Inflammatory monocyte derived respiratory DC (MoRDC) were defined by low to intermediate expression of MHC class II and positive staining for CD11b. Parenchymal DC were defined as MHC Class II^hi/CD11b positive and CD103 negative. Data shown are the average of 9 influenza infected and 7 coinfected mice assessed across two experiments. B–D. Cellular RNA was extracted from lung homogenates on d8 following influenza virus infection. cDNA was synthesized from mRNA by reverse transcription and subjected to qRT-PCR using primer probes specific for IL-15, IL-18, TGFβ, or GAPDH. Fold changes for each animal were calculated based on comparison to the average level of each mRNA detected in PR8 infected animals. Data shown are the average of 15 coinfected and 15 influenza virus infected animals assayed across two independent experiments. A two tailed student’s t test was used to determine significance. * p<0.05