Isolation and Culture of Human ILC2s

Experimental protocols were approved by the USC Institutional Review Board (IRB) and conducted in accordance with the principles of the Declaration of Helsinki. Human blood ILC2s were isolated from total peripheral blood mononuclear cells (PBMCs) to a purity of > 95% on a FACSARIA III system as described previously (29 (link)). Briefly, human fresh blood was first diluted 1:1 in PBS 1X and transferred to SepMateTM-50 separation tubes (STEMCELL Technologies) filled with 12mL Lymphoprep™. Samples were centrifuged for 10 minutes and PBMCs were collected. CRTH2⁺ cells were then isolated using the CRTH2 MicroBead Kit, used according to the manufacturer’s conditions. Samples were then stained and ILC2s were isolated based on the absence of common lineage markers (CD3, CD5, CD14, CD16, CD19, CD20, CD56, CD235a, CD1a, CD123), and the expression of CD45, CRTH2 and CD127. Isolated ILC2s were cultured at 37°C (5x10⁴/mL) with rhIL-2 (10ng/mL) and rhIL-7 (10ng/mL) in complete RPMI (cRPMI). To make cRPMI, RPMI (Gibco) was supplemented with 10% heat-inactivated FBS (Omega Scientific), 100 units/mL penicillin and 100mg/mL streptomycin (GenClone). When indicated, human ILC2s were activated with 50ng/mL rhIL-33 for the indicated times. Human PD-1 blocking antibody or isotype was included in the culture (10µg/ml, BioXCell).