From these data we conclude that high proportions of CD8+ T-cells migrate to the lungs of PVM infected mice and that the appearance of virus-specific CD8+ T-cells in the airways is slightly delayed

compared to influenza virus- or hRSV-infected mice. As PVM-specific CD8+ T-cells migrated relatively late to the lungs of PVM infected mice, we wondered whether migration of other immune cells was delayed also. Quantification of NK cells in the BAL demonstrated a prominent influx of NK cells into the airways of PVM-infected mice at d. 6 of infection, when approximately 50% of total infiltrating lymphocytes were NK cells (Fig. 2A, left panel). In absolute numbers (Fig. 1A, right

panel) NK cell responses in PVM-infected mice peaked between days 8 and 10 of infection and then declined. In comparison, in the airways Ibrutinib clinical trial of influenza strain HKx31-infected mice (Fig. 1A) a large influx of NK cells, representing approximately 60% of total lymphocytes, was detected already at d. 2 p.i. with absolute numbers of infiltrating NK cells peaking at d. 3 of infection. Similar results were obtained in analyses of the BAL of hRSV-infected mice (Supplemenary NVP-BGJ398 in vivo Fig. 1). Both in influenza- and in PVM-infected mice, BAL NK cells displayed an activated phenotype (high CD69) and produced IFNγ upon stimulation ex vivo ( Fig. 2B and C), indicating that they were functional. Thus, PVM-infected mice show a marked influx of NK cells into the airways, although at a later time point than in mice infected with influenza or hRSV. PVM is a natural mouse pathogen and, unlike in case of HKx31, only Dipeptidyl peptidase a few viral particles suffice to establish

severe disease in mice. To determine whether the low numbers of infecting virus particles explains for the shifted kinetics of NK cell responses in PVM compared to HKx31-infected mice, NK cell influx into the airways of PVM-infected mice was compared to that in mice infected with the mouse-adapted influenza strain PR8, which is more virulent than HKx31 and therefore used at 100–1000 fold lower concentration. Still, like HKx31, infection with PR8 (150 EID50) induced a prominent early NK cell influx into the airways (Fig. 2D, d. 2 and 4 p.i). Conversely, mice infected with a high dose of PVM (1250 pfu) lacked NK cells in the BAL at d. 2 p.i., and only minor numbers of NK cells were detected at d. 4 p.i. (Fig. 2D). In conclusion, both CD8+ T-cells and NK cells migrate to the BAL at a much later time point following infection with PVM than with influenza. The relatively late influx of NK cells into the airways of PVM-infected mice is likely to be explained by specific properties of this pneumovirus rather than by the low numbers of viral particles administered to cause infection.