Pegylated Interferon α-2a Triggers NK-Cell Functionality and Specific T-Cell Responses in Patients with Chronic HBV Infection without HBsAg Seroconversion

Abstract

Pegylated interferon α-2a (Peg-IFN-α) represents a therapeutic alternative to the prolonged use of nucleos(t)ide analog (NA) in chronic hepatitis B (CHB) infection. The mechanisms leading to a positive clinical outcome remain unclear. As immune responses are critical for virus control, we investigated the effects of Peg-IFN-α on both innate and adaptive immunity, and related it to the clinical evolution. The phenotypic and functional features of the dendritic cells (DCs), natural killer (NK) cells and HBV-specific CD4/CD8 T cells were analyzed in HBeAg-negative CHB patients treated for 48-weeks with NA alone or together with Peg-IFN-α, before, during and up to 2-years after therapy. Peg-IFN-α induced an early activation of DCs, a potent expansion of the CD56bright NK subset, and enhanced the activation and functionality of the CD56dim NK subset. Peg-IFN-α triggered an increase in the frequencies of Th1- and Th17-oriented HBV-specific CD4/CD8 T cells. Peg-IFN-α reversed the unresponsiveness of patients to a specific stimulation. Most of the parameters returned to baseline after the stop of Peg-IFN-α therapy. Peg-IFN-α impacts both innate and adaptive immunity, overcoming dysfunctional immune responses in CHB patients. These modulations were not associated with seroconversion, which questioned the benefit of the add-on Peg-IFN-α treatment.

Fig 1. Modulation of DC subsets by Peg-IFN-α.

Patients with CHB infection were treated with NA alone (open circles, n = 11–14) or together with Peg-IFN-α (black circles, 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. P-values were calculated using the Wilcoxon test (straight lines) or the Mann-Whitney test (dashed lines). *p<0.05, **p<0.01, ***p<0.001.

Fig 2. Modulation of NK-cell features by Peg-IFN-α.

Patients with CHB infection were treated with NA alone (open circles, n = 12–13) or together with Peg-IFN-α (black circles, 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. P-values were calculated using the Wilcoxon test (straight lines) or the Mann-Whitney test (dashed lines). *p<0.05, **p<0.01, ***p<0.001.

Fig 3. Direct HBV-specific CD8 T-cell response evolution during Peg-IFN-α therapy.

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 (left panel) or together with Peg-IFN-α (right panel). (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 (white bars, n = 7) or together with Peg-IFN-α (grey bars, n = 7). The gray area represents the period of Peg-IFN-α administration.

Fig 4. Frequencies and Th1/Th17 orientation of HBV-specific CD4/CD8 T cells during the course of Peg-IFN-α treatment.

(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). P-values were calculated using the Wilcoxon test (straight lines) or the Mann-Whitney test (dashed lines). *p<0.05, **p<0.01, ***p<0.001.

Fig 5. Peg-IFN-α reverses the unresponsiveness of patients to the stimulation by HBV-derived peptide-loaded pDCs.

PBMCs were stimulated with pDCs loaded with HLA-A*02:01-restricted HBc_18-27 and HBs_335-343 peptides and the amplification of HBV-specific T cells was assessed using HLA-A*02:01-tetramers. (A) Representative dotplots of the tetramer labelling of HBc_18-27- (upper panel) and HBs_335-343- (lower panel) specific T cells (gated on CD8 T cells) at day (D)14 before and on Peg-IFN-α therapy. (B) Evolution of the percentages of HBc_18-27- and HBs_335-343-specific T cells at D0 and D14 of culture during Peg-IFN-α therapy. (C) Comparative levels of HBc_18-27- and HBs_335-343-specific CD8 T cells at D14 between baseline and on-treatment (n = 5). The gray area represents the period of Peg-IFN-α administration.

Fig 6. Correlation between immunological parameters and the evolution of HBsAg in patients treated with Peg-IFN-α.

(A) Evolution of HBsAg during the course of Peg-IFN-α treatment (percentage of W0). P-values were calculated using the Wilcoxon test (straight lines) or the Mann-Whitney test (dashed lines). 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.