Comparison of different inhibitory pathways in hepatitis B virus-specific CD8+ T cell exhaustion

Objectives: Chronic hepatitis B virus (HBV) infection is characterized by a gradual loss of virus-specific CD8+ T cell functionality. Exhausted HBV-specific CD8+ T cells fail to control viral replication, leading to continued liver inflammation, cirrhosis and development of hepatocellular carcinoma. This deprivation of CD8+ T cell effector functions is mediated by multiple mechanisms, such as inhibitory receptors expressed on the cell surface (e.g. PD-1), inhibitory molecules (e.g. IL-10), lack of CD4+ T cell help or direct elimination by natural killer (NK) cells. Previous studies have shown that a blockade of each single inhibitory pathway or an addition of stimulatory cytokines (e.g. IL-12) can at least partially restore HBV-specific CD8+ T cell effector function or survival. It was the aim of this study to compare their relative single and combined impact on HBV-specific CD8+ T cell exhaustion systematically in the same patient cohort. Methods: Peripheral blood mononuclear cells from 13 patients with chronic HBV infection were isolated and cultivated after stimulation with HBV core peptide in different conditions. Specifically, a blockade with anti-PD-L1 or anti-IL-10, a depletion of NK cells and an addition of IL-12 was performed. After 2 weeks, proliferation and cytokine production of CD8+ T cells were measured by flow cytometry. Results: The blockade of the different inhibitory pathways showed an interindividual variability. Altogether, the proliferation of virus-specific CD8+ T cells increased compared to peptide stimulation alone after blockade of PD-L1, IL-10 or depletion of NK cells. Addition of IL-12 had a rather inhibitory effect on virus-specific CD8+ T cell frequency, consistent with the finding that it strongly induced the expansion of NK cells. Furthermore, anti-IL-10 led to the strongest effect on CD8+ T cell proliferation. Production of IFN-gamma was only restored by blockade of IL-10 and depletion of NK cells. While anti-PD-L1 had no effect on cytokine secretion, the addition of IL-12 only resulted in an elevated but unspecific bystander production of IFN-gamma. Conclusion: Our data show that the immunosuppressive cytokine IL-10 may play a key role in HBV-specific CD8+ T cell exhaustion. Blockade of IL-10 dominantly enhanced virus-specific CD8+ T cell proliferation and cytokine production, pointing it out as a potential clinical target for future immunotherapeutic approaches.