Figure 6.

Notch receptors are expressed by human tonsil ILCs. (A) Quantification of relative transcript expression of NOTCH1, NOTCH2, NOTCH3, and NOTCH4 genes by quantitative real-time RT-PCR among freshly purified tonsil-derived stage 3, stage 4A, stage 4B, and stage 5 cells (n = 5; 5 independent experiments) as described in Methods. Gene expression was normalized to 18S internal control. (B) Representative (n = 4; 1 independent experiment) surface flow cytometry analysis of NOTCH1 and NOTCH2 in total tonsillar enriched Lin⁻ lymphocytes following 3 days in vitro with IL-7 and gated on stage 3, stage 4A, stage 4B, and stage 5 cell populations. (C) Representative (n = 6; 2 independent experiments) surface flow cytometry analysis of NOTCH1 and NOTCH2 in total tonsillar enriched Lin⁻ lymphocytes following 3 days in vitro with IL-7. Stage 3/ILCPs were gated as live Lin⁻CD34⁻CD117⁺CD94⁻NKp80⁻CD16⁻KIR2D⁻KIR3DL1/2⁻NKG2C⁻NKp44⁻CD294⁻KLRG1⁻ lymphocytes. ILC2s were gated as live Lin⁻CD56⁻NKp44⁻CD94⁻NKp80⁻CD16⁻KIR2D⁻KIR3DL1/2⁻NKG2C⁻(CD294/KLRG1)⁺ lymphocytes. ILC3s were gated as live Lin⁻CD117⁺NKp44⁺CD94⁻NKp80⁻CD16⁻KIR2D⁻KIR3DL1/2⁻NKG2C⁻CD294⁻KLRG1⁻ lymphocytes. NK cells were gated as any live Lin- lymphocytes expressing CD94, NKG2A, NKp80, CD16, KIR2D, KIR3DL1/2, and/or NKG2C as previously described (4).