Lymphocyte Exhaustion in Hepatocellular Carcinoma: A Dynamic Evolution Across Disease Stages

CARLA FUSTER-ANGLADA^1,2,3Josep Corominas^2,3Aida Marsal^2,3Neus Llarch^2,3,4,5Gemma Iserte^2,3,4Marco Sanduzzi Zamparelli^2,3,4Alejandro Forner^2,3,4,5,6Joana Ferrer^2,3,5,7Victor Holguin^2,7Albert Morales^2,3,8Carolina Saavedra⁹Maria Reig^2,3,4Loreto Boix^2,3*Montserrat Marí^2,3,8*Alba Díaz^1,2,3,5

• ¹HOSPITAL CLÍNIC OF BARCELONA, PATHOLOGY DEPARTMENT, Barcelona, Spain
• ²Barcelona Clínic Liver Cancer (BCLC) group. IDIBAPS, Barcelona, Balearic Islands, Spain
• ³Network Biomedical Research Center (CIBERehd), Madrid, Madrid, Spain
• ⁴Liver Unit, Hospital Clinic of Barcelona, Barcelona, Catalonia, Spain
• ⁵University of Barcelona, Barcelona, Catalonia, Spain
• ⁶Hospital Clinic of Barcelona, Barcelona, Catalonia, Spain
• ⁷Surgical Area, Hospital Clinic of Barcelona, Barcelona, Balearic Islands, Spain
• ⁸Department of Molecular and Cellular Biomedicine, IIBB-CSIC, IDIBAPS, Barcelona, Spain
• ⁹Medical Statistics Core Facility, Institut D'Investigacions Biomédiques August Pi i Sunyer 17 (IDIBAPS), Hospital Clinic, Barcelona, Spain

Background: Immune checkpoint inhibitors (ICIs) have transformed cancer therapy. However, their efficacy in hepatocellular carcinoma (HCC) is limited, highlighting the need to further explore immune microenvironments and novel biomarkers. This study examined lymphocyte populations and immune checkpoint dynamics in early, advanced, and post-progression HCC to better understand immune dynamics in HCC and to help identify predictive biomarkers and immune modulation strategies.Methods: Tumoral and non-tumoral liver tissues were analyzed from HCC patients across early (n=25), advanced (n=22), and advanced-beyond-progression (n=15) stages. Lymphocyte profiling was performed using immunohistochemistry and flow cytometry, focusing on NK cells, T cells, and immune exhaustion markers. An exploratory analysis of this profile and its association with disease progression and recurrence was conducted.Results: Early HCC exhibited higher liver-resident NK (lrNK) cell densities in non-tumor regions, which diminished with advanced stages. Increased CD56+ cell infiltration in the tumor core was associated with recurrence. Tumor region showed elevated PD-1, NKG2A, and CD39 expression in CD4+ and CD8+ T cells, indicating progressive immune exhaustion. Advanced HCC stages demonstrated altered NK cell phenotypes, with reduced cytotoxic activation (CD16) and increased residency markers (CXCR6/CD69) in tumor-isolated lymphocytes. Conclusions: Progressive immune exhaustion and dysregulation of lrNK and T cells in HCC reflect the evolution of the immune microenvironment originating in the tumor and leaking into the nontumoral liver, progressively diminishing the cytotoxic capacity of NK and T cells. CD56+ cell density and immune checkpoint profiles are potential biomarkers for therapeutic response and disease monitoring, underscoring the need for personalized immunotherapy strategies.