Virus Infection Of Hepatic Sinusoidal Endothelial Cells Regulates Hepatic T Cell Recruitment And Activation

Background/Objective:

Animal studies suggest that endothelial cells and not hepatocytes are the site of cytomegalovirus (CMV) latency and reactivation in the liver and the source of secondary viral spread. The aim of this study was to investigate, whether CMV infection modulates the ability of the liver to recruit and activate T cells on the level of hepatic sinusoidal endothelial cells (HSEC).

Materials and Methods:

Recombinant endotheliotropic eGFP-labeled CMV was propagated and purified by ultracentrifugation in tartrate/glycerol gradients. Primary HSEC were isolated from explanted livers, grown to confluence and infected with CMV. Phenotyping of infected HSEC was performed by flow cytometry. T cell subsets were perfused over HSEC monolayers 24 hours after infection under constant flow simulating physiological shear stress. Adhesion and transmigration recorded using phase contrast microscopy. Static transmigration assays through HSEC were used to study selective recruitment and post-migrational phenotype of T cells.

Results:

CMV infection of HSEC resulted in a specific endothelial phenotype different from cytokine-activated endothelium with upregulated surface ICAM-1, dose- and time-dependent secretion of CXCL10 and down-regulation of co-stimulatory molecules. Under flow, adhesion and transendothelial migration of allogeneic resting and activated CD4 T cells, regulatory T cells (Treg) and of virus-specific T cell memory clones were increased in a ICAM-1 and CXCL10-dependent manner. Consistent with these findings, recruited T cells showed a higher expression of CXCR3, CCR5, CD2 and CD11a/CD18 (LFA-1). After migration CD4 T cells demonstrated an effector memory phenotype (CD45RO+CCR7-CD62L-) with increased expression of makers of recent activation (CD69, CD71) and strong IFN-gamma release, which was mediated in part via CD2-LFA3-interaction. Immunhistochemistry from liver allografts with CMV infection confirmed upregulated CD69 expression on liver infiltrates.

Increased CD25-expression of transmigrated CD4 T cells was not a result of recent activation but primarily due to increased recruitment of CD25hiCD127lowFoxP3+ Treg with a suppressive phenotype after migration. In line with these finding transmigrated CD4 T cells demonstrated a strong upgregulation of Th1 (INFG, TNF, SOCS1, TBX21) genes as well as Treg-associated genes (IL2RA, FOXP3) after migration.

Conclusions:

Our data show that viral infections can regulate recruitment and post-migrational fate of several T cell subsets on the level of the hepatic endothelium even in the absence of professional antigen-presenting cells. By promoting adhesion and migration of effector and suppressive T cell subsets, infection of sinusoidal endothelium sets the stage for several key features of hepatic CMV infection: immune infiltration, endothelial damage and CMV persistence.