Gentamycin sulfate

Fluorescence-activated cell sorting-sorted CD3+CD8+CD28+CD45RO−CCR7+ (naive CD8+) T cells were suspended in RPMI medium (Lonza, Breda, The Netherlands) supplemented with 10 mg/ml gentamycin sulfate (Lonza) and 10% fetal calf serum (Thermo Scientific, Breda, The Netherlands) in a volume of 3 ml and seeded at a density of 1 × 10⁶/ml in T25 cm flasks. A final concentration of 50 ng/ml human recombinant IL-15 (Peprotech, London, UK) was added to the cell culture at day 0 and refreshed every 5th day. On day 5, 10, and 15 of culture, cells were harvested and stained for flow cytometry analysis and/or lysed for RNA isolation. To study CD28, CD45RO, and CCR7 expression on naïve CD8+ T cells after IL-15 stimulation, CD3+CD8+CD28+CD45RO−CCR7+ T-cells were sorted as described above and stained with 10 umol/ml eF670 proliferation dye (eBioscience, Vienna, Austria). After 5, 10, and 15 days of culture in the presence of IL-15 (50 ng/ml), cells were harvested, stained, and analyzed by flow cytometer.

After recovery, BAL fluid was centrifuged at 300 x g for 15 min at 4°C, and BACs were suspended in complete medium [RPMI 1640 medium (Lonza, Walkersville, MD, USA) with 2 mM L-glutamine (Sigma Chemical Co., St Louis, MO), 50 mg/ml gentamycin sulfate (Lonza) and 10% heat-inactivated pooled AB human serum (Gemini, Woodland, CA)] at 1 x 10⁶ cells/ml. The average cell viability was 98% based on trypan blue exclusion (Gibco Life Technologies, Grand Island, NY). The BAL fluids were aliquoted and kept at -70°C until used. PBMCs were obtained by centrifugation using lymphocyte separation solution (Lonza), and their viability was 99% based on trypan blue exclusion.

COS-7 cells (African Green Monkey SV40-transformed kidney fibroblast cell line) were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Breda, The Netherlands), 200 mM l-glutamine and 10 mg/ml gentamycin sulfate (Lonza, Breda, The Netherlands) at 37°C in 5% CO₂.

Freshly isolated PBMCs from 3 ANCA-positive GPA-patients and 3 age- and sex-matched HCs were stained with anti-CD4-eF450, anti-CD25-PE and anti-CD45RO-FITC, and sorted on a MoFlo-Astrios (Beckman Coulter) according to Miyara’s classification^{18 (link)} into the following 4 populations: rTreg (CD4⁺CD25⁺CD45RO⁻), aTreg (CD4⁺CD25^HighCD45RO⁺), non-Treg (CD4⁺CD25^LowCD45RO⁻) and responder T cells (Tresp; CD4⁺CD25⁻) (Supplementary Fig. S2). Tresp cells were frozen in liquid nitrogen until use. The 3 other Tregs fractions were expanded in vitro for 2 weeks using anti-CD3/CD28 Dynabeads (Thermo Fisher Scientific) and 200IU of IL-2 (Peprotech, Rocky Hill, USA) in RPMI1640 (Lonza, Breda, The Netherlands) supplemented with 10% human pooled serum (Lonza) and 60 µg/ml gentamycin sulfate (Lonza). Part of the 3 expanded Treg subsets was used in suppression assays (as described below) to determine their suppressive capacity, and another part was frozen and used later to assess cytokine production (as described above).

HEK293-T cells were obtained from ATCC. B16F10 and DC2.4 cells were gifts from the Irvine lab. B16.SIY cells were a gift from the Spranger lab. MC-38 and TC-1 cells were gifts from the Wittrup lab. KP lines were gifts from the Jacks lab. CD8⁺ 58^-/-, Sf9, and Hi5 cells were gifts from the Garcia lab.
HEK 293-T, B16F10, B16.SIY, and MC-38 cells were cultured in Dulbecco’s Modified Eagle’s Medium (ATCC) supplemented with 10% fetal bovine serum (FBS, R&D Systems), 100 U/mL penicillin (Thermo), and 100 μg/mL streptomycin (Thermo). TC-1, DC2.4, and 58^-/^- lines were cultured in RPMI-1640 media (ATCC) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All mammalian cell lines and assay cultures were maintained at 37°C and 5% CO₂. All cells were frequently tested and confirmed negative for mycoplasma contamination. B16F10 cells used for tumor challenge studies also tested negative for rodent pathogens.
Sf9 cells were cultured in Sf-900 III SFM (Gibco) supplemented with L-glutamine, 10% FBS (R&D Systems), and 20 μg/mL gentamicin sulfate (Lonza). Hi5 cells were cultured in Insect-XPRESS (Lonza) supplemented with 10 μg/mL gentamycin sulfate (Lonza). Insect cell lines were maintained at 27°C with shaking at 120 rpm.

Primary human subcutaneous preadipocytes were purchased from Lonza (Alpharetta, GA, USA). Preadipocytes were cultured in preadipocyte growth medium (PGM) supplemented with 10% FBS, 2 mM L-glutamine, 30 μg/mL of gentamycin sulfate, and 15 ng/mL of amphotericin B (Lonza, Alpharetta, GA, USA). Differentiation of human subcutaneous adipocytes was initiated using complete PGM supplemented with insulin, dexamethasone, indomethacin, rosiglitazone, and IBMX (replenished every other day; Lonza, Alpharetta, GA, USA). Adipocytes were matured for 10 days before stimulation with XN.

In order to investigate the role of NF-κB in whey hydrolysate induced cytokine production, THP-1 monocytes were first differentiated into THP-1 macrophages as previously described [42 (link)]. First, THP-1 monocytes were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% decomplemented FBS, 2mM L-glutamine (Lonza, Verviers, Belgium), 1mM sodium pyruvate (Lonza, Verviers, Belgium), 0.05mM 2-mercaptoethanol (Scharlau, Barcelona, Spain), 60 μg/ml gentamycin sulfate (Lonza, Verviers, Belgium), and 2.2 μg/ml amphotericin B (Sigma Aldrich, Zwijndrecht, the Netherlands). Then, cells were seeded in a 24 wells plate at a concentration of 1x10⁶ cells/well (in 0.5 ml medium). To differentiate the cells into macrophages 100 ng/ml PMA was added. After 48 hours, the PMA was removed and cells were washed twice with fresh medium. Cells were cultured for another 24 hours. Then, cells were incubated with either 10 μM of the NF-κB inhibitor celastrol (Invivogen, Toulouse, France) or medium (control) for 30 minutes. Next, 2 mg/ml intact whey protein or its hydrolysates were added. Cells were incubated for 24 hours, after which the supernatant was collected. TNFα and IL-10 levels were measured in the supernatant by ELISA according to the manufacturer’s protocol (eBioscience, San Diego, USA).