Il 23

The protein levels of IFN-γ, IL-6, IL-12, CXCL10, IL-5, CX3CL1, CXCL9, CCL3, and CCL4 in cell supernatants were assessed using a commercial CBA (BD Biosciences, Stockholm, Sweden) performed on a BD FACSCanto II flow cytometer (BD Biosciences) and analyzed using a FCAP Array version 3 software (BD Biosciences) according to the manufacturer’s protocols. Furthermore, the protein levels of IL-15 (Sino Biological, NordicBiosite, Stockholm, Sweden) and IL-23 (Mabtech, Stockholm, Sweden) were measured using commercial ELISA kits according to the manufacturer’s protocols.

T cells were enriched by EasySep™ Human T Cell Isolation Kit (Stemcell) from PBMCs. MAIT, CD4⁺ T cells were purified from those PBMCs by FACS sorting. Ten randomly pooled CAP BAL with a total 50 ml volume or 10 ml control BAL were concentrated by an Amicon® Pro Purification System with 10 kDa Amicon® Ultra-0.5 Device (Millipore) to a final volume of 5 or 1 5 × 10⁵ T cells were cultured in IP10 medium (10% fetal bovine serum + IMDM (Gibco) and stimulated with 1:1 anti-CD3/CD28 beads (Gibco). For differentiation, 0, 5, 10, and 20 μl concentrated BAL fluids (0, 5, and 10 μl for control BAL) were added to the FACS-sorted T cells. For CD14⁺ monocyte-MAIT co-culture assays, 10⁵ blood MAIT cells were cultured with 1 or 2 × 10⁵ FACS-sorted CD14⁺ monocytes pooled from BALs or peripheral blood of CAP patients in the presence of anti-CD3/CD28 beads in IP10 medium for 3 days. For cytokines neutralization assays, 0, 10, and 20 ng/ml IL-1β (Invivogen) or IL-23 (Mabtech) neutralization Abs were added to 10⁵ blood. MAIT cells were cultured with anti-CD3/CD28 beads and 10 μl concentrated BAL fluids in IP10 medium for 3 days (n = 4). Cytokines in the culture supernatants (IL-17A and IFN-γ) were measured by LEGENDplex™ bead-based immunoassays (BioLegend).