Il 1β

Thawed (peripheral blood mononuclear cells) PBMCs were resuspended at 10⁷ cells/ml in 24-well tissue culture plates (5 × 10⁶ cells/well) or in a 75-cm² tissue culture flask (7 × 10⁷ cells/flask) containing AIM medium (Life Technologies, Monza, Italy) supplemented with FLT3L (50 ng/ml; Miltenyi Biotec, Bologna, Italy) to mobilize resident DCs, as previously described (23 (link), 24 (link)). After 24 h (day 1), the peptides were added to the cultures, and DC maturation was induced with TNFα (1,000 U/ml, Miltenyi Biotec), IL-1β (10 ng/ml, Miltenyi Biotec), IL-7 (0.5 ng/ml, R&D Systems), and prostaglandin E2 (1 μM, Calbiochem, Milan, Italy). On day 2, (Fetal Bovine Serum) FBS (Euroclone) was added at a final v/v ratio of 10%. Medium was then replaced at days 4 and 7 with fresh RPMI 1640 (Euroclone, Milan, Italy) enriched with 10% FBS, non-essential amino acids (Euroclone) and sodium pyruvate (Sigma-Aldrich, Milan, Italy). Antigen-specific CD8⁺ T cells were characterized on day 10.

IL‐1β was from Miltenyi; IL‐1α and HGF were from R&D; bFGF and EGF were from TEBU‐BIO; TGF‐β was from Peprotech (Rocky Hill, NJ, USA). ShhN was made by transfecting 293T cells with ShhN in pRK5 (from Genentech, South San Francisco, CA, USA) and after transfection, incubating cells in DMEM containing 0.5% FBS. Prior to the addition of ligands, cells were switched to 0.5% FBS culture medium for 16 h. Ligands were added for 24 h.

On transduction day (day 0), sub-confluent (50–80%) hPSC cultures were dissociated to single cells using TrypLE (Life Technologies) Fig. 2a. Embryoid body formation was initiated with 6–12E+5 viable cells per well of an Aggrewell400 plates (Stem Cell Technologies) leading to 500–1,000 cells per embryoid body following spin aggregation. Lentiviral transduction was performed concomitantly to the aggregation step in CDM supplemented with Y-27632 (10 μM, Sigma), BMP4 (10 ng ml⁻¹, R&D) and protamine sulfate. After 24 h, transduced embryoid bodies were collected and sown in ultralow adherent cell culture plates (Corning) at 1,200 embryoid bodies per 10 cm² dish in CDM with BMP4 and FGF2 (5 ng ml⁻¹). Twenty-four hours later, embryoid bodies were washed and sown in ultralow adherent plates at 600 embryoid bodies per 10 cm² in CellGroSCGM medium (CellGenix) supplemented with TPO (100 ng ml⁻¹, Cellgenix) and SCF (25 ng ml⁻¹, Gibco). At day 10, embryoid bodies were dissociated to single cells using Collagenase-IV and Dispase-II (1 mg ml⁻¹, Gibco) followed by enzyme free cell dissociation buffer (Gibco) treatment. Single cells were cultivated at 2E+5 per ml on tissue culture plates (Corning) for an additional 10 days in CellgroSCGM with TPO (100 ng ml⁻¹) and IL1-β (10 ng ml⁻¹, Miltenyi Biotec). Half of culture media was renewed every 3 days.

The murine thymoma cell line EL4 was cultured in complete DMEM (Hiltensperger et al., 2021 (link)). EL4 cells were costimulated under Th17-skewing conditions for 72 h. Cells were then exposed to PBS or IL-1β (25 ng/ml; Miltenyi) in the presence of actinomycin D (2 µg/ml; Tocris Bioscience) for another 16 h of culture.

4 μm thick kidney sections embedded in paraffin were prepared for immunohistochemistry assays. Sections were rehydrated, and antigens retrieved using heated citrate. Primary antibodies including NLPR3 (Adipogen, USA) and IL-1β (Miltenyi, China) were applied in blocking solution overnight at 4°C. After being incubated with anti-rabbit IgG (1 : 100; Beyotime) for 30 min at 37°C and washed and developed with DAB (Bioss, China), the stained sections were examined using a light microscope (Nikon Eclipse TE2000-U, NIKON, Japan) at 400x magnification. The semiquantitative immunohistochemical analysis was scored using Image-Pro plus 6.0 software in ten randomly selected cortical sections in each tissue section.

Monocyte-derived dendritic cells were stimulated with 50 µg/ml of curdlan (Wako), curdlan microparticles (curdlan-mp) [generated as described previously (14 (link))], and β-1,3 glucan microparticles (glucan-mp) [generated as described above (29 (link), 30 (link))]. mDCs were also stimulated with β-glucans and 2 µM CYTD (Sigma) and/or 10 ng/ml IL-1β (Miltenyi) simultaneously. Cell cultures were incubated for predefined time periods as highlighted in figure legends.