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Hepatitis B Virus-Specific CD8+ T Cells Maintain Functional Exhaustion after Antigen Reexposure in an Acute Activation Immune Environment - PubMed

  • ️Mon Jan 01 2018

doi: 10.3389/fimmu.2018.00219. eCollection 2018.

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Hepatitis B Virus-Specific CD8+ T Cells Maintain Functional Exhaustion after Antigen Reexposure in an Acute Activation Immune Environment

Qin Wang et al. Front Immunol. 2018.

Abstract

Chronic hepatitis B virus (HBV) infection is characterized by the presence of functionally exhausted HBV-specific CD8+ T cells. To characterize the possible residual effector ability of these cells, we reexposed CD8+ T cells from chronically HBV replicating mice to HBV antigens in an acute activation immune environment. We found that after transfer into naive mice, exhausted CD8+ T cells reexpanded in a comparable magnitude as naive CD8+ T cells in response to acute HBV infection; however, their proliferation intensity was significantly lower than that of CD8+ T cells from acute-resolving HBV replicating mice (AR mice). The differentiation phenotypes driven by acute HBV replication of donor exhausted and naive CD8+ T cells were similar, but were different from those of their counterparts from AR mice. Nevertheless, exhausted CD8+ T cells maintained less activated phenotype, an absence of effector cytokine production and poor antiviral function after HBV reexposure in an acute activation immune environment. We thus conclude that exhausted CD8+ T cells undergo a stable form of dysfunctional differentiation during chronic HBV replication and switching immune environment alone is not sufficient for the antiviral functional reconstitution of these cells.

Keywords: CD8+ T cell; cell transfer; functional exhaustion; hepatitis B virus; immune environment.

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Figures

Figure 1
Figure 1

Characterization of hepatitis B virus (HBV)-specific CD8+ T cells in the spleen of chronically HBV replicating mice and acute-resolving HBV replicating mice. C57BL/6 mice were hydrodynamically injected with either pAAV/HBV1.2 or pSM2 plasmid. (A) The levels of serum HBsAg and HBeAg in the plasmid injected mice were monitored at indicated time points. The data were analyzed by analysis of covariance. (B) The serum HBV DNA levels in mice were monitored at indicated time points. The data were analyzed by repeated measures analysis (P < 0.05). (C) Frequencies of HBV core93-specific CD8+ T cells were detected in the spleen of plasmid injected mice at day 21 after hydrodynamic injection by FITC dimer staining. (D,E) Splenocytes were separated from plasmid injected mice at day 21 and were stimulated with core93 or env208 peptide for 5 h in vitro. Intracellular staining was performed and the frequencies of interferon-γ+, interleukin-2+, and tumor necrosis factor-α+ CD8 T cells are shown. Splenocytes from naive mice were used as a negative control. Data are representative of three independent experiments. One-way ANOVA followed by least significant difference test were applied. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Figure 1
Figure 1

Characterization of hepatitis B virus (HBV)-specific CD8+ T cells in the spleen of chronically HBV replicating mice and acute-resolving HBV replicating mice. C57BL/6 mice were hydrodynamically injected with either pAAV/HBV1.2 or pSM2 plasmid. (A) The levels of serum HBsAg and HBeAg in the plasmid injected mice were monitored at indicated time points. The data were analyzed by analysis of covariance. (B) The serum HBV DNA levels in mice were monitored at indicated time points. The data were analyzed by repeated measures analysis (P < 0.05). (C) Frequencies of HBV core93-specific CD8+ T cells were detected in the spleen of plasmid injected mice at day 21 after hydrodynamic injection by FITC dimer staining. (D,E) Splenocytes were separated from plasmid injected mice at day 21 and were stimulated with core93 or env208 peptide for 5 h in vitro. Intracellular staining was performed and the frequencies of interferon-γ+, interleukin-2+, and tumor necrosis factor-α+ CD8 T cells are shown. Splenocytes from naive mice were used as a negative control. Data are representative of three independent experiments. One-way ANOVA followed by least significant difference test were applied. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Figure 2
Figure 2

Analysis of antiviral function of CD8+ T cells from chronically hepatitis B virus (HBV) replicating mice. (A) Scheme of cell transfer and HBV hydrodynamic injection. Splenic CD8+ T cells from naive, chronically or acutely resolving HBV replicating mice were adoptively transferred into naive mice. Recipients were hydrodynamically injected with pSM2 1 day post cell transfer and were monitored for serum viremia. The kinetics of serum HBsAg (B), HBeAg (C), and HBV DNA levels (D) are shown and the statistical differences were analyzed by repeated measures analysis. The asterisks mark the statistically significant differences between CT and NT mice. The hash signs mark the significant differences between CT and AT mice. Data are representative of two independent experiments. *P < 0.05; #P < 0.05; ##P < 0.01.

Figure 3
Figure 3

Analysis of CD8+ T cell absolute numbers after cell transfer and hepatitis B virus hydrodynamic injection. Splenic CD8+ T cells from chronically HBV replicating mice, acute-resolving HBV replicating mice, or naive mice (CD45.2+) were adoptively transferred into naive mice (CD45.1+). Recipients were hydrodynamically injected with pSM2 and were sacrificed at day 28. Representative dot plots show the percentages of donor and recipient CD8+ T cells in the liver (A) and spleen (C). The absolute numbers of recipient and donor CD8+ T cells in the liver (B) and spleen (D) were analyzed by flow cytometry. (E) Representative dot plots show the frequencies of core93-specific CD8+ T cells in the liver and spleen. (F) The absolute numbers of donor and recipient core93-specific CD8+ T cells in the liver and spleen were analyzed by PE dimer staining method. (A,B) Representative results from three independent experiments are shown. Kruskal–Wallis test followed by Dunn’s post hoc tests were applied. (C–F) Representative results from three independent experiments are shown. One-way ANOVA with least significant difference post hoc tests were used. *P < 0.05; **P < 0.01; ns, not significant.

Figure 4
Figure 4

Analysis of the proliferation of engrafted CD8+ T cells after cell transfer and hepatitis B virus hydrodynamic injection. Splenic CD8+ T cells from chronically HBV replicating mice, acute-resolving HBV replicating mice, or naive mice (CD45.2+) were adoptively transferred into naive mice (CD45.1+). Recipients were hydrodynamically injected with pSM2. The kinetic changes of absolute numbers of engrafted CD8+ T cells (A) and core93-specific CD8+ T cells (B) in the liver and the spleen were analyzed by flow cytometry at the indicated time points. (C) Kinetics of Ki-67 expression in donor CD8+ T cells in the liver and the spleen of CT mice were analyzed by flow cytometry. Data are representative of two independent experiments. One-way ANOVA with least significant difference post hoc tests were applied. *P < 0.05; **P < 0.01; ns, not significant.

Figure 5
Figure 5

Analysis of CD8+ T cells phenotype after cell transfer and hepatitis B virus hydrodynamic injection. Splenic CD8+ T cells from chronically HBV replicating mice, acute-resolving HBV replicating mice, or naive mice (CD45.2+) were adoptively transferred into naive mice (CD45.1+). Recipients were hydrodynamically injected with pSM2 and were sacrificed at day 28. The frequencies of CD43+ total (A), donor, and recipient (B) CD8+ T cells in the liver and the spleen were analyzed by flow cytometry. The frequencies of CD44+CD62L− TEM cells and CD44+CD62L+ TCM cells of total (C), donor, and recipient (D) CD8+ T in the liver and the spleen were analyzed by flow cytometry. Data are representative of three independent experiments. Statistics analysis was performed by one-way ANOVA with least significant difference post hoc tests. *P < 0.05; **P < 0.01; ns, not significant.

Figure 6
Figure 6

Analysis of the phenotype of core93-specific CD8+ T cells after cell transfer and hepatitis B virus hydrodynamic injection. Splenic CD8+ T cells from chronically HBV replicating mice, acute-resolving HBV replicating mice, or naive mice (CD45.2+) were adoptively transferred into naive mice (CD45.1+). Recipients were hydrodynamically injected with pSM2 and were sacrificed at day 28. The frequencies of CD43+ total (A), donor, and recipient (B) core93-specific CD8+ T cells examined by PE dimer staining in the liver and spleen were analyzed by flow cytometry. The frequencies of CD44+CD62L− TEM cells and CD44+CD62L+TCM cells of total (C), donor, and recipient (D) core93-specific CD8+ T examined by PE dimer staining in the liver and spleen were analyzed by flow cytometry. Data are representative of three independent experiments. Statistics analysis was performed by one-way ANOVA with least significant difference post hoc tests. *P < 0.05; **P < 0.01; ns, not significant.

Figure 7
Figure 7

Analysis of effector function of hepatitis B virus (HBV)-specific CD8+ T cells after cell transfer and HBV hydrodynamic injection (HI). Intrahepatic lymphocytes and splenocytes were separated from NT, CT, and AT mice at 21 days post HI. Cells were stimulated by HBV core93-100 peptide or env208-216 peptide for 5 h and were stained for intracellular cytokines interferon (IFN)-γ, IL2, and tumor necrosis factor (TNF)-α. The frequencies of IFN-γ, IL2, and TNF-α producing total (A–D), donor, and recipient (E–H) CD8+ T cells in response to HBV core or envelope antigen stimulation in the liver and the spleen were measured by flow cytometry. Data are representative of three independent experiments. Statistics analysis was performed by Kruskal–Wallis test followed by Dunn’s post hoc tests. U.D., undetectable. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

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