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1.  Morphogenesis and maintenance of the 3D-thymic medulla and prevention of nude skin phenotype require FoxN1 in pre- and post-natal K14 epithelium 
Expansion of thymic epithelial cysts represents disruption of an organized three-dimensional (3D) thymic epithelial cell (TEC) meshwork, which is crucial for T-lymphocyte development. Although the FoxN1-null mutant develops a rudimentary 2D-cystic thymus, 2D-thymic cyst-lining resulting from a dGUO culture was reported to be FoxN1-independent, thus, it is unclear whether loss of FoxN1 facilitates cyst formation, and whether FoxN1 regulates the morphogenesis and maintenance of the 3D-thymic microstructure. Using the loxP-floxed-FoxN1 mouse model, we demonstrated that specific deletion of FoxN1 in Keratin (K)-14 promoter-driven TECs induced the loss of 3D-thymic medullary structure by producing a large number of morphologic pulmonary alveolar-like 2D-epithelial cysts, which increased with age. The cyst-lining was positive for differential polarized keratins and had strong Claudin-3,4, but reduced MHC-II, expression. However, an increased % of Claudin-3,4+ TECs, which are presumptive precursors of UEA-1+ and Aire+ mature medullary TECs, failed to promote the development of these mature descendants. Meanwhile, the K14Cre-mediated FoxN1 deletion alone was sufficient to induce a complete hair follicle defect, causing a nude phenotype in the skin, but was not sufficient to cause a complete loss of the thymus. All these changes to occur require deletion of FoxN1 in both prenatal (Cre-recombinase from parents during fertilization) and postnatal (Cre-recombinase from offspring themselves after fertilization) life. These findings provide new insights into FoxN1 regulation of 3D-thymic epithelial morphogenesis and maintenance, the distinct impacts of FoxN1 in the K14 epithelial subset of the thymus and skin, and its postnatal requirement.
PMCID: PMC3060403  PMID: 21109991
K14-Epithelium; FoxN1 gene; thymic cysts; nude skin, loxP-Cre recombination system
2.  Declining expression of a single epithelial cell-autonomous gene accelerates age-related thymic involution 
Aging cell  2010;9(3):347-357.
Age-related thymic involution may be triggered by gene expression changes in lymphohematopoietic and/or non-hematopoietic thymic epithelial cells (TECs). The role of epithelial cell-autonomous gene FoxN1 may be involved in the process, but it is still a puzzle due to shortage of evidence from gradual loss-of-function and exogenous gain-of-function studies. Using our recently generated loxP-floxed-FoxN1(fx) mouse carrying the ubiquitous CreERT (uCreERT) transgene with a low dose of spontaneous activation, which causes gradual FoxN1 deletion with age, we found that the uCreERT-fx/fx mice showed an accelerated age-related thymic involution due to progressive loss of FoxN1+ TECs. The thymic aging phenotypes were clearly observable as early as at 3–6 months of age, resembling the naturally aged (18–22-month-old) murine thymus. By intrathymically supplying aged wild-type mice with exogenous FoxN1-cDNA, thymic involution and defective peripheral CD4+ T-cell function could be partially rescued. The results support the notion that decline of a single epithelial cell-autonomous gene FoxN1 levels with age causes primary deterioration in TECs followed by impairment of the total postnatal thymic microenvironment, and potentially triggers age-related thymic involution in mice.
PMCID: PMC2894280  PMID: 20156205
Thymic aging; thymic epithelium; loxP/CreERT system; spontaneous FoxN1 gene recombination
3.  FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program 
T cell ontogeny is a sophisticated process, which takes place within the thymus through a series of well-defined discrete stages. The process requires a proper lympho-stromal interaction. In particular, cortical and medullary thymic epithelial cells (cTECs, mTECs) drive T cell differentiation, education, and selection processes, while the thymocyte-dependent signals allow thymic epithelial cells (TECs) to maturate and provide an appropriate thymic microenvironment. Alterations in genes implicated in thymus organogenesis, including Tbx1, Pax1, Pax3, Pax9, Hoxa3, Eya1, and Six1, affect this well-orchestrated process, leading to disruption of thymic architecture. Of note, in both human and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the Forkhead-box n1 (FOXN1) gene in the thymic epithelium this essential function is acquired. FOXN1 is a master regulator in the TEC lineage specification in that it down-stream promotes transcription of genes, which, in turn, regulate TECs differentiation. In particular, FOXN1 mainly regulates TEC patterning in the fetal stage and TEC homeostasis in the post-natal thymus. An inborn null mutation in FOXN1 leads to Nude/severe combined immunodeficiency (SCID) phenotype in mouse, rat, and humans. In Foxn1−/− nude animals, initial formation of the primordial organ is arrested and the primordium is not colonized by hematopoietic precursors, causing a severe primary T cell immunodeficiency. In humans, the Nude/SCID phenotype is characterized by congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy, and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder. Aim of this review is to summarize all the scientific information so far available to better characterize the pivotal role of the master regulator FOXN1 transcription factor in the TEC lineage specifications and functionality.
PMCID: PMC3709140  PMID: 23874334
Foxn1 gene; TECs; thymus gland; immunodeficiency; Nude/SCID
4.  Foxn1 Regulates Lineage Progression in Cortical and Medullary Thymic Epithelial Cells But Is Dispensable for Medullary Sublineage Divergence 
PLoS Genetics  2011;7(11):e1002348.
The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development.
Author Summary
The thymus is the specialized organ responsible for generating T cells, which are required to regulate and effect immune responses. The unique functions of the thymus are mediated by a diverse array of specialized epithelial cells found only within this organ. These specialized, functionally mature thymic epithelial cells are generated from immature epithelial progenitor cells present in the fetal and adult thymus through a highly regulated process, termed differentiation, that is tightly controlled by specific genes. Foxn1, a protein that is expressed in thymic epithelial cells, is a transcription factor—a protein that regulates how other genes are expressed. Here, we have investigated the role of Foxn1 in generating mature thymic epithelial cells from immature progenitors. We find that Foxn1 is required throughout this process, from the onset of differentiation in progenitor thymic epithelial cells in the developing fetus to the final differentiation steps through which thymic epithelial cells mature to acquire their full functionality. We further find that Foxn1 controls the expression of a variety of genes with different functions in thymic epithelial cells. Overall, our study defines the role of Foxn1 in thymus development at the cellular level and provides insight into how it mediates these functions.
PMCID: PMC3207875  PMID: 22072979
5.  Deletion of FoxN1 in the Thymic Medullary Epithelium Reduces Peripheral T Cell Responses to Infection and Mimics Changes of Aging 
PLoS ONE  2012;7(4):e34681.
Aging increases susceptibility to infection, in part because thymic involution culminates in reduced naïve T-lymphocyte output. Thymic epithelial cells (TECs) are critical to ensure normal maturation of thymocytes and production of peripheral T cells. The forkhead-class transcription factor, encoded by FoxN1, regulates development, differentiation, and function of TECs, both in the prenatal and postnatal thymus. We recently showed that expression of FoxN1, by keratin 14 (K14)-expressing epithelial cells is essential for maintenance of thymic medullary architecture, and deletion of FoxN1 in K14 promoter-driven TECs inhibited development of mature TECs and reduced the number of total thymocytes. These findings are reminiscent of changes observed during normal thymic aging. In the current report, we compared the effects of K14-driven FoxN1 deletion on peripheral T cell function in response to influenza virus infection with those associated with normal aging in a mouse model. FoxN1-deleted mice had reduced numbers of peripheral CD62L+CD44− naïve T-cells. In addition, during influenza infection, these animals had reduced antigen-specific CD8+ T-cell and IgG responses to influenza virus, combined with increased lung injury, weight loss and mortality. These findings paralleled those observed in aged wild type mice, providing the first evidence that K14-mediated FoxN1 deletion causes changes in T-cell function that mimic those in aging during an immune response to challenge with an infectious agent.
PMCID: PMC3326029  PMID: 22514652
6.  Role of the p63-FoxN1 regulatory axis in thymic epithelial cell homeostasis during aging 
Cell Death & Disease  2013;4(11):e932-.
The p63 gene regulates thymic epithelial cell (TEC) proliferation, whereas FoxN1 regulates their differentiation. However, their collaborative role in the regulation of TEC homeostasis during thymic aging is largely unknown. In murine models, the proportion of TAp63+, but not ΔNp63+, TECs was increased with age, which was associated with an age-related increase in senescent cell clusters, characterized by SA-β-Gal+ and p21+ cells. Intrathymic infusion of exogenous TAp63 cDNA into young wild-type (WT) mice led to an increase in senescent cell clusters. Blockade of TEC differentiation via conditional FoxN1 gene knockout accelerated the appearance of this phenotype to early middle age, whereas intrathymic infusion of exogenous FoxN1 cDNA into aged WT mice brought only a modest reduction in the proportion of TAp63+ TECs, but an increase in ΔNp63+ TECs in the partially rejuvenated thymus. Meanwhile, we found that the increased TAp63+ population contained a high proportion of phosphorylated-p53 TECs, which may be involved in the induction of cellular senescence. Thus, TAp63 levels are positively correlated with TEC senescence but inversely correlated with expression of FoxN1 and FoxN1-regulated TEC differentiation. Thereby, the p63-FoxN1 regulatory axis in regulation of postnatal TEC homeostasis has been revealed.
PMCID: PMC3847336  PMID: 24263106
epithelial cell homeostasis; p63/p53 expression; conditional FoxN1 knockout; thymic aging; senescence
7.  The thymic medulla is required for Foxp3+ regulatory but not conventional CD4+ thymocyte development 
The thymic medulla and an intact mTEC compartment are needed for the development of nTreg cells and negative selection of conventional T cells but not their further maturation.
A key role of the thymic medulla is to negatively select autoreactive CD4+ and CD8+ thymocytes, a process important for T cell tolerance induction. However, the involvement of the thymic medulla in other aspects of αβ T cell development, including the generation of Foxp3+ natural regulatory T cells (nTreg cells) and the continued maturation of positively selected conventional αβ T cells, is unclear. We show that newly generated conventional CD69+Qa2− CD4 single-positive thymocytes mature to the late CD69−Qa2+ stage in the absence of RelB-dependent medullary thymic epithelial cells (mTECs). Furthermore, an increasing ability to continue maturation extrathymically is observed within the CD69+CCR7−/loCCR9+ subset of conventional SP4 thymocytes, providing evidence for an independence from medullary support by the earliest stages after positive selection. In contrast, Foxp3+ nTreg cell development is medullary dependent, with mTECs fostering the generation of Foxp3−CD25+ nTreg cell precursors at the CD69+CCR7+CCR9− stage. Our results demonstrate a differential requirement for the thymic medulla in relation to CD4 conventional and Foxp3+ thymocyte lineages, in which an intact mTEC compartment is a prerequisite for Foxp3+ nTreg cell development through the generation of Foxp3−CD25+ nTreg cell precursors.
PMCID: PMC3620359  PMID: 23530124
8.  FOXN1GFP/w Reporter hESCs Enable Identification of Integrin-β4, HLA-DR, and EpCAM as Markers of Human PSC-Derived FOXN1+ Thymic Epithelial Progenitors 
Stem Cell Reports  2014;2(6):925-937.
Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1GFP/w line as a readout, we developed a reproducible protocol for generating FOXN1-GFP+ thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-β4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1+ TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1+ TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.
•FOXN1-GFP reporter hESC lines were generated•KGF promotes the proliferation of FOXN1-GFP+ cells•FOXN1-GFP+ cells express TEC-associated genes•ITGB4, HLA-DR, and EpCAM can be used to purify FOXN1+ TEC progenitors (219)
Stanley and colleagues generated human embryonic stem cell (hESC) reporter lines in which sequences encoding GFP were inserted into the FOXN1 locus, enabling the isolation of viable thymic progenitors from in vitro differentiation cultures. These reporter lines were used to identify integrin-β4, HLA-DR, and EpCAM as markers of human pluripotent stem cell-derived FOXN1+ thymic epithelial progenitors.
PMCID: PMC4050347  PMID: 24936476
9.  Regulation of medullary thymic epithelial cell differentiation and function by the signaling protein Sin 
The Journal of Experimental Medicine  2010;207(5):999-1013.
Medullary thymic epithelial cells (mTECs) play an important role in T cell tolerance and prevention of autoimmunity. Mice deficient in expression of the signaling protein Sin exhibit exaggerated immune responses and multitissue inflammation. Here, we show that Sin is expressed in the thymic stroma, specifically in mTECs. Sin deficiency led to thymic stroma–dependent autoimmune manifestations shown by radiation chimeras and thymic transplants in nude mice, and associated with defective mTEC-mediated elimination of thymocytes in a T cell receptor transgenic model of negative selection. Lack of Sin expression correlated with a disorganized medullary architecture and fewer functionally mature mTECs under steady–state conditions. Additionally, Sin deficiency inhibited the expansion of mTECs in response to in vivo administration of keratinocyte growth factor (KGF). These results identify Sin as a novel regulator of mTEC development and T cell tolerance, and suggest that Sin is important for homeostatic maintenance of the medullary epithelium in the adult thymus.
PMCID: PMC2867288  PMID: 20404100
10.  Rank Signaling Links the Development of Invariant γδ T Cell Progenitors and Aire+ Medullary Epithelium 
Immunity  2012;36(3-2):427-437.
The thymic medulla provides a specialized microenvironment for the negative selection of T cells, with the presence of autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) during the embryonic-neonatal period being both necessary and sufficient to establish long-lasting tolerance. Here we showed that emergence of the first cohorts of Aire+ mTECs at this key developmental stage, prior to αβ T cell repertoire selection, was jointly directed by Rankl+ lymphoid tissue inducer cells and invariant Vγ5+ dendritic epidermal T cell (DETC) progenitors that are the first thymocytes to express the products of gene rearrangement. In turn, generation of Aire+ mTECs then fostered Skint-1-dependent, but Aire-independent, DETC progenitor maturation and the emergence of an invariant DETC repertoire. Hence, our data attributed a functional importance to the temporal development of Vγ5+ γδ T cells during thymus medulla formation for αβ T cell tolerance induction and demonstrated a Rank-mediated reciprocal link between DETC and Aire+ mTEC maturation.
Graphical Abstract
► Invariant Vγ5+ thymocytes regulate formation of Aire+ medullary thymic epithelium ► Generation of an invariant Vγ5+ T cell population requires thymus medulla development ► Skint-1-mediated Vγ5+ thymocyte development is Aire independent ► Dependency on Tnfrsf11a links γδ T cell and medullary epithelium development
PMCID: PMC3368267  PMID: 22425250
11.  The Wnt Signaling Antagonist Kremen1 is Required for Development of Thymic Architecture  
Wnt signaling has been reported to regulate thymocyte proliferation and selection at several stages during T cell ontogeny, as well as the expression of FoxN1 in thymic epithelial cells (TECs). Kremen1 (Krm1) is a negative regulator of the canonical Wnt signaling pathway, and functions together with the secreted Wnt inhibitor Dickkopf (Dkk) by competing for the lipoprotein receptor-related protein (LRP)-6 co-receptor for Wnts. Here krm1 knockout mice were used to examine krm1 expression in the thymus and its function in thymocyte and TEC development. krm1 expression was detected in both cortical and medullary TEC subsets, as well as in immature thymocyte subsets, beginning at the CD25+CD44+ (DN2) stage and continuing until the CD4+CD8+(DP) stage. Neonatal mice show elevated expression of krm1 in all TEC subsets. krm1− / − mice exhibit a severe defect in thymic cortical architecture, including large epithelial free regions. Much of the epithelial component remains at an immature Keratin 5+ (K5) Keratin 8+(K8) stage, with a loss of defined cortical and medullary regions. A TOPFlash assay revealed a 2-fold increase in canonical Wnt signaling in TEC lines derived from krm1− / − mice, when compared with krm1+ / + derived TEC lines. Fluorescence activated cell sorting (FACS) analysis of dissociated thymus revealed a reduced frequency of both cortical (BP1+EpCAM+) and medullary (UEA-1+ EpCAMhi) epithelial subsets, within the krm1− / − thymus. Surprisingly, no change in thymus size, total thymocyte number or the frequency of thymocyte subsets was detected in krm1− / − mice. However, our data suggest that a loss of Krm1 leads to a severe defect in thymic architecture. Taken together, this study revealed a new role for Krm1 in proper development of thymic epithelium.
PMCID: PMC2270768  PMID: 17162372
12.  Cell-Autonomous Defects in Thymic Epithelial Cells Disrupt Endothelial-Perivascular Cell Interactions in the Mouse Thymus 
PLoS ONE  2013;8(6):e65196.
The thymus is composed of multiple stromal elements comprising specialized stromal microenvironments responsible for the development of self-tolerant and self-restricted T cells. Here, we investigated the ontogeny and maturation of the thymic vasculature. We show that endothelial cells initially enter the thymus at E13.5, with PDGFR-β+ mesenchymal cells following at E14.5. Using an allelic series of the thymic epithelial cell (TEC) specific transcription factor Foxn1, we showed that these events are delayed by 1–2 days in Foxn1Δ/Δ mice, and this phenotype was exacerbated with reduced Foxn1 dosage. At subsequent stages there were fewer capillaries, leaky blood vessels, disrupted endothelium - perivascular cell interactions, endothelial cell vacuolization, and an overall failure of vascular organization. The expression of both VEGF-A and PDGF-B, which are both primarily expressed in vasculature-associated mesenchyme or endothelium in the thymus, were reduced at E13.5 and E15.5 in Foxn1Δ/Δ mice compared with controls. These data suggest that Foxn1 is required in TECs both to recruit endothelial cells and for endothelial cells to communicate with thymic mesenchyme, and for the differentiation of vascular-associated mesenchymal cells. These data show that Foxn1 function in TECs is required for normal thymus size and to generate the cellular and molecular environment needed for normal thymic vascularization. These data further demonstrate a novel TEC-mesenchyme-endothelial interaction required for proper fetal thymus organogenesis.
PMCID: PMC3672159  PMID: 23750244
13.  TSCOT + Thymic Epithelial Cell-Mediated Sensitive CD4 Tolerance by Direct Presentation  
PLoS Biology  2008;6(8):e191.
Although much effort has been directed at dissecting the mechanisms of central tolerance, the role of thymic stromal cells remains elusive. In order to further characterize this event, we developed a mouse model restricting LacZ to thymic stromal cotransporter (TSCOT)-expressing thymic stromal cells (TDLacZ). The thymus of this mouse contains approximately 4,300 TSCOT+ cells, each expressing several thousand molecules of the LacZ antigen. TSCOT+ cells express the cortical marker CDR1, CD40, CD80, CD54, and major histocompatibility complex class II (MHCII). When examining endogenous responses directed against LacZ, we observed significant tolerance. This was evidenced in a diverse T cell repertoire as measured by both a CD4 T cell proliferation assay and an antigen-specific antibody isotype analysis. This tolerance process was at least partially independent of Autoimmune Regulatory Element gene expression. When TDLacZ mice were crossed to a novel CD4 T cell receptor (TCR) transgenic reactive against LacZ (BgII), there was a complete deletion of double-positive thymocytes. Fetal thymic reaggregate culture of CD45- and UEA-depleted thymic stromal cells from TDLacZ and sorted TCR-bearing thymocytes excluded the possibility of cross presentation by thymic dendritic cells and medullary epithelial cells for the deletion. Overall, these results demonstrate that the introduction of a neoantigen into TSCOT-expressing cells can efficiently establish complete tolerance and suggest a possible application for the deletion of antigen-specific T cells by antigen introduction into TSCOT+ cells.
Author Summary
T cells play critical roles in the immune response. While developing in the thymus (from whence T cells and their precursors, thymocytes, derive their name), thymocytes are selected for the ability to recognize harmful antigen (positive selection), while those that respond to antigens present in their own body are eliminated (negative selection). Dogma holds that the thymus is divided into different functional compartments to ensure that these contrasting selection processes occur efficiently: the cortex is thought to be responsible for positive selection and the medulla for negative selection. In this study, we made use of a novel transgenic mouse (carrying a LacZ marker in a small fraction of cells in the cortex) to test whether the cortex is really excluded from negative selection. We were able to show that the introduced LacZ “antigen” present only in the cortical cells leads them to eliminate any LacZ-reactive T cells from the immune repertoire and leads to tolerance of the LacZ “antigen” by the body's immune system. This process is highly efficient, such that a relatively tiny number of antigen molecules present in a small fraction of the cells in the thymic cortex can singularly perform proofreading of all developing thymocytes.
A new study shows that antigen-specific negative selection of developing thymocytes is a property of thymic cortical epithelial cells, challenging the view that this process requires contact with specialized antigen-presenting cells found in the thymic medulla.
PMCID: PMC2494558  PMID: 18684012
14.  Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus 
Thymic epithelial cells (TECs) promote thymocyte maturation and are required for the early stages of thymocyte development and for positive selection. However, investigation of the mechanisms by which TECs perform these functions has been inhibited by the lack of genetic tools. Since the Foxn1 gene is expressed in all presumptive TECs from the early stages of thymus organogenesis and broadly in the adult thymus, it is an ideal locus for driving gene expression in differentiating and mature TECs.
We generated two knock-in alleles of Foxn1 by inserting IRES-Cre or IRES-lacZ cassettes into the 3' UTR of the Foxn1 locus. We simultaneously electroporated the two targeting vectors to generate the two independent alleles in the same experiment, demonstrating the feasibility of multiplex gene targeting at this locus. Our analysis shows that the knockin alleles drive expression of Cre or lacZ in all TECs in the fetal thymus. Furthermore, the knockin alleles express Cre or lacZ in a Foxn1-like pattern without disrupting Foxn1 function as determined by phenotype analysis of Foxn1 knockin/Foxn1 null compound heterozygotes.
These data show that multiplex gene targeting into the 3' UTR of the Foxn1 locus is an efficient method to express any gene of interest in TECs from the earliest stage of thymus organogenesis. The resulting alleles will make possible new molecular and genetic studies of TEC differentiation and function. We also discuss evidence indicating that gene targeting into the 3' UTR is a technique that may be broadly applicable for the generation of genetically neutral driver strains.
PMCID: PMC1906761  PMID: 17577402
15.  The thymic medulla: a unique microenvironment for intercellular self-antigen transfer 
The Journal of Experimental Medicine  2009;206(7):1505-1513.
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.
PMCID: PMC2715082  PMID: 19564355
16.  Inactivation of the RB family prevents thymus involution and promotes thymic function by direct control of Foxn1 expression 
The Journal of Experimental Medicine  2013;210(6):1087-1097.
RB family genes control T cell production and promote thymic involution through reducing Foxn1 expression in thymic epithelial cells.
Thymic involution during aging is a major cause of decreased production of T cells and reduced immunity. Here we show that inactivation of Rb family genes in young mice prevents thymic involution and results in an enlarged thymus competent for increased production of naive T cells. This phenotype originates from the expansion of functional thymic epithelial cells (TECs). In RB family mutant TECs, increased activity of E2F transcription factors drives increased expression of Foxn1, a central regulator of the thymic epithelium. Increased Foxn1 expression is required for the thymic expansion observed in Rb family mutant mice. Thus, the RB family promotes thymic involution and controls T cell production via a bone marrow–independent mechanism, identifying a novel pathway to target to increase thymic function in patients.
PMCID: PMC3674705  PMID: 23669396
17.  Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells 
Journal of immunology (Baltimore, Md. : 1950)  2010;185(8):10.4049/jimmunol.1002151.
The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire+ mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall’s corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire+ mTEC development, use of an inducible ZAP70 transgenic mouse line—in which positive selection can be temporally controlled—demonstrates that the emergence of involucrin+ mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire+ mTECs depends upon RANK signaling, continued mTEC development to the involucrin+ stage maps to activation of the LTα–LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.
PMCID: PMC3826119  PMID: 20861360
18.  DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells 
BMC Immunology  2012;13:58.
Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model.
We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire−/− mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire−/− mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire+/+ mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire+/+ and Aire−/− mTECs but distinct from that of athymic cells and tissues.
These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.
PMCID: PMC3546423  PMID: 23116172
Medullary thymic epithelial cells; Aire; T-DMR
19.  Delayed functional maturation of natural regulatory T cells in the medulla of postnatal thymus: role of TSLP 
BMC Immunology  2006;7:6.
Generation of functional CD4+CD8-CD25+ regulatory T cells (Treg) in the murine thymus depends on FoxP3. Removal of the thymus from neonatal mice has been shown to result in a multiple organ autoimmune disease phenotype that can be prevented by introducing the FoxP3+ Treg population to the animal. It has therefore, been proposed that functional FoxP3+ Treg cells are not made in the neonatal thymus; however, it remains unclear when and where functional FoxP3+CD4+CD8-CD25+ thymocytes are generated in postnatal thymus.
We report that neither FoxP3 mRNA nor protein is expressed in CD4+CD8-CD25+, or CD4+CD8-CD25- thymocytes until 3–4 days post birth, despite the presence of mature CD4+CD8-CD25+/- thymocytes in the thymus by 1–2 days after birth. FoxP3-CD4+CD8-CD25+ thymocytes from day 2 newborn mice show no Treg activity. Interestingly, we are able to detect low numbers of FoxP3+ thymocytes dispersed throughout the medullary region of the thymus as early as 3–4 days post birth. Expression of FoxP3 is induced in embryonic day 17 fetal thymus organ culture (FTOC) after 4–6 days of in vitro culture. Treatment of FTOCs with thymic stromal derived lymphopoietin (TSLP) enhanced expression of FoxP3, and blocking the TSLP receptor reduces FoxP3 expression in FTOC. Furthermore, TSLP stimulates FoxP3 expression in purified CD4+CD8- thymocytes, but not in CD4+CD8+, CD4-CD8+ and CD4-CD8- thymocytes.
Expression of FoxP3 or Treg maturation is ontogenically distinct and kinetically delayed from the generation of CD4+CD8-CD25+ or CD4+CD8-CD25- thymocytes in the postnatal thymus. TSLP produced from medullary thymic epithelia cells (mTEC) contributes to the expression of FoxP3 and the maturation of natural regulatory T cells. Overall, these results suggest that the development of Treg cells requires paracrine signaling during late stages of thymocyte maturation that is distinct from signaling during positive or negative selection.
PMCID: PMC1450317  PMID: 16579866
20.  Aire controls the differentiation program of thymic epithelial cells in the medulla for the establishment of self-tolerance 
The Journal of Experimental Medicine  2008;205(12):2827-2838.
The roles of autoimmune regulator (Aire) in the expression of the diverse arrays of tissue-restricted antigen (TRA) genes from thymic epithelial cells in the medulla (medullary thymic epithelial cells [mTECs]) and in organization of the thymic microenvironment are enigmatic. We approached this issue by creating a mouse strain in which the coding sequence of green fluorescent protein (GFP) was inserted into the Aire locus in a manner allowing concomitant disruption of functional Aire protein expression. We found that Aire+ (i.e., GFP+) mTECs were the major cell types responsible for the expression of Aire-dependent TRA genes such as insulin 2 and salivary protein 1, whereas Aire-independent TRA genes such as C-reactive protein and glutamate decarboxylase 67 were expressed from both Aire+ and Aire− mTECs. Remarkably, absence of Aire from mTECs caused morphological changes together with altered distribution of mTECs committed to Aire expression. Furthermore, we found that the numbers of mTECs that express involucrin, a marker for terminal epidermal differentiation, were reduced in Aire-deficient mouse thymus, which was associated with nearly an absence of Hassall's corpuscle-like structures in the medulla. Our results suggest that Aire controls the differentiation program of mTECs, thereby organizing the global mTEC integrity that enables TRA expression from terminally differentiated mTECs in the thymic microenvironment.
PMCID: PMC2585853  PMID: 19015306
21.  Autophagy in the Thymic Epithelium Is Dispensable for the Development of Self-Tolerance in a Novel Mouse Model 
PLoS ONE  2012;7(6):e38933.
The thymic epithelium plays critical roles in the positive and negative selection of T cells. Recently, it was proposed that autophagy in thymic epithelial cells is essential for the induction of T cell tolerance to self antigens and thus for the prevention of autoimmune diseases. Here we have tested this hypothesis using mouse models in which autophagy was blocked specifically in epithelial cells expressing keratin 14 (K14), including the precursor of thymic epithelial cells. While the thymic epithelial cells of mice carrying the floxed Atg7 gene (ATG7 f/f) showed a high level of autophagy, as determined by LC3 Western blot analysis and fluorescence detection of the recombinant green fluorescent protein (GFP)-LC3 reporter protein on autophagosomes, autophagy in the thymic epithelium was efficiently suppressed by deletion of the Atg7 gene using the Cre-loxP system (ATG7 f/f K14-Cre). Suppression of autophagy led to the massive accumulation of p62/sequestosome 1 (SQSTM1) in thymic epithelial cells. However, the structure of the thymic epithelium as well as the organization and the size of the thymus were not altered in mutant mice. The ratio of CD4 to CD8-positive T cells, as well as the frequency of activated (CD69+) CD4 T cells in lymphoid organs, did not differ between mice with autophagy-competent and autophagy-deficient thymic epithelium. Inflammatory infiltrating cells, potentially indicative of autoimmune reactions, were present in the liver, lung, and colon of a similar fraction of ATG7 f/f and ATG7 f/f K14-Cre mice. In contrast to previously reported mice, that had received an autophagy-deficient thymus transplant, ATG7 f/f K14-Cre mice did not suffer from autoimmunity-induced weight loss. In summary, the results of this study suggest that autophagy in the thymic epithelium is dispensable for negative selection of autoreactive T cells.
PMCID: PMC3377705  PMID: 22719991
22.  DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration 
PLoS ONE  2010;5(2):e9062.
Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.
Methodology/Principal Findings
Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of ΔNP63+ Foxn1+ and Aire+ TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.
Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.
PMCID: PMC2817005  PMID: 20161711
23.  Wnt4 and LAP2alpha as Pacemakers of Thymic Epithelial Senescence 
PLoS ONE  2010;5(5):e10701.
Age-associated thymic involution has considerable physiological impact by inhibiting de novo T-cell selection. This impaired T-cell production leads to weakened immune responses. Yet the molecular mechanisms of thymic stromal adipose involution are not clear. Age-related alterations also occur in the murine thymus providing an excellent model system. In the present work structural and molecular changes of the murine thymic stroma were investigated during aging. We show that thymic epithelial senescence correlates with significant destruction of epithelial network followed by adipose involution. We also show in purified thymic epithelial cells the age-related down-regulation of Wnt4 (and subsequently FoxN1), and the prominent increase in LAP2α expression. These senescence-related changes of gene expression are strikingly similar to those observed during mesenchymal to pre-adipocyte differentiation of fibroblast cells suggesting similar molecular background in epithelial cells. For molecular level proof-of-principle stable LAP2α and Wnt4-over-expressing thymic epithelial cell lines were established. LAP2α over-expression provoked a surge of PPARγ expression, a transcription factor expressed in pre-adipocytes. In contrast, additional Wnt4 decreased the mRNA level of ADRP, a target gene of PPARγ. Murine embryonic thymic lobes have also been transfected with LAP2α- or Wnt4-encoding lentiviral vectors. As expected LAP2α over-expression increased, while additional Wnt4 secretion suppressed PPARγ expression. Based on these pioneer experiments we propose that decreased Wnt activity and increased LAP2α expression provide the molecular basis during thymic senescence. We suggest that these molecular changes trigger thymic epithelial senescence accompanied by adipose involution. This process may either occur directly where epithelium can trans-differentiate into pre-adipocytes; or indirectly where first epithelial to mesenchymal transition (EMT) occurs followed by subsequent pre-adipocyte differentiation. The latter version fits better with literature data and is supported by the observed histological and molecular level changes.
PMCID: PMC2872673  PMID: 20502698
24.  Thymic Epithelial Cell Development and Its Dysfunction in Human Diseases 
BioMed Research International  2014;2014:206929.
Thymic epithelial cells (TECs) are the key components in thymic microenvironment for T cells development. TECs, composed of cortical and medullary TECs, are derived from a common bipotent progenitor and undergo a stepwise development controlled by multiple levels of signals to be functionally mature for supporting thymocyte development. Tumor necrosis factor receptor (TNFR) family members including the receptor activator for NFκB (RANK), CD40, and lymphotoxin β receptor (LTβR) cooperatively control the thymic medullary microenvironment and self-tolerance establishment. In addition, fibroblast growth factors (FGFs), Wnt, and Notch signals are essential for establishment of functional thymic microenvironment. Transcription factors Foxn1 and autoimmune regulator (Aire) are powerful modulators of TEC development, differentiation, and self-tolerance. Dysfunction in thymic microenvironment including defects of TEC and thymocyte development would cause physiological disorders such as tumor, infectious diseases, and autoimmune diseases. In the present review, we will summarize our current understanding on TEC development and the underlying molecular signals pathways and the involvement of thymus dysfunction in human diseases.
PMCID: PMC3929497  PMID: 24672784
25.  Human APECED; a Sick Thymus Syndrome? 
Loss-of-function mutations in the Autoimmune Regulator (AIRE) gene cause a rare inherited form of autoimmune disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, also known as autoimmune polyglandular syndrome type 1. The patients suffer from multiple endocrine deficiencies, the most common manifestations being hypoparathyroidism, Addison’s disease, hypogonadism, and secondary amenorrhea, usually accompanied by typical autoantibodies against the target tissues. Chronic mucocutaneous candidiasis is also a prominent part of the disease. The highest expression of AIRE is found in medullary thymic epithelial cells (mTECs). Murine studies suggest that it promotes ectopic transcription of self antigens in mTECs and is thus important for negative selection. However, failed negative selection alone is not enough to explain key findings in human patients, necessitating the search for alternative or additional pathogenetic mechanisms. A striking feature of the human AIRE-deficient phenotype is that all patients develop high titers of neutralizing autoantibodies against type I interferons, which have been shown to downregulate the expression of interferon-controlled genes. These autoantibodies often precede clinical symptoms and other autoantibodies, suggesting that they are a reflection of the pathogenetic process. Other cytokines are targeted as well, notably those produced by Th17 cells; these autoantibodies have been linked to the defect in anti-candida defenses. A defect in regulatory T cells has also been reported in several studies and seems to affect already the recent thymic emigrant population. Taken together, these findings in human patients point to a widespread disruption of T cell development and regulation, which is likely to have its origins in an abnormal thymic milieu. The absence of functional AIRE in peripheral lymphoid tissues may also contribute to the pathogenesis of the disease.
PMCID: PMC3791424  PMID: 24109480
APECED; AIRE; T cells; autoimmunity; thymus

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