Investigating HCMV-driven adaptive natural killer (NK)-cell reprogramming as a driver of immune dysregulation and liver fibrosis in chronic HBV

• Applicant: Dr. Smaranda Gliga
• Subject Area: Clinical Infectiology and Tropical Medicine
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 574054338

Chronic hepatitis B (HBV) affects more than 250 million people worldwide. Current nucleos(t)ide analogues suppress replication but rarely achieve a functional cure because a stable DNA form (cccDNA) stays hidden inside hepatocyte nuclei. In many regions, people with chronic HBV infection are also infected with human cytomegalovirus (HCMV). HCMV reshapes the innate immune system by expanding a distinct “adaptive” natural-killer (NK)-cell subset that expresses NKG2C and CD57 but lacks the signalling adaptor FcεRIγ. In our Düsseldorf cohort these adaptive NK cells rise in parallel with HBV viraemia and liver stiffness. Although adaptive NK cells display abundant surface CD95 (Fas), initial stimulation with soluble recombinant Fas-Ligand induced virtually no caspase activation. This muted response is consistent with the previously described up-regulation of the anti-apoptotic protein BCL-2 within these cells. Together, the findings point to a rewiring of the Fas axis—from a classical death receptor toward survival- or inflammation-biased signalling. This 12-month pilot project will define how HCMV-driven adaptive NK cells influence immune dysregulation and fibrosis in chronic HBV by addressing three linked objectives: 1. Map the CD95 molecular checkpoint. Adaptive and canonical NK cells from all HBV disease stages will be analysed side-by-side. High-parameter phospho-flow cytometry will quantify active caspase-3/7 (cell death) together with NF-κB and MAPK phosphorylation (inflammatory survival). Complementary Western blots for c-FLIP and BCL-2 will clarify why Fas ligation fails to induce apoptosis. 2. Dissect effector mechanisms against hepatocytes. Flow-sorted adaptive versus canonical NK cells will be co-cultured with HBV-replicating HepaRG cells and primary human hepatocytes. Selective inhibitors of perforin/granzyme, TRAIL or Fas will reveal which killing routes dominate and whether blocking Fas signalling spares hepatocytes from injury. End-points include NK degranulation, granzyme release and target-cell viability. 3. Chart transcriptional programs across the fibrosis spectrum. An existing single-cell RNA-seq atlas (around 30 000 NK cells from controls, chronic HBV infection and chronic HBV hepatitis) will identify gene signatures linked to cytotoxicity, survival and exhaustion. Key surface or intracellular proteins emerging from the analysis will be validated in an independent cohort with a 16-colour flow panel. By integrating HCMV stratification, Fas signalomics and direct hepatocyte assays within a rigorously phenotyped HBV cohort, the study will unravel the molecular wiring that positions adaptive NK cells anywhere between viral containment and tissue damage, an essential knowledge for future attempts to modulate immunity in chronic HBV.

• DFG Programme: Research Grants