Lung dendritic cell characterization and lymphocyte proliferation in response to hMPV infection in TLR4^−/− mice.

<p>TLR4^−/− and WT mice were mock- or hMPV-infected, and sacrificed at week 1 and 2 p.i. to collect lungs. Single cell suspension was obtained and cDCs population enriched using CD11c-tagged magnetic beads isolation. CD40 and CD86 markers were analyzed in cells positive for CD11c and MHC-II by flow cytometry. Histograms showing the expression of costimulatory molecules (CD40, CD86 and MHCII) in cDC from WT mice (open histograms) and cDC from TLR4^−/− mice (dashed line open histograms), and isotype control (shaded histograms), are shown (<b>A</b>). Graph of baseline expression of costimulatory molecules in lung dendritic cells isolated from WT and TLR4^−/− mice (<b>B</b>). Dendritic cells (CD11c positive cells) were isolated from lungs of hMPV or mock-infected mice either WT or TLR4^−/− at day 7 p.i. and loaded with 10 µg/mL of OVA peptide for 2h prior to coculture with T cells. CD4⁺ T cells isolated from spleen of OT II mice were labeled with CFSE and cocultured with DCs at a ratio of 1∶2 (DC: T). T cell proliferation was measured by CFSE dilution (proliferating CD4⁺ cells have a lower CFSE intensity than non-proliferated control cells). Cultures without antigen served as controls. The bar graph shows the percentage of proliferating (CFSE ^low) T cells among the total CD4⁺ T cell population. Data are expressed as mean ± SEM of four mice/group and represent one of two independent experiments (<b>C</b>).