Unveiling Cellular Heterogeneity and Liver Pathophysiology in Liver Transplant Recipients through Multiplex Image Analysis

Liver transplantation represents the only curative treatment for patients with end-stage liver disease. Despite advances in immunosuppressive therapies and surgical techniques, post-transplant complications such as graft dysfunction, fibrosis, and rejection remain major clinical challenges. This study aimed to develop a multiplex immunofluorescence staining and analysis protocol for investigating liver graft biopsies, offering high-resolution insights into cellular and spatial tissue dynamics. Using a cohort of seven liver transplant recipients, biopsy samples were analyzed to profile histological changes of the common post-transplant complications fibrosis, drug-induced liver injury (DILI), and acute T-cell mediated rejection (TCMR). The developed protocol enabled detailed assessment of hepatocytes, cholangiocytes, macrophages, and immune cells across different disease phenotypes using key markers such as yH2Ax, Ki67, p62, TGF-beta, and cleaved caspase 3 to assess proliferation, apoptosis, and tissue damage. Spatial clustering analysis revealed distinct tissue regions associated with specific pathological conditions, particularly identifying areas enriched with proliferating and autophagy-impaired hepatocytes in TCMR. Additionally, spatial and neighboring analysis of CD3+lymphocytes was successful in identifying their crucial role in inflammatory processes during acute rejection and identifying immune- and non-immune mediated origins for tissue damage in DILI. This workflow was validated by applying it to protocol biopsies from clinically healthy patients, revealing early signs of graft injury to be later identified as chronic rejection. The study demonstrates that multiplex staining combined with spatial analysis can provide a more granular understanding of post-transplant liver pathology, offering potential for more personalized immunosuppressive therapies and earlier intervention in graft dysfunction.

Publication History

Article published online:
20 January 2025