A Human Vascularized Liver Tissueoid-on-Chip Platform for Dissecting T cell- and Antibody-Mediated Allograft Rejection

Introduction: Allograft rejection remains a major barrier to long-term liver transplant survival. T cell-mediated rejection (TCMR), characterized by alloimmune T cell infiltration and cytotoxicity, and antibody-mediated rejection (ABMR), driven by donor-specific antibodies (DSA) targeting graft vasculature, represent distinct but equally consequential injury mechanisms. Mechanistic studies of both rejection pathways are limited by the lack of physiologically relevant human models that recapitulate immune-tissue interactions within native-like hepatic microarchitecture. We therefore developed a human vascularized liver tissueoid-on-chip (LToC) platform to model the structural, molecular, and inflammatory hallmarks of both TCMR and ABMR.
Methods: LToCs were generated using primary human intrahepatic portal vein endothelial cells and hepatic progenitor cells maintained under continuous perfusion for 23 days to establish a vascularized liver microenvironment. Baseline characterization confirmed organized hepatic (albumin+, CK18+, CK19+, HNF4α+) and vascular (CD31+) compartments with HLA-I expression. For TCMR modeling, allogeneic T cells (0.1, 1, and 10 x 103 cells/mL) were perfused into LToCs for 24 hours. For ABMR modeling, LToCs were treated with control media, control serum, or DSA-positive serum. Outcomes included cell viability, perfusion assays, immunofluorescence (IgG, C4d, CD31, CD4, CD8, granzyme B, HLA-I, HLA-II), and soluble markers of inflammation and tissue injury.
Results: In the TCMR model, allogeneic T cell perfusion led to pronounced perivascular and parenchymal T cell infiltration, with dose-dependent vascular injury. CD8+ T cells colocalized with dead cells, providing direct evidence of cytotoxic killing. T cell exposure triggered upregulation of HLA-I and HLA-II across endothelial and parenchymal compartments, with CD4+ T cells preferentially localizing to regions of strong HLA-II expression. Granzyme B expression in both compartments confirmed cytotoxic effector activity.

In the ABMR model, DSA exposure induced widespread cytotoxicity across the vascular and parenchymal compartments, leading to complete loss of vascular perfusion and architecture. DSA bound extensively to endothelial surfaces, colocalizing with CD31 and resulting in diffuse C4d deposition. High-magnification imaging revealed fragmented, discontinuous endothelium coated with DSA and C4d, directly linking alloantibody binding to complement-mediated endothelial destruction. Cytokine analysis revealed significant increases in proinflammatory mediators, including MCP-3, CXCL9, IL-6, IL-8, ICAM-1, and VCAM-1.

Conclusion: Our human vascularized LToC platform replicates the key pathological features of both ABMR and TCMR, including DSA binding, complement deposition, endothelial injury, immune cell infiltration, and HLA upregulation. This dual-capability model provides a translational tool for studying humoral and cellular alloimmune mechanisms and for testing targeted rejection therapies in the context of human liver transplantation.