RNA isolation and quantitative real time-PCR
To extract RNA from murine lung cells we used PeqGold RNA Pure according to the manufacturer’s protocol (PeqLab, Erlangen, Germany). For reverse transcription of RNA (1μg), we used the first strand cDNA synthesis kit for RT-PCR (MBI Fermentas, Sat. Leon-Rot, Germany) followed by the amplification by quantitative real-time PCR (qPCR) using SoFast EvaGreen Supermix (Bio-Rad Laboratories, München, Germany). The qPCR itself was performed in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories) with a cycle of 2 min 98°C, 50 cycles at 5 s 95°C, 10 s 60°C, followed by 5 s 65°C and 5 s 95°. The primers and sequences used for mouse were: mHPRT (5’- GCC CCA AAA TGG TTA AGG TT-‘3, 5’-TTG CGC TCA TCT TAG GCT TT-‘3), mICOS (5’- GTG CAG CTT TCG TTG TGG TA –‘3, 5’ - TCA GGG GAA CTA GTC CAT GC –‘3), mIL33 (5’- CCT CCC TGA GTA CAT ACA ATG ACC- ‘3, 5’- GTA GTA GCA CCT GGT CTT GCT CTT –‘3), mFoxp3 (5’- AGA GCC CTC ACA ACC AGC TA –‘3, 5’- CCA GAT GTT GTG GGT GAG TG –‘3), mIL10 (5’- CCA AGC CTT ATC GGA AAT GA –‘3, 5’- TTT TCA CAG GGG AGA AAT CG –‘3), mRora (5’-TCT CCC TGC GCT CTC CGC AC-3’, 5’- TCC ACA GAT CTT GCA TGG A –‘3), mST2 (5’-GCG GAG AAT GGA ACC AAC TA-‘3, 5’- AAG CAA GCT GAA CAG GCA AT –‘3), mAREGB (5’- AGC TGA GGA CAA TGC AGG GTA-‘3, 5’-AGT GAC AAC TGG GCATCT GG-‘3), mPD1 (5’-TCA AGG CATGGT CAT TGG TA-‘3, 5’-TAG GCC ACA CTA GGG ACA GG-‘3).