Efficient Enrichment of Functional ILC Subsets from Human PBMC by Immunomagnetic Selection

Abstract

Abstract Innate lymphoid cells (ILCs) are exceedingly rare but important regulators of homeostatic and disease-associated immune processes. The frequency of ILCs in peripheral blood of healthy humans is ~0.07% of CD45+ leukocytes. ILCs lack specific cell surface markers but can be divided into distinct subsets (ILC1, 2 and 3) based on their differential expression of effector cytokines and master transcription factors. Currently, cell sorting is the most widely used method to isolate ILCs, but it is time consuming, expensive and often results in low purities and recoveries. Pre-enrichment of ILCs would allow for reduced sorting times and improved purities. Accordingly, we have developed a fast immunomagnetic negative selection method to pre-enrich all ILCs subsets from human leukapheresis samples. Briefly, unwanted cells are labelled with antibodies and magnetic particles and placed into an EasySep™ magnet. Unwanted cells are retained in the magnet and the enriched ILC fraction is simply poured off into a new tube. We find that total ILCs (defined as Lineage− CD45+ CD127+) are enriched from a frequency of 0.01 – 0.23% (n=28) to a final frequency of 17 – 86%, an enrichment of 200 – 1500 fold with virtually no loss of ILCs. ILC1 were enriched from 0.01 – 0.2% to 4.5 – 14%. ILC2 were enriched from 0.01 – 0.1% to 5.8 – 51% and ILC3 were enriched from 0.01 – 0.1% to 6 – 16%. This pre-enrichment drastically decreases sort time, allowing sorting over 3.7 × 105 ILCs from 2 × 109 PBMCs in only 12 minutes. Sorted cells maintained their functionality; when stimulated, ILC1s produced IFNγ, ILC2s secreted IL-13 and ILC3s produced IL-22. Our newly developed method of ILC pre-enrichment should aid human ILC research by enabling their rapid isolation when combined with cell sorting