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IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

Figure 5

Design of antagonistic peptides against IL-28 receptor alpha subunit (IL28RA) and their effects on H1N1-stimulated B-cell functions.

(A) Design of inhibitory peptides based on the crystal structures of IL-29 and the IL28RA. A computer prediction of the interaction between IL-29 and the IL28RA is illustrated. Colored fragments represent proposed sites of significant interaction using a proximity model. (B) Example for detailed in silico interaction between peptide 6 and IL-28Rα (peptides 9 and 11). (C) Inhibitory activity of antagonistic peptide 1 against IL-28B binding to IL28RA. ELISA was used to measure the binding of a fixed concentration of IL-28B (100 ng/mL) to IL28RA challenged by increasing concentrations of the inhibitory peptide and control peptide (scrambled version). The data shown is representative of three independently repeated experiments. Whiskers indicate the interquartile range. (D) STAT1 phosphorylation in THP1-derived macrophages treated with peptide 1 and challenged with recombinant IL-28B (100 ng/mL) for 15 min in comparison to scramble peptide control. Data of five individual experiments is shown. Wilcoxon matched-pairs signed rank (WCR)-test was used. (E) STAT1 phosphorylation in THP1-derived macrophages treated with different doses of peptide 1 and challenged with recombinant IL-28B (100 ng/mL) for 15 min in comparison to scramble peptide 1 control. Symbols represent median of three independently repeated experiments. (F) PBMCs from healthy volunteers were pre-treated with peptides for two hours prior to 5-day stimulation with H1N1. In addition recombinant IL-28B (28B) and control peptides (c1, SV40-based peptide; c2, a duck hepatitis B virus based peptide, and various scrambled versions of the peptides) were used. Total IgG in supernatant was determined using an ELISA and fold changes are shown calculated over peptide treatment alone (no H1N1). MWU-test determined statistically significant differences between groups, p<0.05(*). Bars show median and inter-quartile ranges; whiskers indicate 10–90th percentile. (G–H) PBMCs from transplant recipients without successful seroconversion were pre-treated with the IL-28Rα antagonistic peptides (6, 16 and 17) prior to overnight stimulation with H1N1. The mean fluorescence intensity (MFI) of CD86, HLA-DR, and CD69 on B-cells (CD20+) was measured using flow cytometry (G). The amount of H1N1-stimulated IgG release was determined by ELISA (H). Wilcoxon matched-pairs signed rank (WCR)-test determined statistically significant differences between groups.

Figure 5

doi: https://doi.org/10.1371/journal.ppat.1004556.g005