Georg A. Holländer

Population and single cell genomics reveal the Aire-dependency, relief from Polycomb silencing and distribution of self-antigen expression in thymic epithelia

Promiscuous gene expression (PGE) by thymic epithelial cells (TEC) is essential for generating a diverse T cell antigen receptor repertoire tolerant to self-antigens, and thus for avoiding autoimmunity. Nevertheless, the extent and nature of this unusual expression program within TEC populations and single cells are unknown. Using deep transcriptome sequencing of carefully identified mouse TEC subpopulations, we discovered a program of PGE that is common between medullary (m) and cortical TEC, further elaborated in mTEC, and completed in mature mTEC expressing the autoimmune regulator gene (Aire). TEC populations are capable of expressing up to 19,293 protein-coding genes, the highest number of genes known to be expressed in any cell type. Remarkably, in mouse mTEC, Aire expression alone positively regulates 3980 tissue-restricted genes. Notably, the tissue specificities of these genes include known targets of autoimmunity in human AIRE deficiency. Led by the observation that genes induced by Aire expression are generally characterized by a repressive chromatin state in somatic tissues, we found these genes to be strongly associated with H3K27me3 marks in mTEC. Our findings are consistent with AIRE targeting and inducing the promiscuous expression of genes previously epigenetically silenced by Polycomb group proteins. Comparison of the transcriptomes of 174 single mTEC indicates that genes induced by Aire expression are transcribed stochastically at low cell frequency. Furthermore, when present, Aire expression-dependent transcript levels were 16-fold higher, on average, in individual TEC than in the mTEC population.

Essential role for autophagy during invariant NKT cell development

Significance Autophagy is an evolutionarily conserved catabolic process essential to maintaining cellular homeostasis through the breakdown and recycling of damaged organelles and long-lived proteins. We report that autophagy plays an essential cell-intrinsic role in maintaining the survival of a subset of innate-like cells known as invariant natural killer T (iNKT) cells. Autophagy deficiency prevents transition to a quiescent state after population expansion of thymic iNKT cells. Hence, autophagy-deficient iNKT cells accumulate mitochondria and oxygen radicals and subsequently die of apoptosis. Autophagy is an evolutionarily conserved cellular homeostatic pathway essential for development, immunity, and cell death. Although autophagy modulates MHC antigen presentation, it remains unclear whether autophagy defects impact on CD1d lipid loading and presentation to invariant natural killer T (iNKT) cells and on iNKT cell differentiation in the thymus. Furthermore, it remains unclear whether iNKT and conventional T cells have similar autophagy requirements for differentiation, survival, and/or activation. We report that, in mice with a conditional deletion of the essential autophagy gene Atg7 in the T-cell compartment (CD4 Cre-Atg7−/−), thymic iNKT cell development—unlike conventional T-cell development—is blocked at an early stage and mature iNKT cells are absent in peripheral lymphoid organs. The defect is not due to altered loading of intracellular iNKT cell agonists; rather, it is T-cell–intrinsic, resulting in enhanced susceptibility of iNKT cells to apoptosis. We show that autophagy increases during iNKT cell thymic differentiation and that it developmentally regulates mitochondrial content through mitophagy in the thymus of mice and humans. Autophagy defects result in the intracellular accumulation of mitochondrial superoxide species and subsequent apoptotic cell death. Although autophagy-deficient conventional T cells develop normally, they show impaired peripheral survival, particularly memory CD8+ T cells. Because iNKT cells, unlike conventional T cells, differentiate into memory cells while in the thymus, our results highlight a unique autophagy-dependent metabolic regulation of adaptive and innate T cells, which is required for transition to a quiescent state after population expansion.

A regulatory role for TGF-β signaling in the establishment and function of the thymic medulla

Medullary thymic epithelial cells (mTECs) are critical in establishing and maintaining the appropriate microenvironment for negative selection and maturation of immunocompetent T cells with a self-tolerant T cell antigen receptor repertoire. Cues that direct proliferation and maturation of mTECs are provided by members of the tumor necrosis factor (TNF) superfamily expressed on developing thymocytes. Here we demonstrate a negative role of the morphogen TGF-β in tempering these signals under physiological conditions, limiting both growth and function of the thymic medulla. Eliminating TGF-β signaling specifically in TECs or by pharmacological means increased the size of the mTEC compartment, enhanced negative selection and functional maturation of medullary thymocytes as well as the production of regulatory T cells, thus reducing the autoreactive potential of peripheral T cells.

Apoptosis of thymocytes during acute graft-versus-host disease is independent of glucocorticoids

BACKGROUNDnElimination of immature thymocytes resulting in thymic atrophy is characteristic of acute graft-versus-host disease (aGVHD). Because aGVHD has been associated with elevated glucocorticoid (GC) production, and CD4,CD8 double-positive thymocytes undergo rapid apoptosis in response to GCs, we hypothesized that administration of the GC receptor antagonist RU486 (mifepristone) should alter aGVHD-mediated thymocyte apoptosis.nnnMETHODSnThymic development in the presence of aGVHD was studied in a haploidentical nonirradiated murine transplantation model (C57BL/6-->B6D2F1). Recipients were treated with RU486 or vehicle alone. Thymic development was analyzed by flow cytometry at different times post transplant.nnnRESULTSnAcute thymic GVHD was characterized (1) by infiltration of mature donor-derived T cells and (2) by increased apoptosis of immature CD4+CD8+ thymocytes between 1 and 2 weeks after allogeneic transplantation. Contrary to expectations, administration of RU486 had no effect on these two parameters.nnnCONCLUSIONSnOur data suggest that thymic pathology during aGVHD is mediated via a glucocorticoid-independent mechanism of apoptosis as blockade of glucocorticoid receptors did not alter the GVHD-induced thymic phenotype.

Adult Thymic Medullary Epithelium Is Maintained and Regenerated by Lineage-Restricted Cells Rather Than Bipotent Progenitors

Medullary thymic epithelial cells (mTECs) play an essential role in establishing self-tolerance in Txa0cells. mTECs originate from bipotent TEC progenitors that generate both mTECs and cortical TECs (cTECs), although mTEC-restricted progenitors also have been reported. Here, we report inxa0vivo fate-mapping analysis of cells that transcribe β5t, a cTEC trait expressed in bipotent progenitors, during a given period in mice. We show that, in adult mice, most mTECs arexa0derived from progenitors that transcribe β5t during embryogenesis and the neonatal period up to 1xa0week of age. The contribution of adult β5t(+) progenitors was minor even during injury-triggered regeneration. Our results further demonstrate that adult mTEC-restricted progenitors are derived from perinatal β5t(+) progenitors. These results indicate that the adult thymic medullary epithelium is maintained and regenerated by mTEC-lineage cells that pass beyond the bipotent stage during early ontogeny.

Foxn1 regulates key target genes essential for T cell development in postnatal thymic epithelial cells

Thymic epithelial cell differentiation, growth and function depend on the expression of the transcription factor Foxn1; however, its target genes have never been physically identified. Using static and inducible genetic model systems and chromatin studies, we developed a genome-wide map of direct Foxn1 target genes for postnatal thymic epithelia and defined the Foxn1 binding motif. We determined the function of Foxn1 in these cells and found that, in addition to the transcriptional control of genes involved in the attraction and lineage commitment of T cell precursors, Foxn1 regulates the expression of genes involved in antigen processing and thymocyte selection. Thus, critical events in thymic lympho-stromal cross-talk and T cell selection are indispensably choreographed by Foxn1.

Dynamic spatio-temporal contribution of single β5t+ cortical epithelial precursors to the thymus medulla.

Intrathymic T‐cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit β5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that β5t+ TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage‐tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual β5t+ cortical progenitors located at the cortico‐medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio‐temporal dynamics that control the growth of the thymic medulla.

Impaired thymic expression of tissue-restricted antigens licenses the de novo generation of autoreactive CD4+ T cells in acute GVHD.

During acute graft-versus-host disease (aGVHD) in mice, autoreactive T cells can be generated de novo in the host thymus implying an impairment in self-tolerance induction. As a possible mechanism, we have previously reported that mature medullary thymic epithelial cells (mTEC(high)) expressing the autoimmune regulator are targets of donor T-cell alloimmunity during aGVHD. A decline in mTEC(high) cell pool size, which purges individual tissue-restricted peripheral self-antigens (TRA) from the total thymic ectopic TRA repertoire, weakens the platform for central tolerance induction. Here we provide evidence in a transgenic mouse system using ovalbumin (OVA) as a model surrogate TRA that the de novo production of OVA-specific CD4(+) T cells during acute GVHD is a direct consequence of impaired thymic ectopic OVA expression in mTEC(high) cells. Our data, therefore, indicate that a functional compromise of the medullary mTEC(high) compartment may link alloimmunity to the development of autoimmunity during chronic GVHD.

Contribution of CD40-CD154-mediated costimulation to an alloresponse in vivo

BACKGROUNDnCostimulation through CD40-CD154 plays an important role in T-cell activation. Although systemic administration of anti-CD154 antibody prevents or delays rejection of organ allografts in animal models, the molecular mechanisms responsible for this effect are not well defined.nnnMETHODSnWe have previously demonstrated that priming of mice (H2d) with CD40-/- but not with wildtype naive B cells (H2b) leads to alloantigen-specific T-cell hyporesponsiveness in vitro. In the present study, we investigated whether such priming modifies allograft rejection in a major histocompatibility complex-mismatched murine cardiac transplantation model.nnnRESULTSnPriming of hosts with donor-specific CD40-/- B cells delayed rejection of subsequently transplanted wild-type cardiac allografts by 8.0 days (P<0.001). The lack of CD40 on the cardiac graft delayed rejection in unprimed or primed hosts by 3-5 days. Prolongation of graft survival correlated with the failure of infused CD40-/- B cells to express B7.2 and ICAM-1 in vivo.nnnCONCLUSIONSnOur data suggest that CD40-CD154 costimulation contributes to T cell priming to alloantigens in vivo and to a second set rejection phase in which donor antigens are presented to primed T cells.

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.