Hepatitis B virus (HBV) core antigen-specific regulatory T cells confer sustained remission to anti-HBV therapy in chronic hepatitis B with acute exacerbation

Abstract

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are thought to be the result of breakdown of immune tolerance on the natural history of chronic hepatitis B virus (HBV) infection. Immune tolerance to HBV maintained in CH-B patients without hepatitis is under the control of the host's forkhead box p3-expressing regulatory T cells (Tregs). Its breakdown mimics the occurrence of autoimmune diseases. Severe AEs may lead to liver decompensation and mortalities. Consequently, AEs are currently the major therapeutic targets in patient treatment. In this study, we employed the SYFPEITHI scoring system to identify epitopes on HBV core antigen (HBcAg) for the construction of human leukocyte antigen class II tetramers to measure HBcAg-specific Treg frequencies (Tregf). Upregulation of Treg gene profiling accompanied by increased HBcAg-specific Tregf was detected in AE patients with sustained remission (SR) to anti-HBV therapy. Depletion of Tregs from peripheral blood mononuclear cells enhanced proliferation to HBcAg. HBcAg-specific Treg clones inhibited the killing capacity of cytotoxic T lymphocyte clones in an antigen-independent manner. A greater posttherapy increase in HBcAg-specific Tregf correlated with a higher SR rate to anti-HBV therapy. These results suggest that HBcAg-specific Tregs function as suppressor effectors and confer SR to anti-HBV therapy.

Introduction

Hepatitis B virus (HBV) infects more than 350 million people worldwide. All infected subjects are at risk of developing adverse sequelae, such as acute hepatitis and acute fulminant hepatitis, which may lead to liver failure and mortality. Some HBV infections may evolve into chronic persistent infection with a protracted lifelong clinical course that may flare up or develop acute exacerbations (AEs) spontaneously. Severe AE can be associated with massive or submassive hepatic necrosis analogous to acute fulminant hepatitis. Long-term chronic hepatitis B (CH-B) may progress to liver cirrhosis and hepatocellular carcinoma [1], [2], [3], [4], [5], [6]. Since pilot clinical trials of interferon-α (IFN-α) therapy for CH-B patients were reported in 1976 [7], [8], attempts to treat CH-B have exhausted tremendous medical expenditure worldwide. Disappointingly, CH-B patients have so far responded ineffectively, with a low sustained remission (SR) rate to all types of currently available anti-HBV treatment modalities [9], [10], [11], [12], [13]. The need for immediate anti-HBV therapy with nucleoside analogues for patients developing life-threatening AEs, specifically cancer patients receiving chemotherapy and organ transplant recipients treated with immunosuppressants, has reached a consensus [10], [11], [12]. This strategy has achieved great success in rescuing a large proportion of patients from hepatic failure and mortality [10], [11], [12], [13], [14], [15]. Furthermore, this approach seems to prolong survival among decompensated cirrhotic patients [16]. However, all current oral anti-HBV nucleoside analogues can suppress virus replication via the inhibition of HBV DNA polymerase, but are unable to totally remove the covalently closed circular DNA of the HBV genome to achieve complete viral clearance [5], [10], [11], [12], [13], [17], [18]. Consequently, virus breakthroughs, biochemical relapses, and/or HBV e antigen (HBeAg) reversion in serum are frequently encountered when the use of these drugs is stopped. Moreover, long-term use of nucleoside analogues may select for drug-resistant mutant viruses, such as tyrosine-isoleucine-aspartate-aspartate and/or tyrosine-valine-aspartate-aspartate mutations on the tyrosine–methionine–aspartate–aspartate motif of the HBV DNA polymerase and lead to therapeutic failure [5], [10], [11], [12], [13], [19]. Thus, the mechanism by which chronic hepatitis B patients achieve SR or viral clearance after anti-HBV therapy with nucleoside analogues remains unclear [5], [12], [13]. Once the treatment is initiated, lifelong HBV suppression may be necessary [20].

HBV is not directly cytopathic and the immune response of the host appears to mediate the hepatocellular injury and subsequent viral clearance [2], [3], [5], [13], [21]. Accumulating evidence indicates that the immune response of hepatitis B patients may influence the outcome of anti-HBV therapy [22], [23], [24], [25], [26], [27], [28]. We showed that AEs of CH-B are accompanied by increased T-cell responses to hepatitis B core and e antigens (HBcAg/HBeAg) [29] and that HBcAg and/or HBeAg may represent important “targets” for immune-mediated viral clearance [30], [31].

Evidence shows that naturally arising forkhead/winged helix transcription factor box p3 (Foxp3)-expressing CD4⁺CD25^high regulatory T cells (Tregs), namely activated CD4⁺ T cells with expression of interleukin-2 (IL-2) receptor α chain [32], play an important role in the regulation of autoimmune diseases [33], but whether Tregs play a role in the control of infectious diseases and persistent viral infections remains undetermined [34], [35], specifically in patients with chronic viral hepatitis B [36], [37], [38] and C [39]. An immune tolerance response to these viruses is considered important for disease progression [3], [5], [13]. We have shown that HBcAg-specific Tregs play a crucial role in modulating spontaneous AEs on the natural history of perinatally acquired chronic HBV infection [40]. Whether HBcAg-specific Tregs play a role in influencing the outcome of anti-HBV therapy remains unclear. Because AEs are critical events in the natural history of chronic HBV infection and are major therapeutic targets in the treatment of CH-B [1], [2], [3], [5], [10], [11], [12], [13], [14], [15], in the present study we aim to investigate the role of HBcAg-specific Tregs during anti-HBV therapy for CH-B patients with AE.