15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 51 Discussion Recent advances in multiparametric flow cytometry offer new and exciting opportunities for the in-depth characterization of immune cell subsets in research, diagnosis and treatment 28 . However, insufficient standardization in sample handling, multi-colour panel design, and data analysis often hinders data interpretation in longitudinal studies performed by different laboratories. With this study, we aimed to develop harmonized FACS panels for the phenotypic and functional assessment of NK cells. Standardized methods for instrument set-up, sample preparation, gating strategies, and data analysis have been developed, including the testing of the best suitable antibody-fluorochrome combinations, compatible with the optical configuration of three different flow cytometers. The FACS panels were designed with a backbone concept, using lineage antigens and a live/dead dye to effectively exclude the non-NK leukocyte populations and dead cells. By allocating the red laser exclusively for CD56 and CD16 detection, the measurement of an array of additional key NK cell antigens from PBMC was facilitated in panels designed to assess either NK phenotype or functions 29 . Moreover, by separating T and NK cell populations in the NK cell function panel, a precise identification and comparison of NK, T and non-conventional T cells (CD3+CD56+) frequencies and their phenotype and functions could be correlated from a single antibody mix. To enumerate NK cells, T cells and non- conventional T cell subset, we made use of a volumetric flow cytometric counting method or, alternatively, counting beads. The availability of three lasers with the flexibility to use eight colours avoided complexities involving fluorochrome spill over 30 . The TCRγδ antibody was included with the aim of simplifying the identification of NK cell populations from CD3dim or negative subsets under diseased conditions and in αβ-T cell depleted grafts. Moreover, several receptors originally identified in NK cells, are also expressed in γδT cells, such as NKG2D, NKp44, NKp30 or DNAM-1 23 , and regulate in great part the cytotoxic responses that also γδT cells mediate against tumors 31 . Both NK panels had a built in dead cell marker which served as an internal control ensuring the quality and viability of PBMC, besides offering investigators to perform simultaneous cell counting on PBMC samples. The inclusion of CD16 in the panel enables users to gate specifically on the CD56dimCD16+ and CD56brightCD16- subsets in parallel 14 . Further, CD45+CD3-CD56- cells can also be studied. This series of options allows users to further study changes in NK cell subsets in e.g. chronic viral infections including human immunodeficiency virus-1 and human cytomegalovirus infections, where NK cell subsets are redistributed with expansion of CD56 negative cells but also make this panel useful in conditions where there is either an increase of CD56+CD16- cells or a decrease in CD56 expression levels 32 . Furthermore, changes in CD16 and CD107a cell surface levels can be used to determine the occurrence of natural killing and ADCC killing of target cells. Generally, after NK cell activation upon target