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. 2021 Apr 15;17(4):e1009505. doi: 10.1371/journal.ppat.1009505

Fig 6. Impact of TBEV E glycoprotein neutralization on IFN-α and Ig production.

Fig 6

Total PBMC or isolated pDC were left untreated (NS) or stimulated with I-TBEV (dilution 1:12.5) alone or in combination either with two single-chain Ab (scAb) blocking TBEV E glycoprotein, B7 and G7 or with a scAb non-related to TBEV, A10 clone. (A) The release of IFN-α was measured by ELISA in culture supernatants of total PBMC left untreated (NS) or stimulated for 24 hours with I-TBEV alone or in combination either with B7 and G7 or with a non-related scAb A10 clone at the concentration of 0.15 μg/ml, 0.3 μg/ml and 0.6 μg/ml. The results shown were mean values ± standard error of the mean (SEM) of 3 independent experiments. ANOVA p value for IFN-α: 0.006. Based on LSD (equivalent to no adjustments). (B-D) Isolated pDC were left untreated (NS) or stimulated with I-TBEV alone or in combination either with G7 clone (0.6 μg/ml) or with A10 clone (0.6 μg/ml) for 24 hours. The production of IFN-α (B) was measured by ELISA in culture supernatants. The results shown were mean values ± SEM of 3 independent experiments. ANOVA p value for IFN-α: 0.000. Based on LSD (equivalent to no adjustments). (C) pDC were stained with PD-L1, HLA-DR, CD86, CD80, ILT7, BDCA2 and BDCA4. A total of 50.000 cells were analyzed per sample by flow cytometry to evaluate the percentage of pDC sub-populations. A representative pDC sub-population profile out of 3 different experiments conducted separately is shown: P1-pDC (PD-L1+ CD80-) population is indicated in red while P2-pDC (PD-L1+ CD80+) in blue. TNF-α production (D) was tested by cytometric bead assay in 24 hour-collected supernatants. The results shown were mean values ± SEM of 3 independent experiments. ANOVA p value for TNF-α: 0.002. Based on LSD (equivalent to no adjustments). (E-F) IgM and IgG production was measured by ELISA in supernatants collected from PBMC after 10 days of stimulation. The results are mean values ± SEM of 3 independent experiments for IgM and 2 independent experiments for IgG. ANOVA p value for IgM: 0.036. Based on LSD (equivalent to no adjustments).