Il 12

Peripheral blood mononuclear cells (1 × 10⁶ cells/ml) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS and antibiotics. For IL-12 + IL-18 or IL-15 stimulation, PBMCs from HD were seeded at 1.10⁶ cells/ml into 24-well plates and incubated for 48 h with 20 ng/ml of each cytokine (IL-12: R&D Systems; IL-18: MBL International; IL-15: Miltenyi). Golgistop (BD Biosciences) was added for the last 5 h of culture for IL-12 + IL-18 stimulation.

Cells from spleen, liver and BM were isolated as previously described (28 (link)). Antibodies are listed in Supplemental Figure 1. Samples were acquired using LSR Fortessa cytometer (BD Biosciences) and BD FACSDiva software (v.8.0.1, BD Biosciences) and analyzed with FlowJo software (Tree Star). Cell sorting was performed using FACSAria III (BD Biosciences). For the evaluation of IFN-γ expression, cells were left untreated or stimulated with PMA/Ionomycin (Sigma-Aldrich) for 2 h or IL-2 (1,000 U/ml, Hoffmann-La Roche Inc.) and IL-12 (10 ng/ml), or IL-12 (10 ng/ml) and IL-18 (100 ng/ml) (R&D Systems) for 6 h (with the addition of GolgiPlug, from BD Biosciences).

PBMCs were isolated from pSS patients and HCs using Ficoll–Paque gradient centrifugation. CD4⁺CD45RA⁺ naïve T cells were purified from PBMCs using Naïve CD4⁺ T-Cell Isolation Kit II (Miltenyi Biotec, Germany) according to the instructions of the manufacturer, with a purity of more than 95% by flow cytometry. Naïve CD4⁺ T cells were activated with anti-CD3 (5 µg/ml, BD Bioscience) and anti-CD28 (5 µg/ml, BD Bioscience). For the T-cell differentiation, IL-12 (10 ng/ml, R&D systems) plus anti-IL4 (10 µg/ml, PeproTech), IL-4 (2 ng/ml, PeproTech) plus anti-IFNγ(10 µg/ml, BD Bioscience), anti-IL4 (10 µg/ml) plus anti-IFNγ(10 µg/ml) plus TGF-β(5 ng/ml, R&D systems) plus IL-1β(12.5 ng/ml, PeproTech) plus IL-6 (25 ng/ml, PeproTech) plus IL-23 (25 ng/ml, PeproTech), TGF-β1 (5 ng/ml, R&D systems) plus IL-12 (1 ng/ml, R&D systems), or IL-2 (5 ng/ml, R&D systems) plus TGF-β (5 ng/ml, R&D systems) were supplemented for Th1, Th2, Th17, Tfh or Treg differentiation, respectively. T cells were incubated in RPMI-1640 medium (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and at 37°C, 5% CO2 for 3–5 days.

Cytokines and nitrite (an indirect indicator of NO production) were measured in cell culture supernatants from BMM and PuM un-treated or treated with either IFN-γ or AEC_sup at 4, 24 and 48 h after the end of infection with BCG. For ELISA determinations, the commercially, TNF, IL-6, CXCL10/Interferon gamma-induced protein 10 kDa (IP-10), IL-1β (R&D Systems) and IL-12, (Mabtech) were used to determine the cytokine levels in the culture supernatants according to the manufacturer's recommendations. The enzyme-substrate reaction was developed using p-nitrophenyl phosphate (Sigma) for IL-12 and tetramethylbenzidine substrate (R&D Systems) for the rest of determinations. Depending on the substrate used, the optical density was measured in a multiscan ELISA reader (Anthos Labtech Instruments, Salzburg, Austria) at 405 or 450 nm. NO production was determined by measuring nitrite concentration using the Griess reaction according to the manufacturers protocol (Sigma).

Poly(I∶C) was purchased from InvivoGen (SanDiego, CA) and diluted in HBSS for a concentration of 1 µg/µl. Mice were either inoculated with 200 µl HBSS or 200 µg Poly(I∶C) i.p. Mouse IFNβ was purchased from PBL Interferon Source. Cytokines: IL-2 was purchased from BD Biosciences, IL-6 and IL-12 were purchased from R&D, and IL-7 and IL-15 were purchased from PeproTech, INC. Splenocytes were stimulated ex vivo with cytokine concentrations of 1000 U/ml IFNβ, or 10 µg/ml of IL-2, IL-6, IL-7, IL-12, or IL-15 at 37°C for ∼30 minutes.

Cytokine concentrations in murine cell culture supernatants were analyzed by ELISA for the secretion of IL-6, TNF-α, IL-23, and IL-12 (R&D Systems) according to the manufacturer’s instructions.