IL-22–producing ILCs and cNK cells can be discriminated by LFA-1 and CD7 expression. (A) Cord blood CD34⁺ progenitor cells were cultured on EL08-D12 feeder cells with IL-3 (for the first week), IL-7, IL-15, SCF, and FLT3L for 21 days. Only CD56⁺ cells that were negative for CD94, CD7, and LFA-1 expressed IL-22 after IL-1β+23 stimulation. Results are representative of >5 individual donors. (B-C) Differential expression of IL-22 in freshly isolated Lin⁻ (CD3⁻CD14⁻CD19⁻) lymphocytes from human tonsils. Three populations of lymphocytes could be discerned based on NKp44 and CD94 staining. These cells also differed in LFA-1 expression, with all NKp44⁺CD94⁻ cells lacking LFA-1 and the majority of NKp44⁻CD94⁺ cells expressing LFA-1. After IL-1β+23 stimulation, only the NKp44⁺CD94⁻ cells produced IL-22. Flow cytometry plots from a representative donor are shown in panel B and summary data for 3 donors is shown in panel C. (D) Peripheral blood NK cells mostly express LFA-1 and CD7. (E-F) CD56⁺LFA-1⁻ ILC cells (solid lines) express NKp44 and CD161 only, but not other NK associated receptors, including KIR (antibody cocktail for 2DL1, 2DL2/3, and 3DL1), CD16, or CD8. cNK cells (dotted lines) were used as the controls. (G) Cytotoxic proteins granzyme B and K and perforin were not expressed in CD56⁺LFA-1⁻ cells (dotted lines). CD56⁺LFA-1⁺ cNK cells were used as the control (dotted lines). (H) ILC22 cells also do not kill the K562 target cells (1:1 ratio for 6 hours) or express IFN-γ in response to IL-12/18 (10 ng/ml each). Gray-filled histograms are mouse immunoglobulin G. Results are representative of >10 donors. IgG, immunoglobulin G.