Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection

Abstract

Dysfunctional CD8+ T cells present in chronic virus infections can express programmed death 1 (PD-1) molecules, and the inhibition of the engagement of PD-1 with its ligand (PD-L1) has been reported to enhance the antiviral function of these T cells. We took advantage of the wide fluctuations in levels of viremia which are typical of chronic hepatitis B virus (HBV) infection to comprehensively analyze the impact of prolonged exposure to different virus quantities on virus-specific T-cell dysfunction and on its reversibility through the blocking of the PD-1/PD-L1 pathway. We confirm that chronic HBV infection has a profound effect on the HBV-specific T-cell repertoire. Despite the use of a comprehensive panel of peptides covering all HBV proteins, HBV-specific T cells were rarely observed directly ex vivo in samples from patients with chronic infection, in contrast to those from patients with acute HBV infection. In chronic HBV infection, virus-specific T cells were detected mainly in patients with lower levels of viremia. These HBV-specific CD8+ T cells expressed PD-1, and their function was improved by the blocking of PD-1/PD-L1 engagement. Thus, a broad spectrum of anti-HBV immunity is expressed by patients with chronic HBV infection and this spectrum is proportional to HBV replication levels and can be improved by blocking the PD-1/PD-L1 pathway. This information may be useful for the design of immunotherapeutic strategies to complement and optimize available antiviral therapies.

FIG. 1.

Results from ex vivo and in vitro ELISPOT assays for IFN-γ and the identification of the responding T-cell subsets in samples from patients with chronic HBV infection. (A) Ex vivo IFN-γ ELISPOT analysis was performed with samples from seven chronic hepatitis B patients (C1 through C7) studied longitudinally at three time points throughout a 12-month period by using 16 pools of 15-mer peptides covering the overall protein sequence of HBV genotype D. ALT and HBV DNA levels are expressed at the top of each histogram. (B) ELISPOT analysis was also performed after 10 days of in vitro stimulation with the same pools of peptides in the presence of IL-2. Samples from seven chronic hepatitis B patients were analyzed at the same time points as those assessed ex vivo. Levels of IFN-γ-producing cells are represented as numbers of spot-forming units (SFU) per 200,000 PBMC. The number of specific IFN-γ-secreting cells was calculated by subtracting the value for the unstimulated control from the value for the stimulated sample. The positive control consisted of PBMC stimulated with phytohemagglutinin. Each bar represents the response to an individual peptide mixture. The graph at the bottom of each panel indicates the responding T-cell subsets (CD4 and CD8) in the samples from the group of seven chronic hepatitis B patients. After the identification of the immunogenic peptide mixtures by ELISPOT assays, the responding T-cell subsets were defined by intracellular cytokine staining with individual peptide pools either ex vivo (A) or after 10 days of in vitro peptide stimulation (B). Chronic hepatitis B patients were studied at three time points throughout the study period. Each bar represents the percentage of total positive (pos.) tests with the indicated peptide pools. Bars in the insets represent the mean percentages (± standard errors) of positive responses expressed by CD4 and CD8 cells relative to the total (tot.) number of tests. x, x antigen peptide pools; env, envelope peptide pools; pol, polymerase peptide pools.

FIG. 2.

Longitudinal analysis of HBV-specific T-cell responses in relation to the different virological profiles for anti-HBe-positive chronic patients. (A) IFN-γ production by CD4 and CD8 T cells from seven chronic hepatitis B patients (Pt C1 through Pt C7) was tested by ICS after 10 days of in vitro peptide stimulation; levels of HBV DNA were determined at the same time points. Each bar represents the total frequency of IFN-γ-positive cells among the overall CD4⁺ (striped bars) or CD8⁺ (gray bars) population at each individual time point as determined by summing the responses to each individual peptide pool as detected by ICS. Black lines indicate HBV DNA levels. Panel B illustrates the inverse relationship between IFN-γ production and the level of HBV viremia, as shown by linear regression analysis. Each square in panel C illustrates the percentage of IFN-γ-positive T cells among total T cells detected at different levels of viremia (HBV DNA, <1 × 10⁶ copies/ml, 1 × 10⁶ to 10 × 10⁶ copies/ml, and >10 × 10⁶ copies/ml). A statistically significant difference between the levels of IFN-γ production in the first (HBV DNA, <1 × 10⁶ copies/ml) and third (HBV DNA, >10 × 10⁶ copies/ml) groups and between the second (HBV DNA, 1 × 10⁶ to 10 × 10⁶ copies/ml) and third groups of HBV DNA profiles as determined by the Mann-Whitney test was observed.

FIG. 3.

Phenotypic and functional profiles of tetramer-positive CD8 cells from patients with acute and chronic HBV infections. (A) Percentages of CD127⁺ cells and PD-1-positive cells among HBV tetramer-positive CD8 lymphocytes from 12 patients with anti-HBe-positive chronic hepatitis (three of them were tested at different time points) and from seven patients with acute HBV infection tested at two different time points, one corresponding to the acute phase of infection and the other at a later period (during the 2 to 15 months of follow-up after the clinical presentation). Percentages of CD127⁺, tetramer-positive cells in samples from chronic patients were significantly higher than those in samples from patients in the acute stage of infection (P, <0.0005 by Mann-Whitney), and those in samples from patients in the recovery phase of acute hepatitis were significantly higher than those in samples from patients in the acute phase of infection (P, <0.005 by Mann-Whitney); percentages of PD-1-positive, tetramer-positive cells in samples from chronic patients were significantly higher than those in samples from patients in the recovery phase of acute infection (P, <0.001 by Mann-Whitney), and those in samples from patients in the acute phase of infection were significantly higher than those in samples from patients in the recovery phase (P, <0.005 by Mann-Whitney). (B) Representative dot plots for HBV tetramer (Tet)-positive cells from four patients with chronic HBV infection (top) and from two patients in the acute phase of hepatitis and at the time of recovery (bottom) stained with anti-CD8, anti-PD-1, and anti-CD127 monoclonal antibodies. Because of the very low frequency of tetramer-positive cells in chronic HBV patients, a large number of events (>1 × 10⁶) was analyzed by flow cytometry to allow for a reliable phenotypic analysis. Core 18-27, core peptide spanning amino acids 18 to 27; ENV 335-343, envelope peptide spanning amino acids 335 to 343.

FIG. 4.

PD-1 expression on HBV-, CMV-, and influenza virus-specific CD8⁺ T cells. (A) Dot plots representative of PD-1 expression on CMV-, influenza virus (FLU)-, and HBV-specific tetramer (Tet)-positive cells are shown for three representative patients with chronic HBV infection. (B) The PD-1 fluorescence intensities expressed by total CD8⁺ cells and non-HBV-specific (CMV-specific on the left and influenza virus-specific on the right) and HBV-specific tetramer-positive CD8 cells are illustrated for two representative chronic HBV patients. Core 18-27, core peptide spanning amino acids 18 to 27.

FIG. 5.

Effect of anti-PD-L1 antibody on the function of HBV-specific tetramer-positive CD8⁺ T cells. (A) Frequencies of tetramer-positive CD8 cells in samples from six patients with chronic HBV infection were analyzed after 10 days of peptide stimulation in the presence of anti-PD-L1 or control antibodies (P, <0.05 by the Wilcoxon paired test) (top). The mean increase (n-fold) in the frequency of tetramer-positive cells among cells cultured with anti-PD-L1 antibody with respect to the frequency of tetramer-positive cells among cells cultured with the control antibody is shown in the graph at the right. The middle and bottom parts of panel A illustrate the percentages of HBV-specific CD8 T cells able to produce IFN-γ and IL-2 as detected by intracellular cytokine staining upon the restimulation of T-cell cultures derived from 10 days of HBV peptide stimulation in the presence of anti-PD-L1 or control antibodies (difference for IFN-γ by the Wilcoxon paired test, P < 0.05). Pooled data are expressed as mean increases (n-fold) in the percentages of IFN-γ- and IL-2-producing CD8 T cells derived from cultures performed in the presence of anti-PD-L1 antibody with respect to those derived from cultures performed in the presence of the control antibody (graphs at the right). (B) Dot plots representative of frequencies of CD8 cells positive for the tetramer core peptide spanning amino acids 18 to 27 (Tr core 18-27) after 10 days of HBV peptide stimulation in the presence of anti-PD-L1 or control antibodies. (C) Dot plots representative of IFN-γ and IL-2 production by T-cell lines induced by 10 days of HBV peptide stimulation in the presence of anti-PD-L1 or control antibodies in samples from two representative chronic hepatitis B patients (C11 and C8).

FIG. 6.

Effect of anti-PD-L1 antibody on the HBV-specific T-cell function analyzed with peptides spanning the entire HBV protein sequence. We determined the effect of anti-PD-L1 antibody on IFN-γ production by CD4 and CD8 T cells after the expansion of T-cell populations by 10 days of in vitro stimulation with individual peptide pools covering the overall HBV sequence in the presence of anti-PD-L1 or control antibodies. Each individual culture obtained after 10 days of stimulation with each peptide pool was incubated for 18 h in the presence of the corresponding peptide pool or medium alone, and cytokine production was tested by ICS as described in Materials and Methods. Each dot or point represents the increase (n-fold) in IFN-γ production induced by anti-PD-L1 antibody, calculated as the ratio between the percentages of cytokine-producing T cells in cultures expanded with peptide pools in the presence of anti-PD-L1 and those expanded in the presence of control antibodies. For these experiments, polymerase (POL) peptides were pooled in four mixtures, envelope (ENV) peptides were pooled in two mixtures, and core and x peptides were pooled in individual mixtures. Only responses to peptide pools that were increased at least two times by incubation with anti-PD-L1 antibodies are illustrated. □, CD4 cells; ▪, CD8 cells. The panels on the right show dot plots representative of IFN-γ-producing CD4 and CD8 T cells induced by the indicated peptide pools in the presence of anti-PD-L1 or control antibodies. aa, amino acids.