Study of innovative markers associated with hepatitis B virus pathogenesis to guide the development of personalized medical approaches

Hepatitis B virus is a non-cytopathic virus that generally leads to a self-resolving acute infection. Despite this, in unvaccinated children, as well as immune deficient patients, the infection become chronic with conventional NK cells playing a central role, mediating the inactivation of T cells through the upregulation of inhibitory ligands. Among these, Programmed Cell Death Ligand 1 (PD-L1) is one of the most well characterized. Here we will mainly focus on the characterization of HBV-specific T cells, NK cells, and their interactions as well as the impact of PD-L1 on this crosstalk. Two cohorts of chronic HBV (CHB) patients were cultured and stimulated with HBV core overlapping peptide pools (OLPs) for 8 days in AB media. NK cells were depleted from PBMCs via magnetic cell sorting (MACS). Briefly non-NK cells were labelled with an antibody cocktail and passed through a magnetic column, resulting in two distinct population. The retained cells resulted in the positive fraction, which represents the NK depleted PBMCs, whereas the flowthrough resulted in the negative isolated fraction, which represents the isolated NK cells. NK-depleted PBMCs so obtained were stimulated with OLPs for 7 days prior to restimulation with the same OLPs and flow cytometry staining. In selected experiments NK cells were activated with IL-2, IL-12, IL-15, IL-18, treated or not with anti-PD-L1 for 1 hour and cocultured at physiological ratios (~10% of total PBMCs) prior to the restimulation. Intra cytokine staining were performed to evaluate the expansion of HBV-specific T cells. Activated NK cells significantly enhanced the functionality and frequency of HBV-specific compared to bulk-stimulated PBMCs. This effect was preserved in the presence of PD-L1 blockade on NK cells. Patients classified as high responders, based on the level of HBV-specific T cells on upon antigenic stimulation, showed a complete or partial loss of HBV-specific T cell response upon NK depletion, whereas low responders did not display this behaviour. This loss is then partially restored upon addition of activated NK cells regardless of PD-L1 blockade. Moreover, the level of NK activation upon cytokine stimulation negatively correlated with the level of HBV-specific CD8 T cells. Our findings demonstrate that HBV-specific T cells form CHB patients exhibit limited responsiveness to HBV core OLPs stimulation, sign of a significant exhaustion. While NK cells depletion does significantly alter T cell activation, cytokine activated NK cells has been found to be determinant to boost HBV-specific T cells response, particularly in patients with higher exhaustion level. Activated NK cells exhibit increased PD-L1 and inhibitory yet activated phenotype. Overall, these findings suggests that NK cells modulate HBV-specific T cell function in CHB and paved the way for further investigation, to better depict the crosstalk between NK and HBV-specific T cells.