Scgm media

PM21 particles were generated and NK cells were expanded from peripheral blood mononuclear cells (PBMCs) as described previously.23 24 (link) Briefly, PBMCs were depleted of T-cells (EasySep CD3 positive selection kit; STEMCELL Technologies) and then cultured for up to 25 days with 100 U/mL interleukin-2 (IL-2; PeproTech) and 200 µg/mL of PM21 particles in SCGM media (CellGenix) supplemented with 10% non-HI FBS. PM21 particles were derived from CSTX-002 (K562-nmIL21-41BBL) cells, provided by Kiadis Pharma and maintained in RPMI media supplemented with 10% FBS.

Venous blood of healthy volunteers was collected in ethylenediaminetetraacetic acid monovettes (Sarstedt). Polymorphonuclears (PMNs) were subsequently purified as described (67 (link),68 (link)). Immune cells were suspended in SCGM media (CellGenix), counted with a cellometer X2 (Nexcelom) and immediately subject to confrontation assay.
Confrontation of 3 × 10⁶C. glabrata cells with 6 × 10⁶ PMN was performed in one well of a 6-well plate (Corning) in a total volume of 3-ml SCGM media. Following the incubation period, 20-U DNase (Invitrogen) were added to the co-incubation to dissolve DNA based structures, which trapped fungal material. Co-incubations were flooded with five volumes of RNAlater (Ambion). The solution was mixed with an equal volume of 4°C H₂O to lyse neutrophils. Fungal cells were harvested by centrifugation at maximum g for 5 min and subject to immediate RNA isolation.

Healthy donor peripheral blood mononuclear cells (Anne Arundel Medical Blood Donor Center, Annapolis, Maryland, USA; Carter Bloodcare, Woodway, Texas, USA) were isolated by Ficoll density gradient centrifugation and depleted of T cells using CD3 microbeads (Militenyi Biotec, Cologne, Germany). The remaining cells were stimulated on day (D)0 with lethally irradiated K562 feeder cells26 (link) expressing membrane bound IL-15 and 4-1BB ligand at a 1:1 ratio. Cells were maintained in SCGM media (CellGenix, Freiburg, Germany) supplemented with 10% fetal bovine serum, 2 mmol/L GlutaMAX (Thermo Fisher), and 200 IU/mL human interleukin (hIL)-2 (Biological Resources Branch Preclinical Biorepository, National Cancer Institute, Frederick, Maryland, USA). NK-cell purity was verified with flow cytometry using fluorophore conjugated antibodies against CD56 and CD3 (online supplemental table 1). NK cells were transduced on D4 of culture using transiently produced replication incompetent RD114 pseudotyped retroviral particles immobilized on RetroNectin (Clontech Laboratories, Palo Alto, California, USA).