15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 57 matched with the number of target cells (5 x 10 4 target and 5 x 10 4 NK cells from PBMC) at a 1:1 E: T ratio. PBMC without targets and PBMC + CET without targets were included as controls. All treatments and corresponding conditions were performed in triplicate. After 4h incubation at 37°C, 95% humidity and 5% CO 2 atmosphere, cells were pelleted and stained with the NK phenotype (Table 2) or NK function antibody (Table 3) mixes. To assess degranulation by NK cells, anti-CD107a PE (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) was added at the beginning of the assay. Acquisition and data analysis for harmonisation between different flow cytometers Individual acquisition protocols following standardized instrument settings were set up in three centers for three different flow cytometers using the same reference samples (n=6) and the same staining antibody mixtures. In the first step, to compare variability between flow cytometers, reference PBMC samples were isolated and stored as described above for testing. Single-stain controls were acquired for antibody - fluorochrome conjugates in the NK phenotype and NK function panel. Further, to detect expression of NK cell receptors and NK cell function-associated antigens and effector molecules, PBMC were exposed to target cells (A431). FCS files acquired from three flow cytometers were analysed in KALUZA ® . For comparison of different PBMC conditions (fresh versus cryopreserved, no cytokine versus cytokine activated and no CET versus CET stimulated), data acquisition was done using two flow cytometers BD LSRFortessa TM and MACSQuant ® Analyzer X. In this experimental part, the FCS files obtained were analysed by individual laboratories, with KALUZA ® software for analysis of data generated from BD LSRFortessa TM and MACSQuantify TM 2.8 software for data generated from MACSQuant ® Analyzer X. Gating strategy Two different gating strategies were defined, one for the NK phenotype and another for the NK function panel following a modified ISHAGE gating strategy 51 . For the NK phenotype panel, cell doublets were excluded by plotting forward side scatter area and height parameters. Next, CD45 was plotted against CD3/TCRγδ/CD14/CD19 (lineage) and SYTOX® Blue to gate on all live and lineage- CD45+ cells. Thus, selected cells were further plotted against CD56, identifying only viable NK cells. This NK cell gate was used to assess the expression levels of CD16, NKG2A, NKG2C, NKG2D and KIR2D antigens (Supplementary Fig. S2 online). Of note, the gating strategy for the NK cell function panel was essentially the same with one exception, i.e. the inclusion of CD3 and TCRγδ as drop in antigens, excluding them from the backbone markers. With this panel, we differentiated between three cell populations: NK cells (CD45+CD3-/TCRγδ-CD56+) T cells (CD45+CD3/TCRγδ+CD56-) and non-conventional T cell subsets (CD45+CD3+/TCRγδ+CD56+). The gate on the NK cell