NKG2C⁺ NK cells express IFN-γ in response to NKG2C engagement.

(A and B) Viable CD3⁻ CD56^dim NK cells were FACS sorted and stimulated by cross-linking of NKG2C and/or 2B4 for 16 hours. Analysis of intracellular IFN-γ expression was performed by FC after gating on CD56^dim CD57⁺ NKG2A⁻ sKIR⁺ cells from HCMV⁺ donors displaying or not an expansion of NKG2C^hi cells. Gating strategy is depicted in Figure S3D. One representative experiment (A) and mean percentage of IFN-γ producing cells ± SEM (B) are depicted (n≥8). Percentage of IFN-γ producing cells was calculated after subtracting the value observed in unstimulated cells. ***p<0.001 calculated with Mann-Whitney test. (C) Surface expression of CD48 and HLA-E was analyzed by FC on 221 (dashed line) or 221-AEH cells (black line), with isotype control or secondary staining only (solid grey histogram). One representative staining out of two is depicted. (D and E) Viable CD3⁻ CD56^dim NK cells were FACS sorted and stimulated by co-culture with 221 or 221-AEH for 6 hours. Analysis of intracellular IFN-γ expression was performed by FC after gating on CD56^dim CD57⁺ NKG2A⁻ sKIR⁺ NKG2C^hi/+/− cells from HCMV^+/− donors. One representative experiment (D) and mean percentage of IFN-γ producing cells ± SEM (E) are depicted (n = 6). *p<0.05, **p<0.01, calculated with Mann-Whitney test.