15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 45 Three reference samples (Reference sample I-III) were analysed using three different flow cytometers at three different centers by independent operators using an optimized protocol. The performance of the backbone antibodies (white shades) was evaluated using single stain controls for each measured parameter with background subtracted from corresponding negative controls. The drop-in markers for NK phenotype panel (light grey shades) were analysed, when gated on NK cells (CD45+CD3-CD56+). Background staining in NK phenotype panel drop in channels were eliminated using appropriate fluorescence minus one (FMO) controls. For the NK function panel drop in markers; CD107a, CD25 and NKp44, NK cells were stimulated with A431 cells and unstimulated values were subtracted before analysis (dark grey shades). The values shown for each parameter correspond to the average frequency of stained cells from two technical replicates measured in triplicates, following subtraction of appropriate background signal. The statistical comparisons were performed using non-parametric Kruskal-Wallis. Only significant p-values (<0.05) are shown. NK cell phenotype and function is highly preserved in cryopreserved peripheral blood samples With these standardized and harmonized protocols and panels, we initiated a study across the participating partners to compare different handling procedures or culture conditions for PBMC samples to identify the most optimal conditions to study NK cell phenotype and function. For this study, PBMC samples from 12 healthy human blood donors were stained with NK cell phenotype and function panel antibody mixes and were analysed using the BD LSRFortessa TM (6 donors) at VUmc and the MACSQuant ® Analyzer X (6 donors) at Miltenyi (Figure 4). Different PBMC conditions, i.e. freshly isolated PBMC versus cryopreserved PBMC, and cytokine stimulated versus not cytokine stimulated, were compared. To test the ADCC effector function, NK cells were exposed to A431 (EGFR positive cell line) cells and A431 cells coated with cetuximab (CET). The expression levels of NK cell phenotype markers were comparatively stable following cryopreservation and no significant difference were observed between fresh and cryopreserved NK cell phenotypes. (Figure 5, supplementary figures S4 and S5 online, and Tables 5 and 6). Upon stimulation with IL-2 and IL-15, though NKG2D expression levels increased resulting in a more homogenous expression level in fresh NK cells compared to their cryopreserved counterparts, however the increase was not significant (Figure 5C and supplementary figure S5C online). NK cell activation and function was studied by exposure of NK cells to A431 cells in the presence or absence of CET. The NK function FACS panel was used to detect NK cell degranulation (CD107a levels) and ADCC mediated cytotoxicity comparing differences in CD107a and CD16 cell surface levels between PBMC+A431+CET and PBMC+A431 conditions. Results show that neither the overall CD107a expression levels upon exposure to A431 target cells or A431 target cells coated with cetuximab, nor the increase in CD107a expression in the cetuximab condition were affected by cryopreservation (Figure 6A and Supplementary Fig S6A and S7A online and Tables 5 and 6). Total NK cell CD16 expression levels (Figure 6B and Supplementary Fig S6B and S7B online and Tables 5 and 6) and CD16 expression levels in the CD56dim CD16+ NK cell subset (Supplementary Fig S8 online and Tables 5