Increased peripheral CD4+ regulatory T cells persist after successful direct-acting antiviral treatment of chronic hepatitis C

Abstract

Background & aims: CD4⁺ regulatory T cells (Tregs) expand during chronic hepatitis C virus (HCV) infection, inhibit antiviral immunity and promote fibrosis. Direct-acting antiviral agents (DAA) have revolutionized HCV therapy. However, it is unclear if Tregs are normalized after DAA-induced HCV elimination.

Methods: We analyzed Tregs before (baseline), at end of therapy (EOT), 12 and 24weeks (SVR12, SVR24) and long-term (51±14weeks) after EOT in 26 genotype-1-infected patients who were successfully treated with sofosbuvir (SOF) plus interferon (IFN)/ribavirin (n=12) and IFN-free DAA regimens (SOF plus daclatasvir or simeprevir; n=14). Frequency, phenotype and suppressor function of peripheral Foxp3⁺ CD25⁺ CD4⁺ T cells were studied by multi-color flow cytometry and co-culture inhibition assays.

Results: Frequencies and activation status of Foxp3⁺ CD25⁺ CD4⁺ T cells remained elevated above those of normal controls in both treatment groups even long-term after HCV elimination. Co-culture assays indicated a dose-response relationship for functional inhibition of autologous CD4⁺ effector T cells and confirmed that activation of Tregs remained largely unchanged over the observation period. Unlike IFN-free regimens, SOF plus IFN/ribavirin induced a transiently increased frequency of Foxp3⁺ CD25⁺ CD4⁺ T cells at EOT (5.0% at baseline to 6.1% at EOT; p=0.001). These Foxp3⁺ CD25⁺ CD4⁺ T cells co-expressed the activation markers glycoprotein A repetitions predominant (GARP; p=0.012) and tumor necrosis factor receptor superfamily, member 4 (OX-40; p=0.001) but showed unchanged in vitro inhibitory activity.

Conclusion: Although IFN-based DAA therapy induced transient expansion of activated Foxp3⁺ CD25⁺ CD4⁺ T cells, neither IFN-based nor IFN-free DAA regimens normalized frequencies and activation status of Tregs one year after viral elimination. Persistence of immunosuppressive Tregs may thus contribute to complications of liver disease even long-term after HCV cure.

Lay summary: In chronic hepatitis C virus (HCV) infection, CD4⁺ regulatory T cells (Tregs) can reduce antiviral immune responses, promote liver fibrosis and may increase the risk for liver cancer, because they gradually expand during disease. Modern direct-acting antiviral agents (DAA) can "cure" hepatitis C in almost all treated patients. However, our study shows that DAA do not normalize the increased frequency and activation status of Tregs even long-term after HCV elimination. Tregs may persistently modulate functions of the immune system even after "cure" of hepatitis C.

Keywords: CD4(+) regulatory T cells; Chronic hepatitis C virus infection; Directly acting antivirals; Tim-3/galectin-9 pathway.