Impaired hepatitis B vaccine responses during chronic hepatitis C infection: involvement of the PD-1 pathway in regulating CD4(+) T cell responses

Abstract

Vaccination for hepatitis B virus (HBV) in the setting of hepatitis C virus (HCV) infection is recommended, but responses to vaccination are blunted when compared to uninfected populations. The mechanism for this failure of immune response in HCV-infected subjects remains unknown but is thought to be a result of lymphocyte dysfunction during chronic viral infection. We have recently demonstrated that PD-1, a novel negative immunomodulator for T cell receptor (TCR) signaling, is involved in T and B lymphocyte dysregulation during chronic HCV infection. In this report, we further investigated the role of the PD-1 pathway in regulation of CD4(+) T cell responses to HBV vaccination in HCV-infected individuals. In a prospective HCV infected cohort, a poor response rate to HBV vaccination as assayed by seroconversion was observed in HCV-infected subjects (53%), while a high response rate was observed in healthy or spontaneously HCV-resolved individuals (94%). CD4(+) T cell responses to ex vivo stimulations of anti-CD3/CD28 antibodies or hepatitis B surface antigen (HBsAg) were found to be lower in HBV vaccine non-responders compared to those responders in HCV-infected individuals who had received a series of HBV immunizations. PD-1 expression on CD4(+) T cells was detected at relatively higher levels in these HBV vaccine non-responders than those who responded, and this was inversely associated with the cell activation status. Importantly, blocking the PD-1 pathway improved T cell activation and proliferation in response to ex vivo HBsAg or anti-CD3/CD28 stimulation in HBV vaccine non-responders. These results suggest that PD-1 signaling may be involved in impairing CD4(+) T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this negative signaling pathway might improve success rates of immunization in the setting of chronic viral infection.

Fig. 1. CD4⁺ T cell activation status in HCV-infected and healthy subjects who received HBV vaccinations

PBMC were isolated from HCV-infected HBV vaccine non-responders (HBV-NR) (n=29), HCV-infected HBV vaccine responders (HBV-R) (n=32), HCV-resolved subjects (n=6), and healthy subjects (n=10), ex vivo stimulated with anti-CD3/CD28 (left panel) or HBsAg (right panel) for 24 hrs, and assayed by flow cytometry for CD69 expression on CD4⁺ T cells; representative dot plots are shown above and summary data shown below.

Fig. 2. PD-1 / PDL-1 expressions on CD4⁺ T cells in HCV-infected and healthy subjects who received HBV vaccination

PBMC were isolated from HCV-infected HBV vaccine non-responders (HBV-NR) (n=29), HCV-infected HBV vaccine responders (HBV-R) (n=32), HCV-resolved subjects (n=6), and healthy subjects (n=10), ex vivo stimulated with anti-CD3/CD28 or HBsAg for 24 hrs, and assayed by flow cytometric analysis for PD-1 or PDL-1 expressions on CD4⁺ T cells; representative dot plots are shown above and summary data shown below. A) PD-1 expression on CD4⁺ T cells stimulated with anti-CD3/CD28. B) PDL-1 expression on CD4⁺ T cells stimulated with anti-CD3/CD28. C) PD-1 expression on CD4⁺ T cells stimulated with HBsAg. D) PDL-1 expression on CD4⁺ T cells stimulated with HBsAg. Statistical findings are shown between the comparison groups.

Fig. 3. Increase in SOCS-1 expression in CD4+ T cells isolated in HCV-infected HBV vaccine non-responders

Purified CD4+ T cells were isolated by incubation of PBMCs with magnetic beads-conjugated with anti-CD4 antibody, followed by positive selection per the manufacturer's instruction, from three HBV-R and three HBV-NR. Following stimulation with either HBsAg or anti-CD3/CD28 for 24 h as described above, cells were either lysed for immunoblotting (A) or RNA isolation for RT PCR (B) as we have described^,,. SOCS-1 gene expression was detected by quantitative RT-PCR, and β2M gene served as an internal control. Densitometric analysis of immunoblots normalized to actin are shown with error bars below. Data were reproducible in independent experiments using cells isolated from three different HCV-infected subjects per condition.

Fig. 4. PD-1 and PDL-1 expressions are inversely associated with CD4⁺ T cell responses to HBV vaccine in HCV-infected individuals

Each symbol represents an individual expressing cell surface marker for PD-1 inhibitory pathway as well as T cell activation in subjects who had received HBV vaccines and ex vivo stimulated with HBsAg (left panels) or anti-CD3/CD28 (right panels). The inverse association between PD-1 and CD69 (A) and PDL-1 and CD69 (B) expressions on CD4⁺ T cells was evaluated using Pearson Correlation analysis. Pearson correlation and 2-tailed significance are shown in the upper right corner of each analysis.

Fig. 5. Blocking the PD-1 pathway improves CD4⁺ T cell responses in HBV vaccine non-responders with HCV infection

PBMC isolated from HCV-infected subjects who had received HBV vaccinations and failed to seroconvert were incubated with anti-PDL-1 or control antibody overnight, followed by stimulation with either HBsAg or anti-CD3/CD28 antibodies for 5 days. T cell activation and proliferation were evaluated by detection of CD69 expression (A) and CFSE assay (B) in stimulated T cells. Representative percentages of CD69 expression and gating of CFSE in T cells treated with anti-PDL-1 versus control antibody are shown on the left and summary data on the right. Data are reproducible in repeated experiments using cells isolated from three different individuals with HBV-NR in the setting of chronic HCV infection.