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. 2022 Jul 14;14(7):1538. doi: 10.3390/v14071538

Figure 3.

Figure 3

IFNα-NKs resisted to HCV-induced inhibition and showed durable antiviral responses in vitro. (A) Infection rates (percentage of control) of HCV-Huh7.5 cells co-cultured in transwells with IL2/IL15-NKs (gray bars) or IFNα-NKs (lilac bars) that had been previously exposed or not to recombinant HCV for four hours (darker gray or lilac bars). Each dot represents the average of three technical replicates from one donor (n = 4). Mean values ± SD are shown. (B) Percentages of HCV-infected (Venus+) Huh7.5 target cells in co-culture with IL2/IL15-NKs (gray bars) or IFNα-NKs (lilac bars) that had been previously exposed or not to HCV (darker gray or lilac bars). Untreated target cells were used as controls (black bars). Briefly, transwells with NK cells were removed 72 h after the start of the experiment and target cells were maintained in culture in fresh medium until Day 6. Fluorescence intensities of Venus were recorded at 24 h, 72 h, and on Day 6 using flow cytometry. Each dot represents the average of three technical replicates from one donor (n = 4). Mean values ± SEM are shown. (C) Infection rates (percentage of control) of HCV-Huh7.5 either co-cultured with IFNα-NKs (lilac bars) or exposed to 1 µg/mL recombinant IFN-α (gray bars), as calculated by measuring HCV-Ag levels on Days 3, 7, and 12. On Day 7, transwells with IFNα-NKs were re-moved, and the medium was replaced in all wells. Each dot represents the average of three technical replicates from one donor (n = 4). Mean values ± SEM are shown. (D) Representative agarose gel electrophoresis image showing the presence or absence of the HCV intermediate replication product (negative-strand RNA; 150 bp) in HCV-Huh7.5 cells treated as in C. Untreated target cells were used as internal controls. Negative and positive PCR controls were also included in the reaction. Data are representative of four separate experiments performed using NK cells from four different donors. Statistical analyses were performed using unpaired Student’s t-test (A,C) and the two-way ANOVA with Šídák multiple comparison test (B). ** p < 0.01, **** p < 0.0001; n.s., non-significant.