Hepatocyte dependent induction of regulatory T-cell subsets

Introduction:

The liver is continuously exposed to exo- or endogenous antigens and toxins. Therefore, the liver is able to induce a tolerogenic environment. It is known that regulatory T cells have a pivotal role in inducing this immunotolerance. Several liver resident cells, like liver sinusoidal endothelial cells (LSECs) or Kupffer cells (KCs), are able to induce regulatory T cells. Investigations showed that T cells and hepatocytes, the most abundant cell type within liver, are able to communicate directly through fenestrations in the sinusoidal endothelium. Therefore, we studied if the interaction between hepatocytes and T-cells induce regulatory T-cell subsets.

Methods:

Splenic CD4+ T-cells from C57BL/6, FIR x tiger (foxp3-RFP and Il10-eGFP double reporter mice), DEREG (foxp3-eGFP reporter mice), Gzmb ko or CD45.1 congenic mice were purified via MACS sorting and co-cultured with hepatocytes isolated from C57BL/6 mice. Gene expression was measured by nCounter Gene Expression or PCR. Cytokine expression was detected via ELISA. Proliferation of eFluor670 labelled responder T cells was determined by flow cytometry.

Results:

Our results show that hepatocytes induce regulatory T cells which produce IL-10 as well as IFNγ and lack Foxp3 expression. Furthermore this hepatocyte induced T cells produce an increased amount of the serine protease Granzyme B. The Granzyme B, such as the IFNγ and IL10 expression are dependent on Notch signalling. The hepatocyte induced T cells show a high capability to suppress responder T cells in vitro, which is IL-10 independent. In presence of TGFβ hepatocytes induce CD25+Foxp3+Tregs which are also able to suppress responder T cells in vitro.

Conclusion:

We demonstrated that hepatocytes induce IL-10+ and IL10- regulatory T cell subsets that inhibit activation and proliferation of naive CD4+ T cells in vitro thus, suppression occurs independently of IL-10. Without TGFβ in the culture the regulatory T cell subsets are characterized by high Granzyme B expression that is induced via the Notch pathway. As Granzyme B expression in regulatory T cells has been described as one way of suppression we currently perform experiments with Granzyme B knockout mice. Another possible suppressive mechanism we analyse is the depletion of IL-2 and downregulation of CD25 in responder T cells by hepatocyte induced T cells. To get further information about the function of these cell types in vivo, we establish adaptive transfer experiments with hepatocyte induced regulatory T-cell subsets in a mouse model of immune-mediated hepatitis.

Corresponding author: Pfaff, Malte

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