Impact of HBsAg and HBcrAg levels on phenotype and function of HBV-specific T cells in patients with chronic hepatitis B virus infection

Abstract

Objective: Hepatitis B virus (HBV)-specific T cells are main effector cells in the control of HBV infection and hepatitis B surface antigen (HBsAg) is suggested to be a critical factor in the impaired immune response, a hallmark of chronic HBV infection. In addition to HBsAg, other viral markers such as hepatitis B core-related antigen (HBcrAg) are available, but their potential association with HBV-specific immune responses is not defined yet, which will be important if these markers are used for patient stratification for novel therapies aimed at functional HBV cure.

Design: We analysed T cell responses in 92 patients with hepatitis B e antigen negative chronic HBV infection with different HBsAg and HBcrAg levels. Overlapping peptides were used for in vitro response analyses (n=57), and HBV core₁₈-specific and polymerase (pol)₄₅₅-specific CD8⁺ T cells were assessed in human leukocyte antigen (HLA)-A*02 patients (n=35). In addition, in vitro responsiveness to anti-programmed cell death-ligand 1 (anti-PD-L1) was investigated.

Results: HBV-specific T cell responses were not affected by HBsAg levels, but rather by age and CD4⁺ T cell responses were highest in patients with low HBcrAg levels. The phenotypes and functionality of HBV core₁₈-specific and pol₄₅₅-specific CD8⁺ T cells differed, but HBsAg and HBcrAg levels did not affect their profiles. Blocking with anti-PD-L1 could restore HBV-specific T cells, but the effect was significantly higher in T cells isolated from patients with low HBsAg and in particular low HBcrAg.

Conclusion: Our data suggest that age and HBcrAg rather than HBsAg, are associated with HBV-specific T cell responses. Finally, very low antigen levels indicated by HBsAg and in particular HBcrAg may influence T cell response to checkpoint inhibition.

Keywords: T lymphocytes; cellular immunology; chronic hepatitis; hepatitis B; immune response.

Figure 1

Characterisation of total CD4⁺ and CD8⁺ T cells in patients with CHB with different levels of HBsAg and HBcrAg. (A) Concatenated t-SNE plots of CD4⁺ and CD8⁺ T cell subsets (upper panels) and intensities of the indicated markers (lower panels) in 49 patients with CHB categorised according to HBsAg and HBcrAg levels. (B–C) Frequencies of CD4⁺ and CD8⁺ T cell effector memory subsets as well as CD4⁺ and CD8⁺ T cells expressing activation and exhaustion markers grouped according to HBsAg (B) and HBcrAg (C) levels. Statistical significance between each of the two groups was tested by Mann-Whitney test for non-parametric data and by unpaired t-test for parametric data (B–C). *p<0.05; **p<0.01; ***p<0.001. CHB, chronic hepatitis B; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; T_CM, central memory T cell; T_EM, effector memory T cell; T_EMRA, terminally differentiated effector memory T cell; HLA-DR, human leukocyte antigen – DR; KLRG1, killer cell lectin like receptor G1; PD-1, programmed cell death protein 1; t-SNE, t-distributed stochastic neighbor embedding.

Figure 2

HBV-specific CD4⁺ and CD8⁺ T cell responses in patients with different levels of HBsAg and HBcrAg. HBV-specific CD4⁺ and CD8⁺ T cell responses following 10-day in vitro stimulation with HBV overlapping peptide pools. (A) Representative flow cytometry plots of HBV-specific CD4⁺ T cell responses from patients with different levels of HBsAg and HBcrAg. (B–C) HBV-specific IFN-γ⁺ CD4⁺ and IFN-γ⁺ CD8⁺ T cells from 57 patients with CHB categorised based on their HBsAg (B) and HBcrAg (C) levels (total-specific T cell response: sum of 14 peptide pools). Statistical significance between each of the two groups was tested by Mann-Whitney test for non-parametric data and by unpaired t-test for parametric data (B–C). *p<0.05; **p<0.01; ***p<0.001. CHB, chronic hepatitis B; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; IFN, interferon; TNF, tumour necrosis factor.

Figure 3

HBV-specific CD4⁺ and CD8⁺ T cell responses in different age groups. HBV-specific CD4⁺ and CD8⁺ T cell responses following 10-day in vitro stimulation with HBV overlapping peptide (OLP) pools grouped according to the patients’ age. (A) Representative flow cytometry plots of HBV core-specific IFN-γ⁺/TNF⁺ CD4⁺ T cell responses of two patients. (B) Heat map showing mean of frequencies of total cytokine⁺ HBV-specific CD4⁺ and CD8⁺ T cells (age groups: 18–39, n=25; 40–49, n=17; ≥50, n=15). (C) IFN-γ⁺ HBV-specific CD4⁺ and CD8⁺ T cells of different age groups. (D) Correlation analyses of age with frequencies of total IFN-γ⁺ CD4⁺ and CD8⁺ T cells. (E) Mean percentages of multifunctional CD4⁺ and CD8⁺ T cell responses after in vitro expansion with total HBV OLPs (sum of 14 OLP pools). Statistical significance between each of the two groups was tested by Mann-Whitney test for non-parametric data and by unpaired t-test for parametric data (B–C). *p<0.05; **p<0.01. IFN, interferon; TNF, tumour necrosis factor.

Figure 4

Functional and phenotypical characteristics of HBV core₁₈-specific and HBV pol₄₅₅-specific CD8⁺ T cells in patients with CHB. HBV-specific CD8⁺ T cell responses following 10-day in vitro stimulation with core₁₈ or pol₄₅₅ peptides in HLA-A*02 positive patients with CHB. (A) HBV-specific IFN-γ⁺ CD8⁺ T cell responses categorised based on HBsAg or HBcrAg levels. (B) Enriched ex vivo frequencies of naïve, T_CM, T_EM and T_EMRA populations and expression of PD-1, KLRG1, CD39, Eomes, Tbet, PD-1 CD127 and TCF1 by HBV core₁₈ and pol₄₅₅-specific CD8⁺ T cells. (C) Radar plot depicting the mean percentage of ex vivo HBV core₁₈ and pol₄₅₅-specific CD8⁺ T cell expressing different markers obtained from patients with CHB categorised based on HBsAg and HBcrAg levels. Statistical significance was tested by Wilcoxon test and Mann-Whitney test for non-parametric data and by unpaired t-test for parametric data (A–C). ns, not significant; *p<0.05; **p<0.01; ***p<0.001. CHB, chronic hepatitis B; DCM, dead cell marker; FSC, forward scatter; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HLA, human leukocyte antigen; IFN, interferon; PD-1, programmed cell death protein 1; pol, polymerase; SSC, side scatter; TCF1, transcription factor 1; T_CM, central memory T cell; T_EM, effector memory T cell; T_EMRA, terminally differentiated effector memory T cell; TNF, tumour necrosis factor;.

Figure 5

Impact of PD-L1 blockade on HBV-specific T cell responses in patients with CHB. HBV-specific CD4⁺ and CD8⁺ T cell responses following 10-day in vitro stimulation with HBV overlapping peptide (OLP) pools with or without the addition of anti-PD-L1 antibody. (A) IFN-γ expression by HBV-specific CD4⁺ and CD8⁺ T cells of patients with different HBsAg level. Open circles mark three patients with HBsAg of 1000–10 000 IU/mL with the highest T cell response who showed all HBcrAg levels of <3 Log U/mL. (B) Correlation analyses of HBsAg residuals with difference or log2 fold change of HBV-specific CD4⁺ and CD8⁺ T cell responses after expansion in presence of anti-PD-L1. (C) IFN-γ expression by HBV-specific CD4⁺ and CD8⁺ T cells of patients with different levels of HBcrAg. Total-specific T cell response: sum of all 14 OLP pools. Statistical significance was tested by Wilcoxon test for non-parametric data and by paired t-test for parametric data (A, C). *p<0.05; **p<0.01; ***p<0.001. CHB, chronic hepatitis B; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; IFN, interferon; PD-L1, programmed cell death ligand 1.

Figure 6

Impact of PD-L1 blockade on HBV core₁₈-specific and pol₄₅₅-specific CD8⁺ T cell responses. HBV-specific CD8⁺ T cell responses following in vitro stimulation with core₁₈-specific or pol₄₅₅-specific peptides in presence or absence of anti-PD-L1 antibody. (A) Patients with CHB with HBsAg <100 or ≥10 000 IU/mL and (B) patients with CHB with HBcrAg <3 or >3 Log U/mL. Statistical significance was tested by Wilcoxon test. ns, not significant; *p<0.05; **p<0.01. CHB, chronic hepatitis B; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; IFN, interferon; PD-L1, programmed cell death ligand 1; pol, polymerase; TNF, tumour necrosis factor.