Abstract
The liver is a unique lymphoid organ whose microenvironment is biased towards tolerance induction. We previously found that a proportion of CD4+ autoreactive recent thymic emigrants (RTEs) retained in the liver after thymic egress and acquired IL-10 producing capability. To investigate the tolerance of these liver persisting CD4+ RTEs in more detail and to study the liver stromal cell types that facilitate the tolerogenic changes in young T cells, the phenotype and function of liver RTEs were further characterized and the impact of liver sinusoidal endothelial cells (LSECs) and Kupffer cells on RTEs were examined using an in vitro co-culture system. More than 70% of CD4+ CD44hi RTEs in the liver acquired Foxp3-LAG3+ CD49b− regulatory phenotype and function. But higher ratio of apoptosis with enhanced FasL and Bim expression was also found in these CD4+ liver RTEs when compared to those in the lymph nodes and spleen. LSECs played an important role in RTEs’ acquisition of tolerogenic and regulatory phenotype. These results indicate an important role of liver microenvironment in enforcing peripheral tolerance to CD4+ thymic emigrants against self- and gut-derived antigens.
Highlights
Nodes, RTEs in the liver produce higher level of IL-10 upon activation and respond poorly to IL-7-induced survival, indicating that these RTEs acquire tolerogenic properties in the liver microenvironment
To investigate the mechanisms of hepatic tolerogenicity to autoreactive young T cells that just emigrate from the thymus, the current study characterized the phenotype and function of CD4+ liver RTEs in more details and studied the impact of liver KCs and LSECs on RTEs’ acquisition of tolerogenic phenotype
The addition of IL-7 reduced the apoptosis of T cells by half in the wells co-cultured with splenic stromal cells whereas it did not decrease the apoptosis of T cells co-cultured with liver non-parenchymal cells (NPCs) (Fig. 3B). These results indicate the important roles of liver NPCs in inducing IL-10 expression and reducing IL-7 responsiveness in RTEs
Summary
Mouse TGF-β Elisa Kit was purchased from eBioscience (San Diego, CA, USA). For isolation of stromal cells in the spleen, spleens from C57BL/6 mice (CD45.2+) were mechanically disrupted and incubated with 2 mg/ml collagenase IV and 0.5 mg/ml DNase in 8 ml PBS for 30 minutes at 37 °C with constant rotation (200 rpm). For the in vitro suppression assay, CFSE labeled CD4+ CD8− CD25− CD62L+ CD44lo naive T cells from CD45.1+ C57BL/6 mice were either cultured alone or co-cultured with iTregs, or with CD4+ RTEs from the liver or mesenteric lymph nodes of CD45.2+ congenic mice at the ratio of 1:1. RNA was extracted from the CD4+ RTEs from the lymph nodes and liver using TRIZOL (Invitrogen, Grand Island, NY, USA), and cDNA was obtained using the FastQuant RT Kit (TIANGEN, Beijing, China). The following terminology is used to denote the statistical significance: *p < 0.05, **p < 0.01, ***p < 0.005
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