Quadromacs system

Bone marrow-derived mononuclear cells (BM-MNC) were obtained using density gradient centrifugation over Lymphoprep (Axis-Sheld, Oslo, Norway). CD271⁺ BM-MNCs were isolated by immuno-magnetic positive selection using QuadroMACS system and clinical grade CD271 microbead kit (Miltenyi Biotec GmbH, Germany) following manufacturer’s instructions. The unselected and CD271-enriched BM-MNCs were then seeded into T25 culture flasks and 6 well culture plates respectively and cultured in good manufacturing practice (GMP) compliant MSC expansion medium containing 10% FCS (StemMACS, Miltenyi Biotec) at 37 °C in a 5% CO2 incubator. After 3 days, the non-adherent cells were discarded with the replacement of culture medium. The medium was changed twice weekly and cells were passaged to a new flask when the culture reached 80% confluence using standard Trypsin/EDTA (Sigma-Aldrich) treatment. The MSCs derived from BM-MNCs with or without CD271 enrichment were denoted as CD271-MSC and PA-MSC respectively. Each paired PA-MSC and CD271-MSC samples were generated from the same bone marrow donation, seeded at the same density (4 × 10³/cm²) and cultured/passaged under identical conditions. At each passage cell population doubling time was recorded. MSCs at passage 3 were used in all experiments throughout this work.

CD8+ cells in DLN were isolated by magnetic beads conjugated with an anti-CD8 antibody (Miltenyi Biotec) as described in our previous report [36 (link)]. BALF cells were sorted by CD11b+ status using magnetic beads conjugated with an anti-CD11b antibody (Miltenyi Biotec). The magnetically labeled cells were purified using quadroMACS system (Miltenyi Biotec).

Buffy coats from healthy donors were collected after written informed consent (Sanquin, Nijmegen, The Netherlands). Isolation of human peripheral blood mononuclear cells (PBMCs) was performed by dilution of the buffy coat fractions 1:1 with sterile phosphate-buffered saline (PBS) (Sigma Aldrich, Zwijndrecht, The Netherlands) containing 2% fetal bovine serum (FBS) (HyClone™ Fetal Bovine Serum, Fisher Scientific, Loughborough, UK) and loading onto Greiner Bio-One™ LeucoSEP™ Polypropylene Tubes that were pre-loaded with 15 ml Ficoll-Paque plus (GE Healthcare Life Sciences). Cells were centrifuged at 200xg for 5 min followed by centrifugation at 500xg for 10 min. The interface layer, containing PBMCs, was isolated and the cells were washed three times in PBS containing 2% FBS. After washing, cells were diluted in 8 ml MACS buffer (2 mM EDTA, 2% FBS in PBS), after which 1 ml of CD14 microbeads (Miltenyi Biotec, Leiden, The Netherlands) was added per buffy coat followed by an incubation step for 15 min at 4°C and mixing every 5 min. Cells were washed and resuspended in 0.5 ml MACS buffer, and monocytes were isolated using positive selection with the quadroMACS system and LS Columns according to the manufacturer’s protocol (Miltenyi Biotec). Sorted cells were frozen in FBS with 10% dimethyl sulfoxide (DMSO) (Sigma Aldrich) and stored in liquid nitrogen.