Natural Killer Cells Induce CD8⁺ T Cell Dysfunction via Galectin-9/TIM-3 in Chronic Hepatitis B Virus Infection

The antiviral response of natural killer (NK) cells and CD8⁺ T cells is weak in patients with chronic hepatitis B (CHB) infection. However, the specific characteristics of these cells and the association between NK cells and CD8⁺ T cell dysfunction is not well known. In this study, higher galectin-9 (Gal-9) expression was observed in circulating NK cells from CHB patients than from healthy controls and was found to contribute to NK cell dysfunction. In addition, circulating CD8⁺ T cells showed obvious dysfunction and overexpressed TIM-3, the natural receptor of Gal-9, during active CHB infection. Gal-9⁺ and Gal-9^- NK cells from active CHB patients were sorted and cocultured with autologous CD8⁺ T cells. The proportion of tetramer⁺CD8⁺ T cells and the cytokines production of CD8⁺ T cells were lower after cocultivation with Gal-9⁺ than with Gal-9^- NK cells. We showed that in vitro depletion of NK cells increased circulating hepatitis B virus (HBV)-specific CD8⁺ T cell responses in patients with active CHB infection. Because Gal-9 is increased in the serum of CHB patients, CD8⁺ T cells were sorted and cultured with exogenous Gal-9, resulting in lower IFN-γ, TNF-α, CD107a, and granzyme B levels, decreased expression of the activation receptor CD69, increased expression of TIM-3, and a high percentage of early apoptotic CD8⁺ T cells. Blocking Gal-9 or TIM-3 in vitro in a culture of peripheral blood mononuclear cells (PBMCs) stimulated with HBV peptide from active CHB patients restored CD8⁺ T cell function. However, blocking Gal-9 in vitro after removal of NK cells from PBMCs did not rescue CD8⁺ T cells exhaustion. Furthermore, NK and CD8⁺ T cells from active CHB patients were sorted and cocultured in vitro, and the exhaustion of CD8⁺ T cells were alleviated after blocking Gal-9 or TIM-3. In summary, overexpression of Gal-9 on NK cells, which interacts with TIM-3⁺CD8⁺ T cells and likely contributes to antiviral CD8⁺ T cell dysfunction, may be a potential target for the treatment of CHB patients.