HSC-derived CD56⁺CD117^highCD94⁻ NK cells are phenotypically and functionally similar to SLT-derived stage III cells or NK22 cells. (A) HSC-derived CD56⁺CD117^highCD94⁻ NK cells were compared with CD3⁻CD19⁻CD14⁻CD56^+/−CD117⁺ lymphoid fraction of tonsillar mononuclear cells (supplemental Figure 1, gating strategy). Expression of the key receptors was compared, including CD117, NKp44, NKp46, IL-1 receptor, CD161, and CCR6. CD127 (IL-7R) expression is shown with cultures containing IL-7 (left) and when IL-7 is removed from the media for 7 days (right; open histogram). FACS plots for CD127 are gated on the CD56⁺CD117^highCD94⁻ cell fraction. Expression of HSC-derived NK22 cells for AHR (B) and ROR-γτ (C) by quantitative reverse-transcribed PCR. (D) The expression of AHR and ROR-γτ by intracellular FACS staining. Histograms represent isotype controls (gray) or electronic gating on CD56^+/−CD117^highCD94⁻ (solid) or CD56^+/−CD117^lowCD94⁻ (dotted). Tonsillar cells were first gated on the CD3⁻CD14⁻CD19⁻ fraction. HSC-derived cells, which lack T and B cells, were first gated on the CD56⁺ fraction. (E) Intracellular expression of IL-22 and IFN-γ after stimulation of HSC-derived NK cells with either IL-1β + IL-23 or IL-12 + IL-18. Cells were obtained at day 28 of culture and stimulated, and intracellular staining for IL-22 or IFN-γ was performed. Results are representative of 3 donors. (F) Supernatant from HSC-derived NK cells can increase expression of ICAM-1 on MSCs. Shown are the FACS plots of ICAM-1 expression after 48 hours of coculture with media supplemented with IL-1β and IL-23 (closed histogram) or with supernatant from in vitro derived CD56⁺CD117^highCD94⁻ NK cells activated with IL-1β and IL-23 (open histogram). Results are representative of more than 3 donors.