Elsevier

Journal of Hepatology

Volume 61, Issue 3, September 2014, Pages 594-599
Journal of Hepatology

Research Article
TGF-β-dependent induction of CD4+CD25+Foxp3+ Tregs by liver sinusoidal endothelial cells

https://doi.org/10.1016/j.jhep.2014.04.027Get rights and content

Background & Aims

CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction.

Methods

To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3 non-Tregs into Foxp3+ Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-β was tested in Treg conversion assays using T cells with reduced TGF-β sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE).

Results

All tested liver cell types were capable of inducing Foxp3+ Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-β. LSECs featured membrane-bound LAP/TGF-β and the anchor molecule GARP, which is required for tethering LAP/TGF-β to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE.

Conclusions

We demonstrate that LSECs are the major liver cell type responsible for TGF-β dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-β to their membrane.

Introduction

The microenvironment of the liver greatly favours immune tolerance [1], [2]. Indeed, antigens delivered via the portal vein [3] or allogeneic liver transplants [4], [5] do not provoke inflammatory immune responses. Hepatic tolerance is of importance in preventing and controlling inflammatory or autoimmune diseases [1]; however, it may also contribute to the frequent occurrence of persistent hepatitis virus infections [6] or liver cancer [7], [8]. Beyond regulation of local immune responses within the liver, hepatic tolerance can also act systemically, indicated by the finding that the ectopic expression of a neuroantigen in the liver can prevent autoimmune neuroinflammation [9]. The systemic effect of hepatic tolerance can be explained by the capacity of the liver to generate antigen-specific CD4+ Foxp3+ regulatory T cells (Tregs) that have a profound ability to control immune responses [10], [11]. However, the mechanisms and cell types responsible for hepatic Treg generation have not been elucidated.

Liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs) are liver resident antigen-presenting cells constitutively expressing MHC II molecules and capable of stimulating CD4+ T cells [1], [2]. Therefore, these liver cell types are the main candidate cells facilitating Treg generation. The generation of Tregs in the periphery occurs through conversion of CD4+ Foxp3 non-Tregs into CD4+ Foxp3+ Tregs; this conversion is dependent on T cell stimulation in the presence of TGF-β [11]. TGF-β is produced as a pro-form, which is intracellularly processed by furin into latent TGF-β [12]. Latent TGF-β consists of latency-associated peptide (LAP), the N-terminal part of pro-TGF-β, non-covalently associated with mature TGF-β. LAP/TGF-β cannot bind TGF-β receptors; biological activity requires further processing, which is not well characterised [12], [13]. However, it was found that secreted LAP/TGF-β can be tethered to the outer cell membrane through the receptor glycoprotein-A repetitions predominant (GARP, also known as LRRC32) [14], [15]. Such membrane-bound LAP/TGF-β was shown to exhibit biological activity and induce a tolerogenic phenotype and Foxp3-expression in stimulated T cells [16].

In the present study, we demonstrate that LSECs are the major liver antigen-presenting cell type responsible for TGF-β dependent induction of CD4+ Foxp3+ Tregs. The particular Treg-inducing capacity of LSECs was related to their ability to secrete TGF-β and to tether exogenous LAP/TGF-β to their membrane through GARP. In vivo, antigen-specific Tregs induced by LSECs were potent suppressors of autoimmune inflammation in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) [17], which is induced in susceptible B10.PL mice by immunisation to MBP peptide and marked by ascending paralysis. Our findings indicate that Treg induction by LSECs is instrumental for the control of hepatic and systemic inflammatory immune responses.

Section snippets

Mice

B10.PL mice, C57BL/6 mice, tg4 mice [18], CRP-MBP mice [9], Foxp3gfp.KI mice [19], or hCD2-ΔkTβRII mice [20] were bred and kept in the animal facility of the University Medical Centre Hamburg-Eppendorf under specific pathogen-free conditions. F1 mice were generated by mating of Foxp3gfp.KI mice with tg4 mice or tg4 mice with CRP-MBP mice; hCD2-ΔkTβRII mice were backcrossed to tg4 mice. Mice were 6–12 weeks old at start of experiments. Animal experiments were approved by the institutional review

Results

To assess the capacity of antigen-presenting liver cells for Treg induction, we used primary liver DCs, LSECs, or KCs to stimulate CD4+Foxp3 non-Treg cells from the spleen of (tg4 × Foxp3gfp.KI) F1 mice. These mice feature MBP-specific CD4 T cells [18] and Foxp3 and green fluorescent protein (GFP) co-expressing Tregs (Supplementary Fig. 1). Non-Treg cells were sorted based on the absence of the Foxp3-linked expression of GFP [19]. The stimulation of non-Treg cells was performed in serum-free

Discussion

CD4+Foxp3+ Tregs are important suppressors of inflammation and autoimmunity. Tregs can be generated in the thymus, but also in the periphery through TGF-β dependent conversion from conventional CD4+ T cells [11], [23]. The Tregs generated in the thymus and those generated in the periphery feature distinct complementary T cell receptor repertoires [23]. Peripheral Treg generation is of particular importance for maintaining tolerance to antigens that are not represented in the thymus. We have

Financial support

Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 841; HE3532/2-1) and Wellcome Trust (grant 090175/Z/09/Z).

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgements

We are grateful for excellent technical assistance by Marko Hilken, Agnes Malotta, and Christina Trabandt. Cell sorting was performed by the flow cytometry core facility of the University Medical Centre Hamburg-Eppendorf.

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