Jens Derbinski

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.

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.

Promiscuous gene expression and central T-cell tolerance: more than meets the eye

Self-tolerance of the T-cell repertoire is mediated by multiple mechanisms operating both in the thymus (‘central tolerance’) and in peripheral lymphoid and non-lymphoid organs (‘peripheral tolerance’). Based on the recent finding that tissue-specific genes are commonly expressed in the thymus, this strict delineation has been blurred. Medullary thymic epithelial cells have been identified as a unique cell type that expresses a wide range of tissue-specific genes in a ‘promiscuous’ manner and displays these self-antigens for intrathymic repertoire selection. The array of promiscuously expressed genes appears random rather than the result of specific selection. The gains and pitfalls of this tolerance mechanism have important implications for autoimmunity.

Promiscuous gene expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism

Promiscuous expression of tissue-restricted autoantigens in medullary thymic epithelial cells (mTECs) imposes central T cell tolerance. The molecular regulation of this unusual gene expression is not understood, in particular its delineation from cell lineage-specific gene expression control remains unclear. Here, we compared the expression profile of the casein gene locus in mTECs and mammary gland epithelial cells by single cell PCR. Mammary gland cells showed highly correlated intra- and interchromosomal coexpression of milk proteins (the casein genes, lactalbumin-α and whey acidic protein) and one of its transcriptional regulators (Elf5). In contrast, coexpression of these genes in mature CD80hi mTECs was rarely observed and no pattern of gene expression in individual mTECs was discernible. The apparent stochastic expression pattern of genes within the casein locus, the lower mRNA levels compared with mammary gland cells in conjunction with frequent coexpression of insulin in single mTECs clearly delineates the molecular mechanism(s) of promiscuous gene expression from cell lineage-specific gene control.

How thymic antigen presenting cells sample the body's self-antigens

Our perception of the scope self-antigen availability for tolerance induction in the thymus has profoundly changed over the recent years following new insights into the cellular and molecular complexity of intrathymic antigen presentation. The diversity of self-peptide display is on the one hand afforded by the remarkable heterogeneity of thymic antigen presenting cells (APCs) and on the other hand by the endowment of these cells with unconventional molecular pathways. Recent studies show that each APC subset appears to carry its specific antigen cargo as a result of cell-type specific features: firstly, transcriptional control (i.e. promiscuous gene expression in medullary thymic epithelial cells); secondly, antigen processing (i.e. proteasome composition and protease sets); thirdly, intracellular antigen sampling (i.e. autophagy in thymic epithelial cells) and fourthly, extracellular antigen sampling (i.e. immigrating dendritic cells sampling extrathymic milieus). The combinatorial expression patterns of these attributes in distinct APC subsets result in a self-peptide display partly unique to the cortex mediating positive selection and to the medulla mediating tolerance induction.

Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region (“entry site”) to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.

Peptide transporter TAP mediates between competing antigen sources generating distinct surface MHC class I peptide repertoires.

We recently described a category of TAP‐independent peptide‐epitopes that are selectively presented by cells with processing defects in the classical MHC class I (MHC‐I) pathway. Here, we studied the ER‐resident ceramide synthase Trh4 as a prototypic example of these neo‐antigens and found that moderate inhibition of TAP permits cell surface presentation of the Trh4 peptide. The absence of this peptide from WT cells was not related to the binding or stability of the Trh4/Db complexes, or to the availability of MHC‐I heavy chains, but rather to the limited expression of the antigen. Strongly elevated antigen levels were needed to reach comparable peptide display on WT as on TAP‐deficient cells. Our data suggest that the normal influx of TAP‐transported peptides in the ER during routine processing creates an efficient barrier for peptides from alternative processing routes. Impairment of TAP function, as commonly found in cancers and virus‐infected cells, lowers this resistance allowing for MHC‐I presentation of other peptide sources.

Homeodomain-Interacting Protein Kinase 2, a Novel Autoimmune Regulator Interaction Partner, Modulates Promiscuous Gene Expression in Medullary Thymic Epithelial Cells

Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80lo mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo.

Myelin oligodendrocyte glycoprotein induces incomplete tolerance of CD4(+) T cells specific for both a myelin and a neuronal self-antigen in mice.

T‐cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self‐antigen. We previously showed in C57BL/6 mice that part of the CD4+ T‐cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35–55 also recognizes the neuronal antigen neurofilament medium (NF‐M) 15–35. Such bi‐specific CD4+ T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self‐antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF‐M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG35‐55‐reactive CD4+ T cells were increased in MOG‐deficient but not in NF‐M‐deficient mice. We found that presentation of NF‐M15‐35 by I‐Ab on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC‐anchoring residue into NF‐M15‐35 (NF‐M15‐35T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi‐specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self‐antigens.