Impact of Peg-IFN-α on peripheral memory B-cell subsets and plasmablast distribution.

<p>Evolution of B cell subsets was evaluated in patients with CHB before and at different time points during the treatment with nucleos(t)ide analog alone (<i>open circles</i>, n = 11–14) or together with Peg-IFN-α (<i>black circles</i>, n = 8–9). Absolute numbers of (A) natural memory B-cell subsets (left panel), and post-GC memory B cells (right panel), (B) plasmablasts (left panel) and CD27^- IgD^- memory B-cell subsets (right panel). (C) Representative dot plots of the frequency of natural memory (left) and CD27^- IgD^- memory (right) B cells from patients treated with nucleos(t)ide analog alone (<i>upper panel</i>) or together with Peg-IFN-α (<i>lower panel</i>) before and after 24 weeks of treatment. Dot plots are gated on CD19⁺ cells. (D) Accumulation of plasmablasts during Peg-IFN-α therapy. Representative dot plots of the frequency of plasmablasts from patients treated with nucleos(t)ide analog together with Peg-IFN-α before (<i>left panel</i>) and after (<i>right panel</i>) 4 weeks of treatment. Dotplots are gated on CD19⁺IgD^-CD27⁺ cells. The gray area represents the period of Peg-IFN-α administration. Bars represent median. <i>P</i> values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). * p<0.05, ** p<0.01, *** p<0.001.</p

B-cell subsets during Peg-IFN-α therapy.

<p>(A) B-cell gating strategy. The peripheral B-cell subsets were classified according to the most common lineage/differentiation markers CD19, CD10, CD27, CD38, IgD, and IgM. B-cell subsets were defined as: transitional B cells (CD19⁺CD27^-IgD⁺CD10⁺CD38^high); naive B cells (CD19⁺CD27^-IgD⁺CD10^-CD38^low); natural memory B cells (CD19⁺CD27⁺IgD⁺); post-GC memory B cells (CD19⁺CD27⁺IgD^-CD10^-CD38^low); plasmablasts (CD19⁺CD27⁺IgD^-CD10^- CD38^high); CD27^-IgD^- memory B cells (CD19⁺CD27^-IgD^-) and activated B cells (CD19⁺CD27⁺IgD^-CD10⁺CD38^low). Post-GC memory B cells were further subdivided into IgM⁺ and IgM^- switched B cells. Representative dotplots from one patient with CHB infection. (B) Modulation of total B cells by Peg-IFN-α. Frequency (within PBMC) and absolute numbers of total B cells in patients with CHB infection treated with nucleos(t)ide analog alone (<i>open circles</i>, n = 11–14) or together with Peg-IFN-α (<i>black circles</i>, n = 8–9). The gray area represents the period of Peg-IFN-α administration. Bars represent median. <i>P</i> values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). * p<0.05, ** p<0.01, *** p<0.001.</p

Modulation of NK-cell features by Peg-IFN-α.

<p>Patients with CHB infection were treated with NA alone (<i>open circles</i>, n = 12–13) or together with Peg-IFN-α (<i>black circles</i>, n = 8–9). CD56⁺CD3^- total NK cells and CD56^bright/dim NK subsets were analyzed by flow cytometry before and during Peg-IFN-α treatment. (A) Absolute numbers of CD56^bright and CD56^dim NK cells. (B) Basal level of CD69 expression. (C) IFN-γ secretion was evaluated by intracellular staining upon IL12/IL18 stimulation. (D) The cytotoxic activity was evaluated upon IL12/IL18 stimulation after co-culture with K562 by measuring CD107 surface expression. Bars represent median. <i>P</i>-values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). *p<0.05, **p<0.01, ***p<0.001.</p

Direct HBV-specific CD8 T-cell response evolution during Peg-IFN-α therapy.

<p>Direct HBV-specific CD8 T cells measurements following Peg-IFN-α treatment using tetramers. (A) Representative dot plots of the tetramer labeling of HBc_18-27- and HBs_335-343-specific T cells (gated on CD8 T cells) before and 24 weeks after treatment in patients treated with nucleos(t)ide analog alone (<i>left panel</i>) or together with Peg-IFN-α (<i>right panel</i>). (B) Evolution of the basal percentages of HBc_18-27- (left panel) and HBs_335-343- (right panel) specific CD8 T cells in patients with CHB infection treated with nucleos(t)ide analog alone (<i>white bars</i>, n = 7) or together with Peg-IFN-α (<i>grey bars</i>, n = 7). The gray area represents the period of Peg-IFN-α administration.</p

Modulation of B-cell activation signals during Peg-IFN-α therapy.

<p>Patients with CHB infection were treated with nucleos(t)ide analog alone (<i>open circles</i>, n = 11–14) or together with Peg-IFN-α (<i>black circles</i>, n = 7–9). (A,B) Plasma levels of BAFF and APRIL. (C,D) Plasma levels of soluble CD26 and soluble CD30. The gray area represents the period of Peg-IFN-α administration. Bars represent median. <i>P</i> values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). * p<0.05, ** p<0.01, *** p<0.001.</p

Modulation of DC subsets by Peg-IFN-α.

<p>Patients with CHB infection were treated with NA alone (<i>open circles</i>, n = 11–14) or together with Peg-IFN-α (<i>black circles</i>, n = 7–9). DC subsets were analyzed by flow cytometry before and at different time points of treatment. (A) Absolute numbers of pDCs and mDCs. (B) Basal percentages of CD86 expression on pDCs and mDCs. (C) Mean fluorescence intensity of CD86 on pDCs and mDCs. CD86 was not evaluable for W144. The gray area represents the period of Peg-IFN-α administration. Bars represent median. <i>P</i>-values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). *p<0.05, **p<0.01, ***p<0.001.</p

Frequencies and Th1/Th17 orientation of HBV-specific CD4/CD8 T cells during the course of Peg-IFN-α treatment.

<p>(A-B) Frequencies of HBV-specific CD4/CD8 T cells were evaluated before and during Peg-IFN-α treatment upon the stimulation of PBMCs with peptide pools and intracellular TNF-α, IFN-γ and IL-10 cytokine staining from patients treated with NA alone (n = 5–8) or together with Peg-IFN-α (n = 3–5). (A) Frequencies of cytokine-producing HBc-/HBs-specific CD4 T-cell responses (mean values). (B) Frequencies of cytokine-producing HBc-/HBs-specific CD8 T-cell responses (mean values). (C) IL-17A production was analyzed in supernatants of PBMCs stimulated with peptide pools derived from the HBc, HBs, HBx and POL antigens before and during Peg-IFN-α treatment in patients with CHB infection treated with NA alone (n = 10) or together with Peg-IFN-α (n = 9). <i>P</i>-values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). *p<0.05, **p<0.01, ***p<0.001.</p

Clinical features of patients at baseline and during the course of the treatment.

<p>Clinical features of patients at baseline and during the course of the treatment.</p

Correlation between immunological parameters and the evolution of HBsAg in patients treated with Peg-IFN-α.

<p>(A) Evolution of HBsAg during the course of Peg-IFN-α treatment (percentage of W0). <i>P</i>-values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). Bars represent median. *p<0.05, **p<0.01, ***p<0.001 (B) The modulation of NK cells by Peg-IFN-α correlates with pDC activation. Correlation between the percentage of CD69⁺CD56^dim NK cells and the percentage of CD86⁺ pDCs after 48 weeks of treatment (Spearman correlation). (C) The modulation of NK cells by Peg-IFN-α correlates with the decrease in HBsAg. Spearman correlation between the absolute number of CD56^bright NK cells and the decline in HBsAg after 48 weeks of treatment.</p

Peg-IFN-α altered peripheral transitional and naive B-cell subset distribution.

<p>Evolution of B-cell subsets was evaluated in patients with CHB before and at different time points during the treatment with nucleos(t)ide analog alone (<i>open circles</i>, n = 11–14) or together with Peg-IFN-α (<i>black circles</i>, n = 8–9). (A) Absolute numbers of transitional B-cell subsets. (B) Absolute numbers of naive B-cell subsets. (C) Representative dot plots of the frequency of transitional and naive B cells from patients treated with nucleos(t)ide analogue alone (<i>upper panel</i>) or together with Peg-IFN-α (<i>lower panel</i>) before and after 24 weeks of treatment. Dot plots are gated on CD19⁺IgD⁺CD27^- cells. The gray area represents the period of Peg-IFN-α administration. Bars represent median. <i>P</i> values were calculated using the Wilcoxon test (<i>straight lines</i>) or the Mann-Whitney test (<i>dashed lines</i>). * p<0.05, ** p<0.01, *** p<0.001.</p