Verse cytometer

After the 14-d HDM treatment, the mice were euthanized and the lung tissue was removed after perfusion with PBS, cut into small pieces using scissors and digested in 400U/mL collagenase I (Sigma-Aldrich) for 1h in a 37°C shaking incubator (200r.p.m.). The digestion was terminated by diluting with PBS. After lysing red blood cells, the suspension was filtered using a cell strainer (70μM), and then stained with fluorochrome-conjugated antibodies for 30 min. Washed antibody-marked cells were analyzed by BD Verse cytometers or sorted by a FACSAria flow cytometer (BD). BALF cells isolation was described before. After the 14-d HDM or saline treatment, the mice were euthanized. The BALF cells were isolated, stained with fluorochrome-conjugated antibodies and analyzed by BD Verse cytometers. For the caspase-1 activity assay, the cells were stained with FAM-FLICA fluorescent probe (Immunochemistry Technologies) for 40min in a 37°C incubator. And then the cells were washed, stained with antibodies and analyzed by flow cytometry. A description of the gating strategy is provided in Supplementary Figure 1.

Six-parameter flow cytometric analysis was performed on whole blood, IELs, and T cells from LNs according to standard procedures (24 (link)) using a panel of monoclonal antibodies (mAbs) that were originally designed to detect human molecules but that we and others have shown to be cross-reactive with rhesus monkey (3 (link), 19 (link), 25 (link), 26 (link)). The antibodies used were as follows: anti-CD3-FITC (clone SP34), anti-CD4-APC-H7 (clone L200), anti-CD8-PE-Cy7 (clone RPA-T8), anti-CD69-PE (clone FN50), and anti-HLA-DR-PerCP-Cy5.5 (clone G46-6) (all from BD Pharmingen). Isotype antibody was used for negative control of CD69 and HLA-DR expression. Flow cytometric acquisition and analysis of samples were performed on at least 100,000 events on a BD Verse cytometer driven by the FACS Verse software (BD Biosciences). Analysis of the acquired data was performed using FlowJo software (TreeStar, Ashland, OR, USA). The CD4⁺ and CD8⁺ T cell percentages were based on the CD3⁺ T cells, and the activation markers on CD4⁺ and CD8⁺ T cells were based on CD4⁺CD3⁺ and CD8⁺CD3⁺ T cells, respectively.