DEVELOPMENT OF A HIGH-DIMENSIONAL FLOW CYTOMETRY PANEL TO ANALYSE NATURAL KILLER CELLS IN SLE

Background: Natural Killer (NK) cells are an innate immune cell type that has somewhat been overlooked in the context of systemic lupus erythematosus (SLE). SLE patients display a reduced number of NK cells with an activated phenotype and increased capacity to produce IFN-γ, decreased antibody-dependent cellular cytotoxicity (ADCC), and altered natural cytotoxicity (1). NK cell activation is determined by the integration of input from a myriad of activating and inhibitory receptors. Previously, using Nanostring® gene expression technologies, we found our SLE cohort showed decreased gene expression of a number of these receptors (KLRC2, KLRC1, KLRB1, KLRF1, KLRG1, PRF1 and IL2RB) leading us to explore NK cells in SLE in more depth. Objectives: Our aim was to develop a high-dimensional flow cytometry panel to characterise dysregulation of NK cell in SLE, with particular reference to the activating and inhibitory receptors found to be dysregulated in SLE at the gene expression level. Methods: Markers for NK panel were selected to include canonical phenotyping/functional molecules of NK cells with a particular emphasis on receptors found to be lower in our SLE cohort’s gene expression findings. NK panel was designed to minimise spectral overlap, expression and co-expression of markers was taken into consideration. Antibodies were titrated, and voltages optimised to achieve the best separation index for each of the antibodies. The 24-marker panel was run on 52 SLE patients of various disease manifestations, treatments and disease severity. 20 healthy controls were also run for comparison. Results: A 24-marker flow cytometry panel including 19 NK cell antigens was optimised, including basic phenotype (CD3/CD56/CD16/NKp46) and NK differentiation markers (CD57/CD94), activating and inhibitory receptors (NKG2A/NKG2C/NKG2D), costimulatory receptors (CD244/CD226), transcription factors (Eomes/Tbet) and effector molecules (granzyme/perforin). Immunophenotypic high-parameter analysis of SLE and control samples is in progress and results will be presented. Conclusion: Our development of a high-dimensional immunophenotypic panel allows identification of changes in NK cells in SLE including antigen expression levels, subset percentages and potentially of novel subsets. This panel will be used to investigate NK cell changes with disease course/activity, therapeutic response, and to discover potential drug targets for SLE. References: [1]Spada R, Rojas JM, Barber DF. Recent findings on the role of natural killer cells in the pathogenesis of systemic lupus erythematosus. J Leukocyte Biol. 2015;98(4):479-487. doi:10.1189/jlb.4ru0315-081rr Acknowledgments: Westmead Institute for Medical Research Genomics Facility Westmead Institute for Medical Research Flow Cytometry Facility Staff Specialists’ TESL and Trust Fund Committee Disclosure of Interests: None declared