Regulatory B Cells Dysregulated T Cell Function in an IL-35-Dependent Way in Patients With Chronic Hepatitis B

Abstract

Interleukin (IL)-35-secreting B (IL-35+B) cells are critical regulators in autoimmune and infectious diseases and exert suppressive functions in parallel with IL-10-producing B (B10) cells. However, the role of IL-35+B cells in persistent hepatitis B virus (HBV) infection remains unclear. To elucidate the role of IL-35+B cells in the progress of chronic HBV infection, we determined the frequency of IL-35+B cells and their relationship with the classical human regulatory B cell (Breg) subsets, namely, CD19+CD24hiCD38hi and CD19+CD24hiCD27+. Then, the regulatory effect and mechanism of Bregs on effector T cells were investigated in vitro. Here, we found that compared with healthy controls, the frequency of IL-35+B cells was increased in patients with chronic HBV infection and was enriched in human classical Breg subset CD19+CD24hiCD38hi B cells. Moderate correlation was observed between the frequency of IL-35+B cells and alanine aminotransferase levels (Spearman r = 0.401), but only mild correlation was noted between the frequency of IL-35+B cells and HBV DNA level (Spearman r = 0.314). The frequency of IL-35+B cells was negatively correlated with interferon-γ (IFN-γ)-producing CD4+ and CD8+ cells but positively correlated with IL-4-producing T cells. Bregs dysregulated T cell function through an IL-35-dependent mechanism and depended on cell-to-cell contact. In conclusion, IL-35+ B cell was enriched in CD19+CD24hiCD38hi B cell subset during persistent HBV infection and Breg cells exerted dysregulation in T cell function through IL-35 dependent mechanism and depend on cell-to-cell contact.

Clinical trial registration: www.ClinicalTrials.gov, identifier NCT03734783.

Keywords: HBV - hepatitis B virus; IL-35-secreting B (IL-35+B) cells; chronic HBV infection; immune regulation; interleukin 35 (IL-35); regulatory B cells (Bregs).

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Liu, Luo, Zhu, Jiang, Tian, Tang and Liang.

Figures

Figure 1

Serum IL-35 level in patients with chronic hepatitis B. 68 CHB patients were enrolled in this assay. Non-parametric Mann-Whitney U and Non-parametric Kruskal-Wallis ANOVA test were used to evaluate the differences between two groups and among groups more than two, respectively. P value(A) Serum IL-35 level in patients with different degree liver inflammation; (B) Serum IL-35 level in patients with different viral replication level. Data are shown as mean ± s.e.m.

Figure 2

IL-35-producing B cells in patients with chronic hepatitis B. 35 CHB patients (25 of them with ALT ≤200, 17 of them with HBeAg positive and 13 of them with HBV DNA≥2E4) and 15 HCs were enrolled in this assay. Difference between two groups and among groups more than two was test by Non-parametric Mann-Whitney U test and Non-parametric Kruskal-Wallis ANOVA test, respectively. Statistical correlation between variables was calculated by the Spearman rank correlation analysis. P value(A) IL-35-producing B cell was enriched and in parallel with IL-10-producing B cells in patients with chronic hepatitis B; (B) Patients with chronic HBV infection had high frequency of IL-35+B and IL-10-producing B cells cells in the peripheral blood; (C) Frequency of IL-35-producing B cells and IL-10-producing B cells in patients with different HBeAg status; (D) Frequency of IL-35-producing B cells and IL-10-producing B cells in patients with different viral load; (E) IL-35-producing B cells were moderately correlated with ALT level; (F) IL-35-producing B cells were mildly correlated with serum viral load.

Figure 3

Breg subsets in CHB patients and healthy controls (HCs). Two human classical Breg subsets was gated on CD19+ cell. (A) Frequency of CD19+CD24hiCD38hi or CD19+ CD24hiCD27+ Breg subsets in total CHB patients and HCs; (B) Frequency of CD19+CD24hiCD38hi or CD19+ CD24hiCD27+ Breg subset in CHB patients according to different liver inflammation; (C) Frequency of CD19+CD24hiCD38hi or CD19+ CD24hiCD27+ Breg subsets in CHB patients according to different virus replication level; (D) The frequency of IL-35-producing B cells was strongly correlated with the frequency of CD19+ CD24hiCD27+ Breg subset; (E) The frequency of IL-35-producing B cells was strongly correlated with the frequency of CD19+CD24hiCD38hi Breg subset; (F) Higher frequency of IL-35-positive Breg subset in patients with chronic HBV infection. Difference between two groups and among groups more than two was test by Non-parametric Mann-Whitney U test and Non-parametric Kruskal-Wallis ANOVA test, respectively. Statistical correlation between variables was calculated by the Spearman rank correlation analysis. P value<0.05 was considered statistically significant. ns, no significant difference.

Figure 4

Suppression effect on effector cells (ECs). B cells were isolated from PBMC using CD19+ microbead and stimulated with CD40L/CpG/LPS with or without HBVcore peptide. Stimulated B cells were then co-cultured with autologous CD19-depleted PBMCs at the ratio of 2:1 in anti-CD3/CD28 coated plate for 48hours. As a control, CD19-depleted PBMCs were also cultured alone without B cells (CD19-depleted PBMCs non-activated and CD19-depleted PBMCs activated). (A) Suppression effect on IFN-ɤ-producing CD4+T cells; (B) Suppression on IL-4-producing CD4+T cells; (C) Suppression effect on IL-17A-producing CD4+T cells; (D) Suppression on IFN-ɤ-producing CD8+T cells; (E) Suppression on IL-4-producing CD8+T cells. Non-parametric Kruskal-Wallis ANOVA test and Tukey’s Multiple Comparison test was used to analysis the data. P value<;0.05 was considered statistically significant.

Figure 5

IL-35 neutralizing antibody blocking study and trans-well assay. To explore whether IL-35 worked alone or in collaboration with IL-10 in the process of Bregs regulating on T cell function, neutralizing antibody against IL-35 (1μg/ml, R&D Systems) alone or with neutralizing antibody against IL-10 (5 μg/ml, invitrogen) was added in the co-culture system of anti-CD3/anti-CD28 activated CD19-depleted PBMCs and Bregs. (A) Neutralizing antibody against IL-35 can block suppression function of Bregs on CD4+T cytokine secreting function; (B) Bregs suppressed CD4+T cell cytokine secreting depending on cell-to-cell contact. Non-parametric Kruskal-Wallis ANOVA test and Tukey’s Multiple Comparison test was used to analysis the data. P value<0.05 was considered statistically significant. **p < 0.01. ns, no significant difference.