Fig. 3. Human antigen-specific NK cell responses are associated with NKG2C expression.

(A) Non-linear t-SNE (bh-SNE) plots of 14 NKCL from one untreated viremic PLWH, showing distinct clustering for 7 HIV reactive and 7 non-HIV-reactive NKCL, with HIV-reactive NKCL being partly associated with cell clusters expressing high levels of the inhibitory NKG2A or high levels of activating NKG2C and/or inhibitory KIR3DL1. (B) Antigen-specific killing of HIV Env-pulsed BLCL by 6 NKCL in the presence of isotype control or NKG2C blocking antibodies. Bars represent mean+SEM. Statistical significance was tested using Wilcoxon signed-rank test. (C) Representative flow cytometry plots showing NKG2C expression on one NKCL 48 hours after nucleofection with a scramble siRNA control (Ctrl siRNA) or with siRNA targeting NKG2C. (D) Percentages of lysis by 3 NKG2C+ NKCL reacting against HIV Env (left panel) and MFI of NKG2C on the respective NKCL (right panel) from 2 PLWH 48 hours following nucleofection with siRNA control or siRNA targeting NKG2C. (E) Percentages of lysis by 2 NKG2C+ NKCL reacting against influenza NP (A/AnnArbor/6/1960(H2N2)) (left panel) and MFI of NKG2C on the respective NKCL (right panel) from 1 healthy donor 48 hours following nucleofection with siRNA control or siRNA targeting NKG2C. Percentages of NKG2C down-modulation are indicated for each NKCL. Only NKCL displaying responses over 5% specific killing and at least 1.5-fold above the background (unpulsed) after nucleofection with Ctrl siRNA were included in the analysis. (F), (G) Spearman correlation analysis between frequencies of NKG2C⁺ NKCL and specific killing of BLCL pulsed with HIV Env (F) or influenza NP, MP1, or HA (G). Frequencies of NKG2C⁺ NKCL were determined as the proportion of NKCL beyond an arbitrary cut-off line set by at the right-hand side border of the NKG2C histogram in FMO controls (See Fig. S9).