Exploring the liver microenvironment following successful therapy for HCV: gene expression profiling and residual T cells infiltration

Daniel E Millian¹Esteban Arroyave¹Timothy Wanninger¹Santhoshi Krishnan^2,3Daniel Bao⁴Jared R Zhang⁴Arvind Rao^3,5Heidi Spratt⁶Monique R Ferguson⁷Vincent Chen⁸Heather L Stevenson¹Omar A. Saldarriaga^1*

• ¹Department of Pathology, University of Texas Medical Branch at Galveston, Galveston, United States
• ²Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, United States
• ³Department of Electrical and Computer Engineering, Rice University, Houston, United States
• ⁴John Sealy School of Medicine, University of Texas Medical Branch at Galveston, Galveston, United States
• ⁵Department of Computational Medicine and Bioinformatics, Department of Radiation Oncology, Department of Biostatistics, Department of Biomedical Engineering, University of Michigan, Ann Arbor, United States
• ⁶Department of Biostatistics and Data Science, University of Texas Medical Branch at Galveston, Galveston, United States
• ⁷Department of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston, United States
• ⁸Department of Internal Medicine, University of Michigan, Ann Arbor, United States

Background & aims. Direct-acting antivirals (DAAs) revolutionized hepatitis C (HCV) treatment. Yet, some patients present persistent inflammation and still face adverse outcomes, including cirrhosis and hepatocellular carcinoma, despite achieving sustained virologic response (SVR). Approach & Results. This study examined liver biopsies from 22 patients before and after DAA treatment to assess gene and protein expression changes linked to disease progression. Pre-treatment, patients exhibited two distinct profiles: one characterized by high pro-inflammatory and antiviral gene expression (pre-hot), and another with weaker expression (pre-cold). Patients with pre-hot profiles were initially associated with poor outcomes (OR = 14.0, 95% CI: 1.31–178.5; p = 0.049), but this lost significance after adjustment for baseline disease severity (adjusted OR = 8.04, 95% CI: 0.18–2123.07; p = 0.328). Baseline MHAI scores (OR = 1.90, 95% CI: 0.72–6.34) and fibrosis stage (OR = 1.65, 95% CI: 0.44–9.97) trended toward predicting poor outcomes but were not significant. Post-treatment, liver enzymes decreased, inflammatory scores improved, and type I interferon pathways were restored, yet 14 of 17 patients (82.3%, 95% CI: 64.2–100%) retained persistent lymphocytic infiltrates. In parallel, spectral imaging further revealed post-treatment shifts in macrophage populations, with a significant decrease in inflammatory subsets (CD14+, CD14+/Mac387+, p<0.05) and an increase in anti-inflammatory subsets (CD16+, CD16+/CD163+, CD16+/CD68+, p<0.05). Analysis of T cell–related marker expression before and after treatment shows that mixed lymphocytic infiltration (CD3+, CD4+, CD8+, CD45RO+) persists within the liver despite viral clearance, with high inter-patient variability likely limiting statistical significance. Conclusions. Even after achieving SVR and normalization of gene expression, the liver retained a heterogeneous T cell infiltrate, suggesting that persistent immune activity could continue to influence the risk of adverse outcomes. Lay Summary: Direct-acting antiviral (DAA) therapy has dramatically improved the treatment of chronic HCV, making it curable for most people. This study determined gene and protein expression differences in the liver before and after treatment of HCV. Pre-hot profiles and higher baseline inflammation or fibrosis trended toward poor outcomes, but none remained statistically significant after adjustment. Despite SVR, patients showed persistent heterogeneous lymphocytic infiltrates, suggesting that persistent immune activity could continue to influence the risk of adverse outcomes.