n. with 5 × 106 pfu RSV in 50 μl, or with 1 × 105 EID50 HKx31 or 150 EID50 PR8 in 30 μl PBS as described , or with the indicated doses of PVM in 30 μl PBS. All animal experiments were approved by the Committee on Animal Experiments of the University of Utrecht. Mice were sacrificed by injection of sodium pentobarbital and bronchoalveolar lavage (BAL) was collected by three times lavage with
1 ml PBS containing 10 μM EDTA. Thereafter, lungs were perfused with PBS, excised, minced and incubated in PBS containing collagenase (2.4 mg/ml; Roche Applied Science) and DNase (1 mg/ml; Roche Applied Science) for 30 min at 37 °C, passed through a cell strainer and lymphocytes were purified using lympholyte-M (Cederlane). For mRNA isolation, the right lung was placed in 1 ml TRIzol (Invitrogen). Fluorochrome-conjugated antibodies were purchased from eBioscience [CD69 (H1.2F3), CD49b (DX5), TCRβ (H57-597), NKp46 (29A1.4), Selleck Ibrutinib CD62L (MEL-14), IFNy (XMG1.2), CD8 (53-6.7), CD11c (N418), CD19 (MB19-1), CD4 (RM4-5), MHC-II (m5/114.15.2)] or BD Pharmingen [Siglec-F (E50-2440)]. PE-labeled MHC class I tetramers were prepared in collaboration with D. Busch (TU-Muenchen), by refolding H2-Kd heavy chains and human β2m in the presence of synthetic influenza-derived NP147–155 (TYQRTRALV), hRSV M282–90 (SYIGSINNI) or PVM
P261–269 (CYLTDRARI). Cell surface markers were stained as described . For tetramer stainings, cells were incubated Vandetanib with 1 μg tetramer for 1 h at 4 °C and then stained Florfenicol for surface markers. To measure IFNγ production, BAL cells were stimulated 1:1 with YAC cells for 4 h (NK cell activation) or with 2 μM P261–269 for 6 h (CD8+ T-cell stimulation) in 100 μl RPMI medium containing 10% FCS, glutamax, antibiotics and 30 μM β-mercaptoethanol, and 10 μM monensin and then stained as described . Cells were analyzed on a FACS Calibur or Canto II (BD Biosciences) using FlowJo software (Tree Star). Mouse
BM-DC were expanded for 6 days in RPMI medium with 15% GM-CSF (culture supernatant of X63Ag cells), activated overnight with 100 ng/ml LPS and then pulsed for 1 h with 2 μM P261–269. Mice were immunized intravenously (i.v.) with 5 × 106 peptide-loaded BM-DC in 200 μl PBS. FI-PVM was prepared as described  and was administered in 100 μl s.c. Mice were infected with PVM, 3–5 weeks after immunization. Total lung RNA was purified using TRIzol (Invitrogen) and cDNA was transcribed (iScript cDNA Synthesis Kit; Bio-Rad Laboratories). PVMSH RT-PCR was performed as described  in an iCycler (Bio-Rad Laboratories), 95 °C for 10 min and then 45 cycles of 95 °C for 15 s and 60 °C for 60 s. Copy numbers per lung were calculated from a standard curve generated using serially diluted PVM-SH cDNA. RT-PCR for IL-4, IFNγ and GAPDH were performed using the TaqMan Gene Expression Assays (Applied Biosystems) Mm00445259, Mm00801778 and Mm99999915.