Granzyme b clone gb12

The left lung lobe was excised post BAL, crudely dissociated using the GentleMACS™ tissue dissociator (Miltenyi Biotech, Germany) and digested upon incubation at 37°C in buffer containing 1 mg/mL collagenase Type XI and 80 units/mL Bovine Pancreatic DNase Type IV (both Sigma-Aldrich, Dorset, UK). For intracellular cytokine staining (ICS) cells were stimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL) with BD GolgiStop™ (BD Biosciences) for 3 h at 37°C. Lung and BAL cells were incubated with anti-mouse CD16/CD32 (FC Block, BD Biosciences) prior to staining for cell surface markers: CD3e (clone 500A2), CD4 (clone RM4-5), CD8a (clone 53-6.7), NK1.1 (clone PK136), NKp46 (clone 29A1.4) and CD69 (clone H1.2F3) (all BD Biosciences). Cells were washed and stained with Live/Dead fixable dead cell stain kit (Invitrogen), followed by incubation with BD Fix/Perm solution (BD Biosciences). For ICS, cells were stained for IFN-γ (clone XMG1.2, BD Biosciences) and granzyme B (clone GB12, Invitrogen) in BD PermWash™ (BD Biosciences). Data were acquired using a BD LSR II digital flow cytometer (BD Biosciences) and BD FACS Diva software. Analysis was performed using FlowJo 9.3.1.2 software.

Cell degranulation was determined by CD107a and intracellular granzyme B staining. Briefly, CD4⁺ T cells were co-cultured with target cells at a 10:1 ratio. 5μL APC-CD107a (clone H4A3, BD Biosciences) antibody was added at the beginning of culture. After 1 hour, monensin and Brefeldin A (Thermo Fisher Scientific) were added according to manufacturer’s instructions to block intracellular protein transport and cells were incubated for another 5 hours. Cells were collected, surface stained, and fixed and permeabilized in Cytofix/Cytoperm solution (BD Biosciences) for 30 minutes. Cells were then intracellular stained for granzyme B (clone GB12, Invitrogen) in permeabilization buffer and analyzed by a flow cytometer. For blocking experiments, 10μg/mL recombinant anti-HLA-E (Thermo Fisher Scientific), anti-CD48 (Thermo Fisher Scientific), or isotype antibody was added to target cells for 30 minutes at 37°C prior to co-culture and additionally during the 6-hour co-culture.