Sonic Hedgehog regulates thymic epithelial cell differentiation

José Ignacio Saldaña,Anisha Solanki,Ching-In Lau,Hemant Sahni,Susan Ross,Anna L Furmanski,Masahiro Ono,Georg Holländer,Tessa Crompton

doi:10.1016/j.jaut.2015.12.004

Abstract

Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

Highlights

The thymus provides a specialised environment for the production of T cells
Transcripts for the transcriptional activator Gli1 were low in both thymic epithelial cell (TEC) subpopulations whereas transcripts for Gli2 and Gli3, whose products can act either as transcriptional activators or repressors, were detected at significantly higher concentrations in cTEC when compared to mTEC (Fig. 1B)
The use of Gli Binding Site (GBS)-green fluorescence protein (GFP) reporter mice, and ShhÀ/À, ShhcoKO and Gli3À/À mouse mutants confirmed that Sonic Hedgehog (Shh) is required for normal TEC differentiation, that it is produced by TEC and that developing TEC transduce Hh signals

Summary

Introduction

The thymus provides a specialised environment for the production of T cells. Thymic epithelial cells (TECs) are an essential component of the thymic stroma, and are required to support T cell development. Two broad categories of TEC, which are believed to arise from a common progenitor, have been defined by their localisation, function and cell surface markers [1,2]. Cortical(c)TEC provide Dl4 for T cell fate specification, and present MHC þ peptide ligands for positive selection. They are defined as EpCam1þ, CD40þ, CD205þ, Ly51þ and MHCIIþ, and express genes for antigen presentation, including Cathepsin-L, Prss and b5t. Medullary (m)TEC are specialised for negative selection, and are defined as surface EpCamþ, CD40þ, CD205À, Ly51À and MHCIIþ cells that react with the lectin UEA-1. While TEC provide multiple essential signals for T cell development, they require signals from thymocytes for their maturation

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