CD4+ T cells reverse surface antigen persistence in a mouse model of HBV replication

Abstract

Hepatitis B virus (HBV) is a leading causative agent of viral hepatitis. A preventative vaccine has existed for decades, but only limited treatment options are available for people living with chronic HBV. Animal models for studying HBV are constrained due to narrow viral tropism, impeding understanding of the natural immune response to the virus. Here, using a vector to overcome the narrow host range and establish HBV replication in mice, we identified the role of helper T cells in controlling HBV. We show that helper T cells promote the B cell's ability to generate antibodies that remove HBV and its associated surface antigen from the blood and that transfer of purified helper T cells from HBV-immunized mice can reverse the accumulation of virus and antigen, furthering our understanding of the immune response to HBV.

Keywords: B lymphocytes; T lymphocytes; antibody function; hepatitis B virus.

Fig 1

HBsAg is spontaneously resolved in AAV-HBV mice by HBsAb. AAV-HBV-transduced BALB/c mice received 10 doses of depleting (α-CD8, α-CD20), blocking (α-MHCII), or control antibody injections beginning before transduction and ceasing at week 4 post-transduction. (A) Serum HBsAg was measured over time by ELISA. (B) Serum HBsAb was measured at week 13 by ELISA. (C) Splenic IgM and IgG antibody-secreting cells (ASCs) specific for HBsAg were measured by dual-color ELISPOT at the experiment endpoint (week 13). (D) Correlation of HBsAg vs anti-HBsAg IgG-producing ASCs. (E) Representative flow cytometry plots of lymphocytes in blood at 4 and 7 weeks following depletion of CD8⁺ or CD20⁺ cells (gated on single cells/CD45⁺). (F) Serum HBeAg was measured at weeks 1 and 12 by ELISA. (G) Serum HBsAg was measured by ELISA at weeks 1 and 3 post-transduction in mice receiving α-FcγR. (H) Serum HBV DNA was determined by qPCR at week 3 in mice administered α-FcγR. n = 8–10 mice per group. *P < 0.05, **P < 0.01. Mann-Whitney test, one- or two-way ANOVA, or Spearman correlation was used to determine statistical significance.

Fig 2

Transient CD4⁺ T cell depletion induces HBsAg persistence. CD4⁺ T cell activation was evaluated following AAV-HBV transduction. (A) CD69 and (B) intracellular IFN-γ (T_H1) and IL-4 (T_H2) were measured by flow cytometry at 3 and 7 days post-AAV-HBV transduction. Populations in (A) were gated on single cells/lymphocyte size exclusion/CD4⁺/CD69⁺ and populations in (B) were gated on single cells/lymphocyte size exclusion/CD4⁺/IFN-γ⁺ or IL-4⁺; n = 5. AAV-HBV mice received 10 doses of α-CD4 or vehicle (PBS) beginning before transduction and continuing until cessation at week 4. Serum (C) HBsAg and (D) HBsAb were measured over time by ELISA in AAV-HBV transduced mice depleted of CD4⁺ T cells. (C, right) CD4⁺ T cell depletion determined by flow cytometry from whole blood at week 7. (E) Splenic IgM and IgG antibody-secreting cells (ASCs) specific for HBsAg were measured by dual-color ELISPOT at the experimental endpoint (week 18). (F, G) Lymphocyte count from spleen (week 18) as determined by flow cytometry. CD4, CD8, and CD19 were gated on single cells and lymphocyte size exclusion. (G) CXCR5⁺CD4⁺ event count determined by flow cytometry. Population gated on single cells and lymphocyte size exclusion. (H) Percent PD-1⁺ splenocytes were gated on single cells and lymphocyte size exclusion. n = 7–10 mice per group. A–B and C–H are separate experiments. *P < 0.05, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by Student’s t-test or two-way ANOVA.

Fig 3

HBsAg clearance in BALB/c mice depends on the CD40/CD40L axis. AAV-HBV mice received 10 doses of CD40L-blocking antibody (α-CD40L) or vehicle (PBS) beginning before transduction and continuing until cessation at week 4. (A) Serum HBsAg was measured biweekly in CD40L-blocked AAV-HBV mice by ELISA. (B) Splenic IgM and IgG antibody-secreting cells (ASCs) specific for HBsAg were measured by dual-color ELISPOT at the experimental endpoint (week 7). (C) Serum HBsAb was measured by ELISA at week 7. (D) Serum HBeAg was measured by ELISA at weeks 1 and 7 post-AAV-HBV transduction. All graphs are from the same experiment. n = 10 mice per group. *P < 0.05, **P < 0.01, ****P < 0.0001. Statistical significance was determined by the Mann-Whitney test or two-way ANOVA.

Fig 4

Total splenocyte transfer reverses HBsAg persistence in AAV-HBV mice. HBsAg persistence was induced in AAV-HBV-transduced mice by transient CD4 depletion, beginning before transduction and ending on week 4 for a total of 10 doses of CD4-depleting antibody. Following the repopulation of CD4⁺ T cells, splenocytes from naïve or immunized mice were transferred into HBsAg-persistent recipients (week 11). (A) Serum HBsAg was measured by ELISA over time in AAV-HBV transduced mice. (B) HBV DNA was isolated from serum at week 17 post-transduction and measured by qPCR. (C) Serum HBsAb was measured at week 15 by ELISA. (D) Splenic IgG1 and IgG2a antibody-secreting cells (ASCs) specific for HBsAg were measured by dual-color ELISPOT at week 18. (E) CD4⁺ T cell count from blood (week 16) as determined by flow cytometry. CD3 and CD4 were gated on single cells and lymphocyte size exclusion. n = 4. (F) HBeAg was measured in serum at weeks 12 and 14 post-transduction. (G) Liver HBV gene expression was measured at week 17 by RT-qPCR. (H) Serum HBcAb as determined by ELISA at week 17. Groups shown are PBS, no splenocyte transfer (–), naïve splenocyte recipients (naïve), and immunized splenocyte recipients (Imm). All graphs are from the same experiment. n = 6–8 mice per group. *P < 0.05. Statistical significance was determined by Student’s t-test, one-way ANOVA, or two-way ANOVA.

Fig 5

CD4⁺ T cell transfer from HBsAg-immunized mice reverses HBsAg persistence in AAV-HBV mice. HBsAg-persistent AAV-HBV mice generated by transient CD4 depletion (two doses of α-CD4) beginning before transduction received purified CD4⁺ T cells or B cells from HBsAg-immunized mice at week 9 post-transduction. (A) CD4⁺ T cell repopulation before cell transfer was measured by flow cytometry at week 8 post-AAV-HBV transduction. (B) B cell (left) and CD4⁺ T cell (right) purity following magnetic column separation was determined by flow cytometry. (C) Serum HBsAg was measured over time by ELISA. (D) HBV DNA from serum was measured at week 17 post-transduction by qPCR. (E) Splenic IgM and IgG antibody-secreting cells (ASCs) specific for HBsAg were measured by dual-color ELISPOT at week 17. (F) HBsAb was measured in serum at week 17 post-transduction by ELISA. (G) Liver HBV gene expression was determined by RT-qPCR at week 17. (H) Serum HBeAg was measured by ELISA at weeks 11 and 14 post-transduction. (I) Serum HBcAb was measured by ELISA at week 17. All graphs are from the same experiment. n = 8 mice per group *P < 0.05, ***P < 0.001. Statistical significance was determined by Student’s t-test, one-way ANOVA, or two-way ANOVA.