Peripheral NK cell phenotypic alteration and dysfunctional state post hepatitis B subviral particles stimulation in CHB patients: evading immune surveillance

Abstract

Background: The relationship between chronic hepatitis B (CHB) infection and natural killer (NK) cell dysfunction is well-established, but the specific role of HBV viral antigens in driving NK cell impairment in patients with CHB remains unclear. This study investigates the modulatory effects of hepatitis B virus subviral particles (HBVsvp, a representative model for HBsAg) on the phenotypic regulation (activating and inhibitory receptors), cytokine production and cytotoxic potential of peripheral blood mononuclear cell-derived natural killer cells (PBMCs-derived NK cell), which contributes to NK cell dysfunction in CHB infection, potentially serving as an effective HBV immune evasion strategy by the virus.

Methods: NK cells were isolated from peripheral blood of patients with CHB (n=5) and healthy individuals (n=5), stimulated with HBVsvp. Subsequent flow cytometric characterization involved assessing changes in activating (NKp46 and NKG2D) and inhibitory (CD94) receptors expression, quantifying TNF-α and IFN- γ cytokine secretion, and evaluating the cytotoxic response against HepG2.2.15 cells with subsequent HBVsvp quantification.

Results: In CHB patients, in vitro exposure of PBMCs-derived NK cell with HBVsvp (represent HBsAg model) significantly reduced NK cell-activating receptors expression (P = 0.022), increased expression of CD94 ⁺ NK cells (p = 0.029), accompanied with a reduced TNF-α - IFN-γ cytokine levels, and impaired cytotoxic capacity (evidenced by increased cell proliferation and elevated HBVsvp levels in co-cultures with HepG2.2.15 cells in a time-dependent), relative to healthy donors.

Conclusion: These findings suggest that HBVsvp may induce dysfunctional NK cell responses characterized by phenotypic imbalance with subsequent reduction in cytokine and cytotoxic levels, indicating HBVsvp immunosuppressive effect that compromises antiviral defense in CHB patients. These data enhance our understanding of NK cell interactions with HBsAg and highlight the potential for targeting CD94 inhibitory receptors to restore NK cell function as an immunotherapeutic approach. Further clinical research is needed to validate these observations and establish their utility as reliable biomarkers.

Keywords: CD94 inhibitory receptor; HB subviral particles (HBVsvp); HbsAg; NKp46 and NKG2D activating receptors; chronic hepatitis B (CHB); cytokine; immunotherapy; natural killer (NK) cells.

Figure 1

Indicates the absence of the HBX gene fragment (465 bp) in the SVP sample, suggesting the presence of empty SVP particles that serve as a model for HBsAg.

Figure 2

Displays flow cytometry dot plot analysis was employed to identify and gate NK cell populations from CHB patients and healthy donors, allowed for the gating zone to encompass both small and large lymphocyte-sized cells (R1) (gated area) distinct from other peripheral blood cells. This illustrates the gating method for NKs sorting; (A) Dot plot histogram for initial gating of lymphocyte region on FS/SS. (B) Identification of NKs. NKs were described as cells that were resident in (R2) and were (CD3 FITC negative/CD56+CD16 PE positive). (C) To set the cut-off positive for isotype control, use a quadrant plot. It was established that [R1 and R2] was the sorting gate.

Figure 3

Presents flow cytometric scatter plot analysis to assess the percentage and purity of natural killer cells within the peripheral blood mononuclear cell fraction. (A) post-Ficoll density gradient separation, the abundance of NK cells, characterized by the CD56+CD16+ and CD3- phenotype, displaying variable levels across the evaluated samples. (B) Quantification of NK cell purity showed an average of 64.97% for the CD56+CD16+ and CD3- population in the selected samples.

Figure 4

Presents a flow cytometry histogram analysis of the phenotypic expression profile of inhibitory and activating receptors on NK cells from CHB patients and healthy controls following stimulation with HBVsvp particles. The frequency analysis includes the expression levels of (A) the activating receptors NKG2D and (B) NKp46, as well as (C) the inhibitory receptor CD94 on gated NK cells. This provides insights into the functional state of NK cells in the context of HBV infection. The analysis was conducted using monoclonal antibodies directly conjugated with allophycocyanin (APC) and fluorescein isothiocyanate (FITC).

Figure 5

The bar graph illustrates the effect of HBVsvp-Stimulated NK cells on the viability of HepG2.2.15, highlighting changes over various time intervals. Data are presented as the mean ± SEM of three independent replicates. Statistical significance was assessed using two-way ANOVA to analyze the effects of time and treatment.

Figure 6

The bar graph explain the pattern effects of NK cells derived from CHB patients upon stimulation with HBVsvp on HepG2.2.15 cells cell viability over an extended period; The data are presented as the mean ± SEM of three independent replicates for each set of results. Two-way ANOVA tests were conducted to assess statistical significance, with p-values less than 0.001 (***). ns, non-significant.

Figure 7

The bar graph illustrates the effects of HBVsvp stimulated NK cells derived from both healthy and CHB patients on the viability of HepG2.2.15 cells over time. Upon stimulation with HBVsvp, a distinct pattern of cell viability is observed. The data are presented as the mean ± SEM of three independent replicates for each set of results. Two-way ANOVA tests were conducted to assess statistical significance, with p-values less than 0.001 (***), and 0.05 (*).

Figure 8

The bar graph Illustrates the comparative assessment of HBVsvp levels in the supernatant from HepG2.2.15 cocultured with NK cells stimulated by HBVsvp, highlights the temporal effects on HBVsvp production across various conditions: obtained from (A) healthy donors; (B); CHB patients. (C) healthy donors VS CHB at 24, 48, 72, and 96 hours. The data are presented as the mean ± SEM of three independent replicates for each set of results. Two-way ANOVA tests were conducted to assess statistical significance, with p-values less than 0.0001 (****), and 0.01 (**) considered statistically significant.

Figure 9

The bar graph displays the comparative levels of TNF-α secretion from NK cells of healthy donors and CHB patients post-HBVsvp pulsation. It elucidates the effects on TNF-α production across various conditions: (A) healthy donors; (B) CHB patients; and (C) a comparison between healthy donors and CHB patients. Data represents the mean ± SD from three independent experiments. The data are presented as the mean ± SEM of three independent replicates for each set of results. Unpaired two-tailed Student’s t-tests were conducted to assess statistical significance, with p-values less than 0.0001 (****), and 0.001 (***) considered statistically significant.

Figure 10

Shows a bar graph comparing the levels of IFN- γ secreted from NK cells from healthy donors and CHB patients After pulsation with HBVsvp, highlighting the effects of various NK cell conditions on IFN- γ production. across various conditions: (A) healthy donors; (B) CHB patients; (C) comparison between healthy donors and CHB patients. The data are presented as the mean ± SEM of three independent replicates for each set of results. Unpaired two-tailed Student’s t-tests were conducted to assess statistical significance, with p-values less than 0.0001 (****), 0.01 (**), and 0.05 (*) considered statistically significant.