Rosettesep

PBMCs where isolated from buffy coats using ficoll density gradient isolation (Lymphoprep, Nycomed-Pharma AS, Oslo, Norway). To isolate human CD4+ CD25hi Treg, first CD4+ T cells were enriched using the RosetteSepTM (StemCell™ Technologies, Vancouver, Canada) human CD4⁺ T cell enrichment cocktail according to the manufacturers description, followed by high purity CD4⁺CD25^high cell sorting using a BD FACSAria cell sorter (BD Biosciences, Erembodegem, Belgium). For this purpose, cells were labeled with CD4-BV510 and CD25-Pe-Cy7(M-A251; BD Biosciences, New Jersey, USA). This typically resulted in >96% pure CD4⁺CD25^hi cells and more than 90% of these cells were FOXP3⁺. Without ex vivo expansion, high purity sorted Treg (or PBS, vehicle control) were injected intradermally in the mouse skin at 4 spots closely around the human skin graft, in a total volume of 100 µl PBS.

Fresh healthy donor NK cells were purchased from AllCells with either CD56 positive selection or CD56 negative selection (Allcells, cat#PB012-P or PB012-N). For 2D migration experiments, NK cells were enriched from peripheral blood using RosetteSep (StemCell Technologies) from healthy adult donors. T cells, B cells and monocytes were isolated from PBMCs (AllCells) using Mojosort magnetic cell separation system from Biolegend via CD3 positivity (Biolegend, cat#480133), CD19 positivity (Biolegend, cat#480105), CD14 positivity (Biolegend, cat#480093). PBMC purity was assessed using flow cytometry: CD3-APC (Biolegend, cat#300411), CD14-BV421 (Biolegend, cat#325627), CD45-FITC (BD Bioscience, cat#347463), CD56-PE (BD Bioscience, cat#555516), CD20-PE (BD Bioscience, cat#555623). For donor NK cell lysis of PANC-1 clusters, primary donor NK cells were purchased from AllCells then expanded using irradiated K562–4-1BBL-mbIL-21 (names “CSTX002”) cells kindly provided by Dr. Dean Lee according to his protocol^{58 (link)}.

On day 6 PBMC, either GAD65 AA 114–122 or FLU peptides stimulated for 4 days, additionally cultured for two days in the presence of IL-2 (100 IU/ml), were co-cultured with pulsed or unpulsed APCs at 1:3 (PBMC:APCs) ratio in 96 well round bottomed culture plates (Corning Incorporated, Corning, NY 14831–001, USA) for 2 and half hours in RPMI 10% FBS complete medium (see above) additionally supplemented with GolgiStop reagent (1:500 dilution, BD Biosciences). An experimental positive control was set-up by NK cell isolation from PBMC of a HD volunteer previously obtained with the RosetteSep method (Stem Cell Technologies, Vancouver, Canada) and FicollPaque Plus (Lympholyte, Cedarlane Laboratories, Burlington, Ontario, USA). Isolated NK cells have been routinely checked for the percentage of CD3^-CD56⁺ cells by FACS analysis and those with purity greater than 90% were cultured with 200 IU/ml of recombinant human IL-2 (Sigma-Aldrich) at 37°C and used up to 5 days after isolation as effectors in degranulation assay. Isolated NK cells were then co-cultured with K562 cells (American Type Culture Collection, ATCC), a tumoral cell line known to induce NK cell degranulation according to standard protocols, as control target [41 (link)], and with either GAD65 AA 114–122 peptide pulsed and unpulsed APCs.

NK cells were isolated from heparinized whole blood from malaria-naïve healthy donors with RosetteSep (Stemcell Technologies, Canada) and density gradient separation via Ficoll (Bio-Strategy Lab, Australia) centrifugation according to manufacturer’s protocols.