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-A(b) on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC-anchoring residue into NF-M15-35 (NF-M15-35 T20Y) 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.

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 CD80(lo) 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.

In vivo monitoring of urea cycle activity with (13)C-acetate as a tracer of ureagenesis.

BACKGROUND: The hepatic urea cycle is the main metabolic pathway for detoxification of ammonia. Inborn errors of urea cycle function present with severe hyperammonemia and a high case fatality rate. Long-term prognosis depends on the residual activity of the defective enzyme. A reliable method to estimate urea cycle activity in-vivo does not exist yet. The aim of this study was to evaluate a practical method to quantify (13)C-urea production as a marker for urea cycle function in healthy subjects, patients with confirmed urea cycle defect (UCD) and asymptomatic carriers of UCD mutations. METHODS: (13)C-labeled sodium acetate was applied orally in a single dose to 47 subjects (10 healthy subjects, 28 symptomatic patients, 9 asymptomatic carriers). RESULTS: The oral (13)C-ureagenesis assay is a safe method. While healthy subjects and asymptomatic carriers did not differ with regards to kinetic variables for urea cycle flux, symptomatic patients had lower (13)C-plasma urea levels. Although the (13)C-ureagenesis assay revealed no significant differences between individual urea cycle enzyme defects, it reflected the heterogeneity between different clinical subgroups, including male neonatal onset ornithine carbamoyltransferase deficiency. Applying the (13)C-urea area under the curve can differentiate between severe from more mildly affected neonates. Late onset patients differ significantly from neonates, carriers and healthy subjects. CONCLUSION: This study evaluated the oral (13)C-ureagenesis assay as a sensitive in-vivo measure for ureagenesis capacity. The assay has the potential to become a reliable tool to differentiate UCD patient subgroups, follow changes in ureagenesis capacity and could be helpful in monitoring novel therapies of UCD.

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.

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/D(b) 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.

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 CD80(hi) 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.

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-alpha and whey acidic protein) and one of its transcriptional regulators (Elf5). In contrast, coexpression of these genes in mature CD80(hi) 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.

Clinical course of 63 patients with neonatal onset urea cycle disorders in the years 2001-2013.

BACKGROUND: Urea cycle disorders (UCDs) are rare inherited metabolic defects of ammonia detoxification. In about half of patients presenting with a UCD, the first symptoms appear within a few days after birth. These neonatal onset patients generally have a severe defect of urea cycle function and their survival and outcome prognoses are often limited. To understand better the current situation of neonatal onset in UCDs, we have performed a multicentre, retrospective, non-interventional case series study focussing on the most severe UCDs, namely defects of carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), and argininosuccinate synthetase (ASS). METHODS AND RESULTS: Data of 63 patients were collected (27 patients with ASS deficiency, 23 patients with OTC deficiency, and 12 patients with CPS1 deficiency, one patient definite diagnosis not documented). The majority of patients (43/63, 68 %) had an initial ammonia concentration exceeding 500 µmol/L (normal < 100), of which most (26/43, 60.5 %) were also encephalopathic and were treated with hemodialysis. In patients surviving the initial crisis, recurrence of hyperammonemic events within the first 1.5 years of life occurred frequently (mean 3.6 events, range 0-20). Of all patients, 16 (25.4 %) died during or immediately after the neonatal period. CONCLUSION: We observed in this cohort of neonatal onset UCD patients a high rate of initial life-threatening hyperammonemia and a high risk of recurrence of severe hyperammonemic crises. These corresponded to a high mortality rate during the entire study period (30.2 %) despite the fact that patients were treated in leading European metabolic centers. This underlines the need to critically re-evaluate the current treatment strategies in these patients.

Genome-wide gene expression profiling of homeodomain-interacting protein kinase 2 deficient medullary thymic epithelial cells.

The establishment of central tolerance essentially depends on the promiscuous gene expression (pGE) of a plethora of tissue restricted antigens by the medullary thymic epithelial cells. The antigens are presented to developing thymocytes in the thymus to select for non-self reactive T-cell receptors in order to prevent autoimmune reactions in the periphery. However the molecular regulation of tissue-restricted antigen expression is still poorly understood. The only regulator known to play a role in the transcriptional regulation so far is the autoimmune regulator (AIRE). AIRE is thought to act in a multi-protein complex, promoting transcription, elongation and splicing of target genes. Yet the full composition of this Aire-associated multi-protein complex and its mode of action remain to be elucidated. Here we describe the experimental details and controls of the gene array analysis on the impact of the homeodomain-interacting protein kinase 2 (Hipk2) on promiscuous gene expression in medullary thymic epithelial cells based on the analysis of newly generated TEC-specific Hipk2 conditional knockout mice. The changes in gene expression are presumably mediated through a regulatory effect of Hipk2 on AIRE as published in the study by Rattay and colleagues in the Journal of Immunology [1]. The gene array data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE63432).

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.

Foxp3+ CD25+ regulatory T cells specific for a neo-self-antigen develop at the double-positive thymic stage.

Thymus-derived regulatory T cells (Tregs) expressing CD4, CD25, and the transcription factor Foxp3 play major roles in preventing autoimmunity. The Treg population is enriched in T cells expressing high-avidity self-reactive T cell receptors, and thymic epithelial cells expressing self-antigens (Ag) have been implicated in their induction and/or selection. However, the thymic selection events leading to Treg lineage commitment remain unclear. We followed the thymic development of self-Ag-specific Tregs in double-transgenic mice coexpressing a neo-self-Ag, hemagglutinin (HA) under the control of a neural tissue-specific promoter, and a transgenic class II-restricted T cell antigen receptor specific for HA111-119. Our data show that the promiscuous expression of the HA transgene in thymic epithelial cells is involved in the selective induction and/or expansion of HA-specific Foxp3(+) Treg thymic precursors as early as the double-positive stage.

Linking signalling pathways, thymic stroma integrity and autoimmunity.

Medullary thymic epithelial cells (mTECs) are indispensable for self-tolerance to peripheral organs by virtue of their expression of a host of tissue-restricted self-antigens. The full extent of this promiscuous gene expression is confined to functionally mature mTECs. Consequently, any interference with signalling pathways directing the differentiation and/or proliferation of this mature subset will affect the scope of central tolerance and potentially predispose to autoimmunity. In a recent study, tumour necrosis factor receptor-associated factor 6 (TRAF6) has been identified as an essential component of a new signalling pathway directing mTEC development.