Rhil 4

Monocytes were purified from PBMCs of six kiwi-allergic patients and six healthy donors by positive selection using CD14 dynabeads (Dynal Biotech ASA, Invitrogen, Oslo, Norway), following the manufacturer's protocol. DCs were derived from monocytes by culturing the CD14⁺ fraction in complete medium RPMI (Invitrogen) with l-glutamine, 10% heat-inactivated fetal bovine serum (Lonza, Amboise, France), and 100 mg/mL antibiotics (streptomycin and penicillin; Invitrogen) following the manufacturer's instructions, and with 200 ng/mL rhIL4 and 100 ng/mL rhGM-CSF (Immunotools, Friesoythe, Germany), for 4–5 days at 5% CO₂ and 37°C, as described previously (Gomez et al. 2012 (link)). Immature DCs (imDCs) derived from monocytes were incubated in complete medium at 5 × 10⁵ cells/mL in 24-well plates (Falcon BD Labware, Le Pont de Claix, France) with Act d 2, dAct d 2, and N-gly at 20 μg/mL. LPS at 10 μg/mL (Sigma) was used as a positive control. After 72 h of stimulation at 37°C in 5% CO₂, treated and nonuntreated DCs were recovered and maturation was assessed by CD83 (Immunotech, Marseille, France), CD80 and CD86 upregulation (Immunotools), in an Accuri cytometer (BD Accuri cytometers, Ann Arbor, MI). The maturation index (MI) was calculated as the ratio between stimulated and unstimulated DCs. A response was considered positive when MI > 2.

Primary human lung fibroblasts, fetal (Sigma) were cultured in DMEM/F12 medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% P/S, 0.5% L-glutamine and 0.2% amphotericin B and were maintained at 37°C in a humidified incubator containing 5% CO₂. Prior to stimulation, 1 × 10⁵ fibroblasts were seeded per well of 6 well-plates and rested in 0.1% FBS overnight. Fibroblasts were then stimulated for 48 h with fresh medium supplemented with rh IL-4 (1 pg/ml and 5 pg/ml, Immunotools) and rh IL-13 (10 ng/ml, Immunotools) or 1:5 diluted conditioned ILC2 supernatants derived from ex vivo stimulated human ILC2s (collected between d11 to d15 or after 3 days of restimulating adjusted numbers of expanded ILC2s). When indicated, anti-human IL-4 (10 ng/ml, eBioscience), anti-human IL-13 (20 ng/ml, BioLegend) or combinations of these were added. Conditioned supernatant derived from stimulated feeder cells alone or 1:5 diluted Yssel's medium (1% human serum AB, 1% P/S) supplemented with the cytokine cocktails used for ILC2 expansion (PHA, IL-2, IL-33 and IL-25) or restimulation (IL-2, IL-33, and IL-25) served as control. Fibroblasts up to passage eleven were used.

The THP-1-derived iDCs were generated as stated in Section 4.2. On day 5, for further differentiation into mDCs, the medium was removed, and fresh medium containing 100 ng/mL (=900 U/mL) rhGM-CSF (ImmunoTools, #11343125), 200 ng/mL (=4600 U/mL) rhIL-4 (Immuno-Tools, #11340045), 20 ng/mL (=400 U/mL) TNF-α (PromoKine, #C63719), and 200 ng/mL ionomycin (Sigma-Aldrich, #I0634) was added. The cells were cultivated for 48 h at 37 °C, 5% CO₂. For flow cytometry analysis, adherent cells were detached with accutase (Sigma-Aldrich, #A6964).

After washing IC‐coated plate, 1.5 × 10⁵ cells/mL of detached MDMs were seeded, either on IC‐coated wells or control wells without coated antibodies and treated with rh‐M‐CSF (BioLegend, San Diego, CA, USA), rh‐IL‐4, and rh‐IL‐13 (ImmunoTools, Friesoythe, Germany) M2a cytokine mix (Figure S1A‐B).

Human peripheral blood mononuclear cells were isolated and handled according to institutional ethical guidelines (Østfold Hospital Trust, Norway), as described elsewhere (25 (link)). Briefly, cells were isolated by density gradient centrifugation for 25 min at 1,500 × g using Lymphoprep TM (Axis-Shield Diagnostics Ltd.) at room temperature and washed four times with PBS to remove the platelets. CD14⁺ cells were separated on an LS column (Miltenyi Biotec) using human CD14 MicroBeads (Miltenyi Biotec). Cells were seeded in 24-well plates (1 × 10⁶ cells/well) and maintained in complete RPMI medium (RPMI supplemented with 10% fetal bovine serum FBS, 1% penicillin streptomycin, 2 mM l-glutamine, and 50 μM 2-mercaptoethanol, all from Sigma-Aldrich) with 25 ng/mL rhIL-4 and 50 ng/ml rhGM-CSF (ImmunoTools) for 4 days followed by replacement with fresh IL-4- and GM-CSF-supplemented complete medium and cultivation for another 3 days.

Peripheral blood monocytes in the PBMC fraction were resuspended in pre-warmed adhesion medium (RPMI supplemented with 5% human AB serum, non-essential amino acids, sodium pyruvate, HEPES and 1% penicillin/streptomycin) at a concentration of 10 Mio cells/mL and seeded onto Corning^® CellBIND plates (Sigma-Aldrich, St. Louis, Missouri, USA) for 1.5 h at 37 °C in a humidified atmosphere with 5% CO₂. Non-adherent cells were washed off and enriched monocytes incubated with differentiation medium (10% FCS, 1% penicillin/streptomycin and 20 ng/mL rh M-CSF (Peprotech, New Jersey, USA, #AF-300)) for 6 to 8 days. Human MDM were activated with 20 ng/mL recombinant human (rh) IFN-γ (Immunotools, Friesoythe, Germany #11343534) and 100 ng/mL LPS (Sigma-Aldrich, #L2880) or 20 ng/mL rh IL-4 (Immunotools, #11340043). To ensure successful activation, accutase-detached macrophages were stained with PE-labelled monoclonal antibodies for CD80 (20 µg/mL, PE-conjugated, BD Biosciences, Franklin Lakes, New Jersey, USA, #557227) and CD206 (20 µg/mL, PE-conjugated, BD Bioscience, #555954) or respective isotype controls (20 μg/mL, PE-conjugated) for 30 min.