Bruno Kyewski

Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self

Expression of peripheral antigens in the thymus has been implicated in T cell tolerance and autoimmunity. Here we identified medullary thymic epithelial cells as being a unique cell type that expresses a diverse range of tissue-specific antigens. We found that this promiscuous gene expression was a cell-autonomous property of medullary epithelial cells and was maintained during the entire period of thymic T cell output. It may facilitate tolerance induction to self-antigens that would otherwise be temporally or spatially secluded from the immune system. However, the array of promiscuously expressed self-antigens appeared random rather than selected and was not confined to secluded self-antigens.

Promiscuous gene expression in thymic epithelial cells is regulated at multiple levels

The role of central tolerance induction has recently been revised after the discovery of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent of tissue representation afforded by this mechanism and its cellular and molecular regulation are barely defined. Here we show that medullary thymic epithelial cells (mTECs) are specialized to express a highly diverse set of genes representing essentially all tissues of the body. Most, but not all, of these genes are induced in functionally mature CD80hi mTECs. Although the autoimmune regulator (Aire) is responsible for inducing a large portion of this gene pool, numerous tissue-restricted genes are also up-regulated in mature mTECs in the absence of Aire. Promiscuously expressed genes tend to colocalize in clusters in the genome. Analysis of a particular gene locus revealed expression of clustered genes to be contiguous within such a cluster and to encompass both Aire-dependent and –independent genes. A role for epigenetic regulation is furthermore implied by the selective loss of imprinting of the insulin-like growth factor 2 gene in mTECs. Our data document a remarkable cellular and molecular specialization of the thymic stroma in order to mimic the transcriptome of multiple peripheral tissues and, thus, maximize the scope of central self-tolerance.

Positive and negative selection of the T cell repertoire: what thymocytes see (and don't see)

The fate of developing T cells is specified by the interaction of their antigen receptors with self-peptide–MHC complexes that are displayed by thymic antigen-presenting cells (APCs). Various subsets of thymic APCs are strategically positioned in particular thymic microenvironments and they coordinate the selection of a functional and self-tolerant T cell repertoire. In this Review, we discuss the different strategies that these APCs use to sample and process self antigens and to thereby generate partly unique, idiosyncratic peptide–MHC ligandomes. We discuss how the particular composition of the peptide–MHC ligandomes that are presented by specific APC subsets not only shapes the T cell repertoire in the thymus but may also indelibly imprint the behaviour of mature T cells in the periphery.

Antigen presentation in the thymus for positive selection and central tolerance induction

Understanding how thymic selection imparts self-peptide–MHC complex restriction and a high degree of self tolerance on the T cell repertoire requires a detailed description of the parameters that shape the MHC ligand repertoire of distinct thymic antigen-presenting cells and of how these cells communicate with T cells. Several recent discoveries pertaining to cortex-specific pathways of antigen processing, the heterogeneity of thymic dendritic cells and the intercellular transfer of self antigens have uncovered surprising and unique aspects of antigen presentation in the thymic microenvironment. Here, we discuss these new findings in the context of how individual thymic stromal cell types support T cell selection in a cooperative rather than a redundant manner.

Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells

Intrathymic expression of tissue-specific self antigens may be involved in immunological tolerance and protection from autoimmune disease. We have analyzed the role of T-cell tolerance to proteolipid protein (PLP), the main protein of the myelin sheath, in susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Intrathymic expression of PLP was largely restricted to the shorter splice variant, DM20. Expression of DM20 by thymic epithelium was sufficient to confer T-cell tolerance to all epitopes of PLP in EAE-resistant C57BL/6 mice. In contrast, the major T-cell epitope in SJL/J mice was only encoded by the central nervous system-specific exon of PLP, but not by thymic DM20. Thus, lack of tolerance to this epitope offers an explanation for the exquisite susceptibility of SJL/J mice to EAE. As PLP expression in the human thymus is also restricted to the DM20 isoform, these findings have implications for selection of the autoimmune T-cell repertoire in multiple sclerosis.

Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters

Promiscuous expression of tissue-specific self-antigens in the thymus imposes T cell tolerance and protects from autoimmune diseases, as shown in animal studies. Analysis of promiscuous gene expression in purified stromal cells of the human thymus at the single and global gene level documents the species conservation of this phenomenon. Medullary thymic epithelial cells overexpress a highly diverse set of genes (>400) including many tissue-specific antigens, disease-associated autoantigens, and cancer-germline genes. Although there are no apparent structural or functional commonalities among these genes and their products, they cluster along chromosomes. These findings have implications for human autoimmune diseases, immuno-therapy of tumors, and the understanding of the nature of this unorthodox regulation of gene expression.

Self-representation in the thymus: an extended view

The thymus has been viewed as the main site of tolerance induction to self-antigens that are specifically expressed by thymic cells and abundant blood-borne self-antigens, whereas tolerance to tissue-restricted self-antigens has been ascribed to extrathymic (peripheral) tolerance mechanisms. However, the phenomenon of promiscuous expression of tissue-restricted self-antigens by medullary thymic epithelial cells has led to a reassessment of the role of central T-cell tolerance in preventing organ-specific autoimmunity. Recent evidence indicates that both genetic and epigenetic mechanisms account for this unorthodox mode of gene expression. As we discuss here, these new insights have implications for our understanding of self-tolerance in humans, its breakdown in autoimmune diseases and the significance of this tolerance mode in vertebrate evolution.

Bone marrow as a priming site for T-cell responses to blood-borne antigen

Although bone marrow is known as a primary lymphoid organ, its potential to serve as a secondary immune organ has hardly been explored. Here we demonstrate that naive, antigen-specific T cells home to bone marrow, where they can be primed. Antigen presentation to T cells in bone marrow is mediated via resident CD11c+ dendritic cells. They are highly efficient in taking up exogenous blood-borne antigen and processing it via major histocompatibility complex class I and class II pathways. T-cell activation correlates with dendritic cell–T cell clustering in bone marrow stroma. Primary CD4+ and CD8+ T-cell responses generated in bone marrow occur in the absence of secondary lymphoid organs. The responses are not tolerogenic and result in generation of cytotoxic T cells, protective anti-tumor immunity and immunological memory. These findings highlight the uniqueness of bone marrow as an organ important for hemato- and lymphopoiesis and for systemic T cell–mediated immunity.

The thymic medulla: a unique microenvironment for intercellular self-antigen transfer

Central tolerance is shaped by the array of self-antigens expressed and presented by various types of thymic antigen-presenting cells (APCs). Depending on the overall signal quality and/or quantity delivered in these interactions, self-reactive thymocytes either apoptose or commit to the T regulatory cell lineage. The cellular and molecular complexity underlying these events has only recently been appreciated. We analyzed the ex vivo presentation of ubiquitous or tissue-restricted self-antigens by medullary thymic epithelial cells (mTECs) and thymic dendritic cells (DCs), the two major APC types present in the medulla. We found that the ubiquitously expressed nuclear neo–self-antigen ovalbumin (OVA) was efficiently presented via major histocompatibility complex class II by mTECs and thymic DCs. However, presentation by DCs was highly dependent on antigen expression by TECs, and hemopoietic cells did not substitute for this antigen source. Accordingly, efficient deletion of OVA-specific T cells correlated with OVA expression by TECs. Notably, OVA was only presented by thymic but not peripheral DCs. We further demonstrate that thymic DCs are constitutively provided in situ with cytosolic as well as membrane-bound mTEC-derived proteins. The subset of DCs displaying transferred proteins was enriched in activated DCs, with these cells being most efficient in presenting TEC-derived antigens. These data provide evidence for a unique, constitutive, and unidirectional transfer of self-antigens within the thymic microenvironment, thus broadening the cellular base for tolerance induction toward promiscuously expressed tissue antigens.

An IRF8-binding promoter variant and AIRE control CHRNA1 promiscuous expression in thymus.

Promiscuous expression of tissue-restricted auto-antigens in the thymus imposes T-cell tolerance and provides protection from autoimmune diseases. Promiscuous expression of a set of self-antigens occurs in medullary thymic epithelial cells and is partly controlled by the autoimmune regulator (AIRE), a nuclear protein for which loss-of-function mutations cause the type 1 autoimmune polyendocrine syndrome. However, additional factors must be involved in the regulation of this promiscuous expression. Here we describe a mechanism controlling thymic transcription of a prototypic tissue-restricted human auto-antigen gene, CHRNA1. This gene encodes the α-subunit of the muscle acetylcholine receptor, which is the main target of pathogenic auto-antibodies in autoimmune myasthenia gravis. On re-sequencing the CHRNA1 gene, we identified a functional bi-allelic variant in the promoter that is associated with early onset of disease in two independent human populations (France and United Kingdom). We show that this variant prevents binding of interferon regulatory factor 8 (IRF8) and abrogates CHRNA1 promoter activity in thymic epithelial cells in vitro. Notably, both the CHRNA1 promoter variant and AIRE modulate CHRNA1 messenger RNA levels in human medullary thymic epithelial cells ex vivo and also in a transactivation assay. These findings reveal a critical function of AIRE and the interferon signalling pathway in regulating quantitative expression of this auto-antigen in the thymus, suggesting that together they set the threshold for self-tolerance versus autoimmunity.