1 4 dtt

Peripheral blood mononuclear cells (PBMCs) were isolated from blood collected in CPT tubes according to the manufacturer´s protocol. BAL samples were kept on ice, filtered through a 100 μm nylon filter (Syntab) and centrifuged at 400 × g for 15 min. EBBs were processed as described earlier.6 In brief, EBBs were collected in RPMI 1640 with 2% fetal calf serum (FCS). Biopsies were washed in HBSS (Sigma–Aldrich) for 5 min and incubated with 5 mM 1,4‐DTT (Sigma–Aldrich) for 15 min. Incubations were performed on a rocker at 30 rpm at room temperature. Single biopsies were transferred to a 48‐well plate and incubated with 0.25 mg/ml Collagenase II and 0.2 mg/ml DNase (both Sigma–Aldrich) in RPMI 1640 for 60 min at 37°C. After digestion, biopsies were filtered through a 40 μm cell strainer (BD). LN aspirates were filtered through a 40 μm nylon cell strainer, centrifuged at 300 × g for 10 min. Lysis of red blood cells (RBC) was performed with 1 × RBC lysis buffer (University laboratories, Karolinska Hospital, Solna) for 5 min and subsequently centrifuged at 300 × g for 5 min. Cells were counted manually and Trypan Blue was used to assess viability.