Hepatitis D Virus-Specific CD8+ T Cells Have a Memory-Like Phenotype Associated With Viral Immune Escape in Patients With Chronic Hepatitis D Virus Infection

Abstract

Background & aim: Hepatitis D virus (HDV) superinfection of patients with chronic HBV infection results in rapid progression to liver cirrhosis. Little is known about HDV-specific T cells and how they contribute to the antiviral immune response and liver disease pathogenesis.

Methods: We isolated peripheral blood mononuclear cells from 28 patients with chronic HDV and HBV infection, identified HDV-specific CD8+ T-cell epitopes, and characterized HDV-specific CD8+ T cells. We associated these with HDV sequence variations and clinical features of patients.

Results: We identified 6 CD8+ T-cell epitopes; several were restricted by multiple HLA class I alleles. HDV-specific CD8+ T cells were as frequent as HBV-specific CD8+ T cells but were less frequent than T cells with specificity for cytomegalovirus, Epstein-Barr virus, or influenza virus. The ex vivo frequency of activated HDV-specific CD8+ T cells correlated with transaminase activity. CD8+ T-cell production of interferon gamma after stimulation with HDV peptides correlated inversely with HDV titer. HDV-specific CD8+ T cells did not express the terminal differentiation marker CD57, and fewer HDV-specific than Epstein-Barr virus-specific CD8+ T cells were 2B4+CD160+PD1+, a characteristic of exhausted cells. Approximately half of the HDV-specific CD8+ T cells had a memory-like PD1+CD127+TCF1hiT-betlow phenotype, which associated with HDV sequence variants with reduced HLA binding and reduced T-cell activation.

Conclusions: CD8+ T cells isolated from patients with chronic HDV and HBV infection recognize HDV epitopes presented by multiple HLA molecules. The subset of activated HDV-specific CD8+ T cells targets conserved epitopes and likely contributes to disease progression. The subset of memory-like HDV-specific CD8+ T cells is functional but unable to clear HDV because of the presence of escape variants. ClinicalTrials.gov, Numbers: NCT02511431, NCT00023322, NCT01495585, and NCT00001971. GenBank accession, Number: MK333199-333226.

Keywords: Lymphocyte; Mutation; Transcription Factor; Virus Escape.

Conflict of interest statement

Disclosures: The authors have no conflict of interest.

Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. CD8+ T-cell epitope mapping in chronic HDV infection.

(A) T-cell lines were generated by stimulating PBMC from a chronic HDV patient with pools of overlapping HDV peptides. IFN-γ production was analyzed by flow cytometry after restimulation with or without pool 4 of overlapping 15mer HDV peptides (left panels) or individual peptides from that pool (right panels). (B-D) T-cell lines of an HLA-B*35:01+ patient (B), an HLA-B*0701+ patient (C) and an HLA-B*2705+ patient (D) were tested against shorter peptides to determine the CD8+ T-cell epitope. An epitope (marked in red) is defined as the peptide eliciting half-maximal cytokine responses at the lowest peptide concentration.

Figure 2:. Characterization of HDV-specific CD8+ T-cell epitopes using in vitro expanded T-cell lines.

(A-C) The location of HDV-specific CD8+ T-cell epitopes in the aminoterminal (A), middle (B) and carboxyterminal HDV amino acid sequence (C) is shown. The HLA molecule presenting the epitope is indicated on the left, an underlined HLA molecule indicates that an HLA/peptide multimer was generated. The previously published epitopes KLEDENPWL, RRKALENKK and RRDHRRRKAL are included for reference purposes. (D) Number of patients with T-cell lines responding to the indicated peptides in the HDV peptide pools. Arrows indicate responses that were mapped (after further culture of theT cell lines) to the indicated HLA-restricted minimal optimal epitopes. (E) Inverse correlation (Spearman correlation) between the frequency of IFNγ-producing HDV-specific CD8+ T cells in the T-cell lines and the level of viremia in patient serum. The T-cell lines were established from PBMC obtained at week 4 post lonafarnib/ritonavir treatment.

Figure 3:. Frequency and phenotype of HDV,- HBV-, CMV-, EBV- and Flu-specific CD8+ T-cells in chronic HDV infection.

(A, B) PBMC were depleted of non-CD8 T cells with magnetic beads, antibody-stained and analyzed by flow cytometry following the indicated gating strategy (A) to identify HLA/epitope multimer+ cells as HDV-specific CD8+ T cells (B). FSC-H: forward scatter height; FSC-A: forward scatter area; SSC-A: side scatter area; FVS: fixable viability stain. (C-F) Frequency of HDV-specific multimer+ cells (C) and their subpopulations of CD45RO+CCR7- (D), CD45RO+CCR7+ (E), and CD45RO-CCR7- cells (F). Statistics: Kruskal-Wallis test with Dunn’s multiple comparisons.

Figure 4:. Most HDV-specific CD8+ T cells have a memory-like PD-1+CD127+ phenotype, and are activated, but not terminally differentiated.

(A) HDV-, (B) HBV-, (C) EBV- and (D) Flu-specific antigen-experienced CD8+ T cells were assessed for expression of PD-1 and CD127 (left panels), PD-1 and CD38 (middle panels) and PD-1 and CD57 (right panels). Statistics: Friedman test with Dunn’s multiple comparisons. (E) Correlation between serum AST activity and frequency of CD38+ cells in the HDV-specific (left panel) or HBV-specific (right panel) CD8+ T-cell population using Spearman correlation. N.s., not significant.

Figure 5:. HDV-specific CD8+ T cells are less exhausted than CMV- and EBV-specific CD8+ T cells.

(A) Frequency of HDV-, HBV-, CMV-, EBV- or Flu-specific effector memory (CCR7- CD45RO+) CD8+ T cells expressing either 2B4, CD160, PD-1 or Tim-3 (pie arcs) or combinations thereof (pie slices, corresponding to individual panels in B). (B-C) Virus-specific effector memory CD8+ T cells were identified by flow cytometry and assessed for expression of the surface marker combinations as indicated by bar graphs (B, median with interquartile range) and a coolplot (C). Statistics: Students T test and partial permutation test using SPICE. Only statistically significant differences to HDV-specific CD8+ cells are displayed. *P<.05, ** P<.01, ***P<.001.

Figure 6:. The memory-like phenotype of HDV-specific CD8+ T cells is associated with the presence of viral sequence variants with reduced HLA binding affinity.

(A-C) Frequency of HDV-specific CD8+ T cells expressing the transcription factors TCF1(A) or T-bet (B). Statistics: Mann-Whitney test. (C) Expression level (median fluorescence intensity, MFI) of the activation marker CD38 on HDV- and Flu-specific CD8+ T cells. (D) The memory-like TCF1hi phenotype of HDV-specific CD8+ T cells is associated with the presence of viral sequence variants with reduced HLA binding affinity. HDV-infected patients were divided into two groups with either TCFhi or TCF1lo HDV-specific CD8+ T cells for the respective epitope. For each group, peptides were synthesized according to the prototype genotype 1 HDV sequence and the corresponding autologous sequence present in the patient’s serum. Peptides with prototype or autologous sequence were compared in HLA-binding assays. A low IC50 reflects a high HLA-binding affinity. The following autologous sequences were associated with reduced HLA binding (high IC50) and the presence of TCF1hi cells: EENLWLGNI (HLA-B*18:01); QGFPWDMLF, RGFPWDILF (HLA-B*35:01); RRKALENKS (HLA-B*27:01); FPWDILFPS (HLA-B*07:02); TGRQGFPW (HLA-B*58:01). The following autologous sequences were associated with preserved HLA binding (low IC50) and the presence of TCF1lo cells: FPWDILFPA, QGFPWDLLF and FPWDLLFPA, FPWDMLFPA (HLA-B*35:01); The corresponding prototype sequences are shown in Table 1. Statistics: Wilcoxon matched-pairs signed rank test (P=.0312) and Mann-Whitney test (P=.0043).