Immunomagnetic enrichment of human ILC2 cells from peripheral blood leukopak

Abstract Group 2 innate lymphoid cells (ILC2s) are essential for airway tissue repair, protection against helminth infection, metabolic homeostasis, and the development of allergic airway diseases. They are widely distributed throughout the human body in lung, intestine, palatine tonsils, skin and peripheral blood, but are extremely rare within a given tissue. In healthy individuals, ILC2s comprise 0.01–0.04% of leukocytes in the peripheral blood. Currently, flow cytometric cell (FACS) sorting is the only method to purify ILC2s, which is time-consuming due to their low frequency. We have adapted EasySep™ to pre-enrich ILC2s from peripheral blood leukopaks, and thereby shorten subsequent FACS analysis and sorting times. Non-ILC2 cells in an unprocessed leukopak were immunomagnetically labeled using a cocktail of antibody complexes and magnetic particles. The sample was then placed in a magnet for 10 minutes, the labeled cells were retained, and the unlabeled cells, containing the ILC2 population, were recovered. ILC2s were enriched from 5 x108unprocessed cells in 30 minutes. Enriched cells were counted and evaluated by FACS. ILC2s, defined as lineage negative (CD1a− CD3− CD11c− CD14− CD19− CD34− CD123− TCRαβ− TCRγδ− BDCA2− FcɛR1−), CRTH2+ CD161+CD127+, were enriched 268±167 fold, with 74± 34% recovery (means ± SD, n=7). EasySep™ pre-enrichment of ILC2s reduced subsequent FACS analysis and sorting time compared to non-enriched samples. Importantly, ILC2s enriched with EasySep™ were functional, as shown by production of the type 2 cytokine IL-13 upon stimulation with IL-33 and IL-2. Overall, EasySep™ is a simple and rapid method to pre-enrich ILC2s from leukopak for subsequent FACS analysis or sorting and functional analyses.