# THYMIC STROMAL ALTERATIONS AND GENETIC DISORDERS OF IMMUNE SYSTEM

EDITED BY: Claudio Pignata and Ana E. Sousa PUBLISHED IN: Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88919-717-0 DOI 10.3389/978-2-88919-717-0

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## **THYMIC STROMAL ALTERATIONS AND GENETIC DISORDERS OF IMMUNE SYSTEM**

Topic Editors: **Claudio Pignata,** Federico II University, Italy **Ana E. Sousa,** Universidade de Lisboa, Portugal

The pathogenic mechanisms underlying primary T-cell disorders are mainly related to molecular alterations of genes whose expression is intrinsic to hematopoietic cells. However, since the differentiation process requires a crosstalk among thymocytes and the thymic microenvironment, molecular alterations of genes, involved in the differentiation and functionality of the stromal component of the thymus, may lead to a severe T-cell defect or failure of central tolerance, as well. The first example of severe combined immunodeficiency (SCID) not related to an intrinsic alteration of the hematopoietic cell but rather of the thymic epithelial component is the Nude/ SCID phenotype, inherited as an autosomal recessive disorder, whose hallmarks are the T-cell defect and the absence of the thymus. The clinical and immunological phenotype is the human equivalent of the murine Nude/SCID syndrome, which represents the first spontaneous SCID identified in nude mice in 1966. For over 3 decades studies of immune system in these mice enormously contributed to the overall knowledge of cell mediated immunity, in the assumption that the athymia of these mice was solely responsible for the T-cell immunological defect. This syndrome is due to mutations of the transcription factor FOXN1, belonging to the forkhead-box gene family, which is mainly expressed in the thymus and skin epithelial cells, where it plays a critical role in differentiation and survival. An alteration of the thymic structure is also a feature of the DiGeorge syndrome (DGS), which has been long considered the human counterpart of the nude mice phenotype. This syndrome is frequently associated to a deletion of the 22q11 region, which contains approximately 30 genes, including the TBX1 gene, which is responsible for most of the clinical features of DGS in humans and mice. In this syndrome common manifestations are cardiac malformations, speech delay, hypoparathyrodism and immunodeficiency, even though the immunological hallmarks of the T-cell defect in DiGeorge syndrome are profoundly different from those reported in human Nude/SCID. The divergence of the phenotype among these 2 entities raised the possibility that the FOXN1 transcription factor represents the real key stromal molecule implicated in directing the hematopoietic stem cell toward a proper T-cell fate. Thymic stromal component of the primary lymphoid organ is also required to negatively select the autoreactive clones, a process driven by the expression of tissue specific antigens (TSA) by medullary thymic epithelial cells (mTECs). The expression of genes encoding TSA antigens is mediated by autoimmune regulator (AIRE) gene, encoding a transcription factor expressed in mTECs. Molecular alterations of this gene are associated to autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a rare autosomal disorder, which may be considered the prototype of an autoimmune disease due to the failure of central tolerance homeostasis.

All these "experiments of nature" led to unravel novel pathogenic mechanisms underlying inherited disorders of immune system and, of note, to clarify the pivotal role of epithelial cells in the maturation and education process of T-cell precursors.

**Citation:** Pignata, C., Sousa, A. E., eds. (2015). Thymic Stromal Alterations and Genetic Disorders of Immune System.

T. Petteri Arstila and Hanna Jarva

Lucia De Martino, Donatella Capalbo, Nicola Improda, Federica D'Elia, Raffaella Di Mase, Roberta D'Assante, Ida D'Acunzo, Claudio Pignata and Mariacarolina Salerno

*56 Immunodeficiency in DiGeorge syndrome and options for treating cases with complete athymia*

E. Graham Davies

*65 Lymphopenia-induced proliferation in Aire-deficient mice helps to explain their autoimmunity and differences from human patients*

Kai Kisand, Pärt Peterson and Martti Laan

## Thymic stromal alterations and genetic disorders of immune system

## **Claudio Pignata1\*, Roberta D'Assante1 and Ana E. Sousa2**

1 Department of Translational Medical Sciences, Federico II University, Naples, Italy

2 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

\*Correspondence<EMAIL_ADDRESS>
**Edited and reviewed by:**

Ellis L. Reinherz, Dana-Farber Cancer Institute, USA

**Keywords: severe combined immunodeficiency, FOXN1, central tolerance, medullary thymic epithelial cells, DiGeorge syndrome, rag defects, APECED**

In this specialty section of the journal, we host a topic focused on thymic stromal alterations and genetic disorders of immune system. The thymus is a specialized organ of the immune system where, through stage-specific differentiation of hematopoietic progenitor cells, fully mature and self-tolerant T cells origin. The process is strictly dependent on the link between the thymic stromal cells (TSCs), which allow the selection of a functional and self-tolerant T-cell repertoire, and the thymus tridimensional architecture. Indeed, the interaction between the developing thymocytes and the stromal cells is crucial for the development of both T cells and TSCs (1, 2). In both human and mice, the primordial thymic epithelial cells (TECs) are yet unable to fully support the T-cell development and only after the transcriptional activation of the *Forkhead-box n1* (*FOXN1*) gene, this essential function is acquired. Most of the information concerning the T-cell development came out from studies on mice carrying null mutation in *FOXN1* gene. In humans, as detailed in the Romano et al. review, 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 (3). As extensively approached in the Villa et al. review, the intercellular cross-talk is also essential to support the maturation of Foxp3C natural regulatory T cells. In Omenn syndrome (OS), caused by hypomorphic Rag alterations, an infiltration of peripheral tissues by activated T cells and immune dysregulation have been found (4). The authors discuss on abnormalities of thymic microenvironment in OS with a special focus on the defective maturation of TECs, and impairment of central tolerance.

The commonest association of thymic stromal deficiency resulting in T-cell immunodeficiency is the DiGeorge syndrome (DGS), discussed in the Davies review. In this syndrome, however, the immunological impairment is highly variable, ranging from normal to a severe immune defect in rare individuals, thus suggesting that partial thymic hypoplasia may occur or that extrathymic sites of differentiation play a role in the process (5). The difference in the immunological defects between DGS and the Nude/SCID phenotypes implies that FOXN1 controlled genes are mandatory for a fully mature T-cell development process rather than the integrity of the thymus itself.

It is known that autoimmune regulator (*AIRE*) gene plays a central role in the induction of central tolerance, and different mechanisms of action have been hypothesized for this process. According to the most reliable theory, AIRE directly induces the production of tissue-specific antigens (TSA) (6). However, recent evidence suggests that another mechanism for negative selection of self-reactive thymocytes may be due to AIRE-induced differentiation of medullary TECs, and regulation of the expression of intrathymic chemokines directed to antigens presenting cells (APCs),such as thymocytes and dendritic cells (7). In their reviews, Laan and Peterson and Kisand et al. give an overview on what is known about the different mechanisms through which AIRE induces central tolerance.

The process aimed at the elimination of potential self-reactive T cells in the thymus is crucial for preventing the onset of autoimmune diseases. As discussed in the Akiyama et al. paper, medullary epithelial cells play a central role in the process through the regulation of gene expression, and, in particular, of those genes encoding for the TNF family cytokines,RANK ligand,CD40 ligand,and lymphotoxin. These genes promote the differentiation of AIRE- and TSA-expressing mTECs (8).

The mechanism by which a single *AIRE* gene can influence the transcription of such a large number of TSA within mTECs has been discussed in the Matsumoto et al. paper. Two models have been proposed. The first one implies a direct transcriptional control of *AIRE* on TSA, while the second one is based on the role of *AIRE* on the maturation program of mTECs (9).

The clinical and immunological phenotype of patients affected with autoimmune polyendocrinopathy ectodermal dystrophy (APECED), as reviewed by Petteri Arstila and Jarva, is characterized by multiple endocrine deficiencies, the most common manifestations being hypoparathyroidism, Addison's disease, hypogonadism, and secondary amenorrhea, usually associated with the presence of autoantibodies toward the target tissues (10). However, the phenotype and, therefore, the underlying pathogenic mechanism, are even more complex, in that Chronic Mucocutaneous Candidiasis is also a prominent part of the disease. This clinical entity is related to abnormalities in the Th17-related cytokines, which are mostly involved in immune defenses against Candida (11). Finally, high titers of neutralizing autoantibodies against type I interferons, which have been shown to downregulate the expression of interferon-controlled genes, have been documented (12).

In this Research Topic, De Martino et al. focus their attention on the complexity of the APECED phenotype in that a wide variability of the clinical expression, in the presence of the same genotype alteration, has been found (13). They suggest that additional mechanisms, in addition to AIRE function, are involved in the pathogenesis of the disease. This might be helpful to understand not only the molecular basis of APECED but will also help improve diagnosis, management, and therapeutic strategies to treat this complex disease.

## **REFERENCES**

the transcription model or the maturation model? *Front Immunol* (2013) **4**:210. doi:10.3389/fimmu.2013.00210

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 16 January 2015; accepted: 11 February 2015; published online: 25 February 2015.*

*Citation: Pignata C, D'Assante R and Sousa AE (2015) Thymic stromal alterations and genetic disorders of immune system. Front. Immunol. 6:81. doi: 10.3389/fimmu.2015.00081*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2015 Pignata, D'Assante and Sousa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## FOXN1: a master regulator gene of thymic epithelial development program

#### **Rosa Romano1 , Loredana Palamaro1 , Anna Fusco1 , Giuliana Giardino1 , Vera Gallo1 , Luigi Del Vecchio2 and Claudio Pignata1\***

1 Department of Translational Medical Sciences, "Federico II" University, Naples, Italy

2 Department of Biochemistry and Medical Biotechnology, CEINGE Institute, "Federico II" University, Naples, Italy

## **Edited by:**

Ana E. Sousa, Universidade de Lisboa, Portugal

### **Reviewed by:**

Graham Anderson, University of Birmingham, UK Maria L. Toribio, Spanish National Research Council, Spain

### **\*Correspondence:**

Claudio Pignata, Department of Translational Medical Sciences, "Federico II" University, Via S. Pansini 5, Naples 80131, Italy e-mail<EMAIL_ADDRESS>
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) driveT 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, includingTbx1, 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 downstream 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.

**Keywords: Foxn1 gene,TECs, thymus gland, immunodeficiency, Nude/SCID**

## **INTRODUCTION**

The thymus is the primary lymphoid organ with the unique function to produce and to maintain the pool of mature and functional T cells. This process is strictly dependent on specialized functions of thymic stromal cells (TSCs) and requires the thymus peculiar tridimensional (3D) architecture, which allows a proper intercellular cross talk (1). For a long time, the difficulty in the isolation and characterization of the thymic cellular components has limited studies on the peculiar role of individual stromal components. Novel experimental tools, including stromal cell isolation by phenotype-based cell sorting (2), dissociation and reaggregation of stromal cell subsets (3, 4), or global gene expression analysis and the evaluation of the pattern of self-antigen expression within the individual thymic epithelial cells (TECs) subset (5), allowed to acquire important knowledge on the cellular and molecular basis of thymus organogenesis and TECs functionality.

The recent discovery of disease models associated to genetic alterations of molecules implicated in thymus specification and TECs differentiation,provided new and conclusive insights regarding the pathways, the genes, and the molecular mechanism governing these processes and stromal functionality.

## **THE THYMUS ARCHITECTURE: REQUIREMENT OF A 3D STRUCTURE FOR A PROPER LYMPHO-EPITHELIAL CROSSTALK**

The thymus provides the microenvironment essential for the development of T cells. T cell progenitors originate in the bone marrow, enter into the thymus (6, 7) and, through a series of well defined and coordinated developmental stages, differentiate, undergo selection process, and mature into functional T cells. The steps in this process are tightly regulated through a complex network of transcriptional events, specific receptor-ligand interactions, and sensitization to trophic factors, which mediate the homing, proliferation, survival, and differentiation of developing T cells (1, 8, 9).

The thymus is organized in two lobes, which are already present in mice at 21 days of thymic organogenesis and is completely organized at 1 month of post-natal life. The lobes are divided in three areas: a cortical and the dark cortical area, with a high number of lymphoid cells and epithelial cells, cortical thymic epithelial cells (cTECs); a light medullary area with a low number of mature T cells, named medullary TECs (mTECs), Hassall's bodies (HB), macrophages, dendritic cells (DCs), B lymphocytes, and rarely myoid cells. Eventually, there is a transitional area, named cortico-medullary junction (CMJ), characterized by abundant blood vessels (10).

The unique function of the thymus in the establishment and maintenance of the T cell pool is intimately linked to this peculiar thymus architecture and to the specialized functions of the TSCs.

## **LYMPHO-EPITHELIAL CROSS-TALK REQUIRED FOR THYMOCYTE AND TECs DIFFERENTIATION**

An important feature of the thymic microenvironment is its 3D organization, consisting of an ordered architecture of TSCs, that represents a heterogeneous mixture of distinct cell types, including cTECs, mTECs, fibroblasts, endothelial cells, DCs, and macrophages (11). Among these stromal elements, TECs are the most abundant cell types, which form a delicate 3D cellular network spanning throughout both the thymic cortex and the medulla. The requirement for the 3D-supporting stroma appears to be unique to the T cell development, as the *in vitro* differentiation program of other hematopoietic lineages, including B and NK cells, does not require a 3D structure (12).

Thymocyte development is not a cell-autonomous process, and the transition to the next stage in development relies on the proper interaction of HSCs with thymic stroma. The 3D configuration of the thymus maximizes this interaction, allowing intercellular cross-talk integral to the development of both T cells and TSCs (13). Paralleling the T cell precursor proliferation and differentiation program, immature TECs undergo a developmental sequence, resulting in the establishment of mature cTECs and mTECs organized in this 3D network. Several studies on mutant mice with an abnormal organization of thymic epithelium substantiated the concept that a reciprocal signaling between thymocytes and TSCs is required, not only for the production of mature T cells but also for the development and organization of the thymic microenvironment in a bi-directional fashion (14, 15). Mice showing a blockage of the T cell development process, in the absence of T cell receptor (TCR)-expressing cells, have a defective organization of the thymic medulla, as well (16, 17). Of note, under this condition, thymic medullary organization can be restored by the addition of mature T cells, which follows stem cell transplantation (17, 18). In adult CD3etg26 mice, lacking intra-thymic T cell precursors, a severe alteration of the cortical thymic architecture has been documented (19), even though a restoration of the architecture and TEC development in these mice can occur. Recently, the injection of either fetal or adult T-committed precursors into adult CD3etg26 mice leads to the reconstitution of thymic microenvironment, as indicated by thymocyte differentiation, organization of functional cortical and medullary areas, and generation of Foxp3+ Treg and Aire+ mTECs (20). These data suggest that adult TECs maintain the receptivity to cross talk with thymocytes despite a prolonged absence of T cell precursors. Moreover, the absence of both thymocytes and of the 3D framework may result in changes of the keratin genes expression, thus inducing the cTECs and mTECs to undergo a de-differentiation process and to reacquire the precursor K5+K8+ cellular phenotype. Taken together, these findings suggest that signals from early CD4–CD8– DN T cell precursors and/or their immediate progeny provide necessary signals to promote the formation of the

thymic cortex, while, later in ontogeny, the differentiation of TECs into a medullary phenotype are clearly dependent on the presence of CD4+CD8− and CD4−CD8+ single positive (SP) thymocytes (21–23). However, the precise molecular nature of the signals provided by developing thymocytes, which lead to the generation of the thymic stromal compartment are still incompletely defined.

Eventually,a better understanding of the developmental process through which a normal thymus structure is built, is essential for a better comprehension of the intimate mechanisms which take place within the thymus to promote the T cell development *in vivo*. This knowledge may also be useful in designing future therapeutic strategies, as alterations of the thymus structure and function may result in serious health consequences, including immunodeficiency or autoimmunity.

## **mTECS AND cTECS ARE SPECIALIZED CELLS PLAYING A DIFFERENT ROLE IN THE T CELL EDUCATION PROCESS**

T cell ontogeny is a sophisticated process, which takes place through discrete stages during which developing thymocytes dynamically relocate in different thymic areas, following a corticomedullary gradient.

The initial colonization of the thymus anlagen by migrant lymphoid progenitors occurs at an early stage, embryonic day 11.5 (E11.5) in mice and 8 week of gestation in humans (24, 25). Studies documented that chemokines CC ligand (CCL)21 and CCL25 play a major role in the early stage of fetal thymus colonization (26, 27). Indeed, mice deficient for these chemokines or for the cognate receptors, showed a significant reduction in the number of thymocytes compared to normal mice (28). In post-natal thymus, lymphoid progenitor cells through their cell surface adhesion molecules, such as platelet-selectin glycoprotein ligand 1, interact with P-selectin, expressed on the TECs, and thanks to this interaction they are allowed to migrate from the blood into the thymic parenchyma, in correspondence of the area around the CMJ [**Figure 1**; (29)].

Entered thymocytes started to intensely proliferate and to acquire T cell hallmarks. In this phase, T cell proliferation and differentiation are triggered by a potent combination of signals provided by cTECs. Delta-like 4 (DL4), which is an essential, nonredundant ligand for Notch1 during thymic T cell development, and IL-7 are critically involved in the activation of signaling pathways, leading to the proliferation and migration of thymocytes (30–32). In particular, these intra-thymic ligands induce the development of DN CD25+ cells, which migrate toward the subcapsular region of thymic cortex (33). Several chemokine receptors have been suggested to guide the migration of immature thymocytes, such as CXCR4, CCR7, and CCR9 [**Figure 1**; (34)]. In the thymic cortex DN thymocytes begin V(D)J rearrangement of their TCRβ gene. Successfully rearranged TCRβ protein, assembled with the pre-TCRα chains, forms the pre-TCR complex. Membrane expression of pre-TCR complex, along with the Delta-Notch interaction, provides the signal necessary to induce the expression of the coreceptors CD4 and CD8, as well as V-J rearrangement of the TCRα genomic region. Subsequently, DP thymocytes with a functional TCR-αβ receptor are generated [**Figure 1**; (35)].

Thymic cortex is also the area where takes place the positive selection of DP thymocytes. Positive selection is the process by

allows the relocation of DN thymocytes from the cortex to the subcapsular region. DP thymocytes bearing TCR and capable of binding to self-MHC

thymocytes are deleted through the negative selection, a process mediated by dendritic cells and Aire-expressing mTECs.

which developing thymocytes, that recognize and bind with mild avidity peptide-major histocompatibility complex on cTECs surface, get a rescue signal through their TCR and are allowed to further maturate to the CD4+CD8− or CD4−CD8+ SP stage. Only a small fraction (1–5%) of DP cells survive to positive selection. By contrast, the majority of DP cells, that bind with too low affinity to MHC complex, are programed to undergo death by neglect (36, 37).

Cortical thymic epithelial cells have a crucial role in the positive selection process of T cells within thymus cortex (38). Recent studies have found that cTECs exclusively express a specific form of proteasome, referred as thymoproteasome, which contains a peculiar catalytic subunit, the β5-thymus (β5t) (39). β5t subunits exhibit an unique peptidase activity, compared to other β5 subunits found in common immunoproteasome, which leads to the production of a set of self-peptides with a high affinity for class I MHC molecules (40). Moreover, β5t-deficient mice show a severe decrease in the number of CD8+ SP thymocytes, but no alteration in the CD4+ number or in the thymic architecture. In addition, the small fraction of CD8+ T cells, positively selected by β5t-deficient cTECs, show altered immune responses toward several stimuli. Taken together these results suggest that the thymoproteasome is essential for the production of self antigens involved in the positive selection of functional CD4−CD8+ T cells (41).

As for the positive selection of CD4+ T cells, two other proteins predominantly expressed in cTECs, the lysosomal protease Prss16 and Cathepsin L, have been demonstrated to be essential to generate an immunocompetent repertoire of CD4+CD8− T cells [**Figure 1**; (42, 43)].

TCR engagement by peptide-MHC complex also triggers the expression of the chemokine receptor CCR7 in positively selected thymocytes. Thanks to the chemotactic attraction between CCR7 and its ligands, CCL19 and CCL21, expressed on the mTECs, developing thymocytes are relocated from cortex to the medulla [**Figure 1**; (44, 45)].

In order to create a repertoire of mature T cells able to recognize foreign antigens and, at the meantime, to ignore self antigens, SP thymocytes have to undergo the negative selection process in the thymic medulla. Both mTECs and DCs, play a pivotal role in this last stage of thymocyte development, which is critical to establish the central tolerance and, eventually, to prevent autoimmunity. In contrast to cTECs, mTECs are characterized by a high expression of clustered tissue-restricted autoantigens (TSAs), the so called promiscuous gene expression (46). To date, the autoimmune regulator (AIRE) transcription factor represents the only molecule, so far identified, which contributes to the mTECs function and, in particular, to the molecular regulation of the promiscuous gene expression [**Figure 1**; (47)]. However, not all TSAs are regulated in an AIRE-dependent manner, suggesting that other molecular mechanisms, such as epigenetic mechanisms, may be involved in mTECs function regulation. TSAs associated with class II MHC molecules are presented directly by mTECs or indirectly by DCs to developing thymocytes (48). T cells which recognize with a high avidity self antigens are deleted. Remarkably, only a few number of mTECs express a given TSAs (about 50–500 per thymus), and lead to apoptosis by negative selection of a few thymocytes (37, 49, 50). A possible explanation is that the high motility of thymocytes within the thymic medulla during a period of 4–5 days, allows each of them to interact with mTECs (51). DCs play a similar role in the negative selection process. They are attracted in the thymic medulla by the chemochine XCL1 (lymphotactin), produced by mTECs in an AIRE-dependent manner. Differently from mTECs, DCs are not able to produce TSAs and the TSAs expressed mostly derive from the phagocytosis of apoptotic mTECs (52, 53). mTECs and DCs not only contribute to the establishment of central tolerance through the deletion of self-reactive T cells, but, also, through the generation of regulatory T cells (Tregs) (54, 55, 65, 153), which act in the periphery by suppressing autoreactive T cells, which have escaped to the process of the central tolerance.

A body of evidence documents that the expression of an autoreactive TCR leads to the entry of the thymocyte into the Treg lineage. Tregs, that are about 5–10% of peripheral T cells CD4+, constitutively express the CD25 molecule and share several immunological features, in humans and mice (56, 57). These cells specifically express the transcription factor FOXP3 (Foxp3 in mice) that plays a pivotal role in Tregs differentiation and function (58). The Foxp3 promoter region and the conserved non-coding sequence 2 (CNS2) (known as TSDR, the Treg-specific-demethylated-region) are fully methylated in immature thymocytes (59, 155). At the beginning of Treg development, an appropriate TCR/CD28 signal is needed to make available the *Foxp3* promoter through shift of the Protein Inhibitors of Activated STAT 1 (PIAS1), a signal cascade, which results in the NF-κB-mediated transcription of genes playing a role in Treg differentiation (60, 61).

## **THYMIC FORMATION: NEW INSIGHTS IN EPITHELIAL LINEAGES SPECIFICATION**

In the mouse, mTECs and cTECs originated from the third pharyngeal pouch endoderm and the thymus anlage are located next to that of the parathyroid. The expression of Forkhead-box transcription factor n1 (Foxn1) approximately at E11.5 is crucial for the subsequent epithelial differentiation, since in its absence, the colonization of the anlage by T cell progenitors from the bone marrow fails (62) and the subsequent T cell development and TECs formation is aborted, resulting in a severe immunodeficiency (63, 64, 66, 154).

The maturation process of TECs during thymic organogenesis could be divided in two genetic phases. The first stage is independent from the *Foxn1* expression and consists in the induction and outgrowth of the thymic epithelial anlage from the third pharyngeal pouch, through the expression of genes including the *Eya1* and *Six* (67), *Hoxa3* (68), and *Tbx1* (69, 70). During the second genetic phase, epithelial patterning and differentiation take place and the *Foxn1* expression drives the immature epithelial cells to differentiate into functional cTECs and mTECs (71).

## **FOXN1-INDIPENDENT GENETIC STAGE OF TEC DIFFERENTIATION**

In the first phase of the thymus organogenesis an interaction between epithelial and mesenchymal cells occurs, while at the later phase lympho-epithelial interaction predominates (72). In mice, at about E10.5 the mesenchymal cells are able to respond to the endodermic signals, which induce the development of the primordial thymic epithelium (73, 74). Subsequently, at about E12.5, the thymic rudiment is colonized by progenitors come from the fetal liver, thus resulting in a tight epithelialthymocyte interaction within the mesenchymal derived capsule. This thymic rudiment contains the EpCam+Plet1+ epithelial population (72, 75), which includes a common thymic epithelial precursor (TEPC), from which both cTECs and mTECs will be subsequently generated (72, 76).

Through studies on animal models carrying molecular alterations of distinct genes, the key role of several transcription factors involved in the thymus organogenesis and TEC-sublineage specification process, have thus far been identified (77). In particular, several genes, including *Tbx1* (69, 70), *Pax1*, *Pax3*, *Pax9* (78–80), *Hoxa3* (68), *Eya1*, and *Six1* (67) have been shown to play a central role in the thymus ontogeny. Indeed, their molecular alteration affects this well-orchestrated process, leading to disruption of the thymic architecture. Abnormalities of the paired box (Pax) family transcription factors Pax1 or Pax9 result in a blockage of the thymus organogenesis (79, 81). Mutations in the Hox transcription factor family member, Hoxa3, expressed on both thymic epithelium and mesenchymal cells, result in athymia (68). Furthermore, the homozygous loss of *Tbx1*, related to the DiGeorge syndrome phenotype, leads to thymic a/hypoplasia in humans (69, 82), while mice heterozygous for a null allele of *Tbx1* show a mild phenotype without thymus anomalies (83). Therefore, the expression of *Tbx1* both in the pharyngeal core mesoderm and in the pharyngeal endoderm is required for a proper thymus development. However, it remains to be elucidated whether the expression of *Tbx1* in the TECs occurs and whether the gene participates in the TECs development (4).

## **FOXN1-DEPENDENT GENETIC STAGE OF TEC DIFFERENTIATION**

In both humans and mice, the primordial TECs are yet unable to fully support T cell development and only after the transcriptional activation of the *FOXN1* gene in the thymic epithelium this essential function is acquired. *FOXN1* is a master regulator in the TEC lineage development in that it promotes down-stream the transcription of genes implicated in the thymus organogenesis and TECs full differentiation.

Forkhead-box n1 transcription factor belongs to the FOX transcription factor family implicated in a variety of biochemical and cellular processes, including development, metabolism, aging, and cancer (84, 85). During the post-natal life, *Foxn1* is selectively expressed only in thymic and skin epithelia, where it regulates the expression of several molecular targets to maintain the balance between growth and differentiation (86, 87). The signals required for *FOXN1* expression, and its activity, are still unclear, even though the wingless (Wnt) proteins (88) and bone morphogenetic protein (BMP) signaling have been shown to regulate *FOXN1* expression (89). Even though the complete pattern of *FOXN1* expression over the time and its role are not yet completely defined, studies on mouse and human model of gene alterations enormously helped unravel important issues on its role. Mutations in *Foxn1* gene lead to alymphoid cystic thymic dysgenesis due to a defective TECs differentiation process (63, 90). In both mice and humans *FOXN1* abnormalities lead to a hairless phenotype (87, 154).

In the *Foxn1*-dependent step of thymus organogenesis, precursor epithelial cells differentiate into mature and functional cTECs and mTECs from the same bi-potential TEC progenitor (4, 72, 76). It has been reported, that *Foxn1* is differentially expressed during the TE-lineage specification, since it is expressed in all TECs during the pre-natal life, but not in all TECs postnatally, indicating that the gene is highly developmentally regulated. There is a body of evidence documenting different effects of *Foxn1* expression in mTEC and cTECs. Particularly, studies on K5- and K18-CreERTmediated *Foxn1*-deleted mouse models suggested that during the post-natal life, the loss of *Foxn1* affected mTECs, characterized by the expression of K5 and K14 keratins type. Conversely, the loss of *Foxn1* did not affect cTECs, which express the keratins K8 and K18 (91, 92). Taken together, these data suggest that cTECs and mTECs are not equally *Foxn1*-dependent in the post-natal life.

Recent reports highlighted a central role for *Foxn1* in TECs homeostasis in the adult thymus and its necessary role for the functionality and survival of adult TEC progenitors (92), expressing K5+ and K14+ markers. This role in adult thymus seems to be exerted in cooperation with other stem cell-related genes, such as *p63*. Of note, the transcription factor p63, encoding for multiple isoforms (93), plays a pivotal for the development of stratified epithelia of several tissues, such as epidermis, breast, prostate, and thymus (94). In the thymus, the p63 protein drives the proliferation of epithelial progenitor cells (94, 95). Therefore, it has been hypothesized that *p63* and *Foxn1* could act synergistically through the formation of a *p63*-*Foxn1* regulatory axis aimed at regulating TECs homeostasis. However, the molecular mechanism through which the proliferation regulator *p63* and differentiation regulator *Foxn1* collaborate in this axis are still unclear.

## **FOXN1-MEDIATED GENE EXPRESSION FOR TEC DIFFERENTIATION**

*Forkhead-box n1* is directly or indirectly implicated in the transcriptional regulation of a panel of genes involved in thymus development and function.

*Pax1* is a key regulator of TEC differentiation/survival balance. *Pax1* is expressed in the third pharyngeal pouch from E9.5 during the thymus ontogeny, while in the post-natal thymus only in cTEC (96). Even though the regulation of *Pax1* is still unclear, from E11.0 its expression requires *Hoxa3* (68). Of note, the loss of *Hoxa3* impairs the intrinsic ability of the neural crest cell population to differentiate and/or to lead to the differentiation of the

tissues of pharyngeal arch and pouch. Indeed, in *Hoxa3* mutant mice the thymus is absent and thyroid hypoplasia has been documented (68). Moreover, the first step of thymus development is the expansion of mesenchymal neural crest in the posterior part of the third pharyngeal pouch. Prior to this event, in the *Hoxa3* mutant embryos a marked reduction in *Pax1* expression has been shown. Similarly, *Pax1* mutant mice also show thymic hypoplasia, suggesting a role for *Hoxa3* in maintaining *Pax1* expression in these cells (68). In the thymic primordium, *Pax1* expression is under the control of *Foxn1* (71). This finding indicates that *Foxn1* and *Hoxa3* are both involved in the network of molecular signals that regulates *Pax1* expression, thus demonstrating the existence of a molecular and/or functional interaction between *Hoxa3* and *Foxn1* [**Figure 2**; (71)]. In keeping with this, Hoxa3+/−Pax1−/− compound mutant mice display a few phenotypic hallmarks of the Foxn1R/R mouse model, which expresses low-dose of *Foxn1*, such as hypomorphic post-natal thymus, and reduced levels of MHC class II expression on the TECs surface (80). These data suggest two alternative hypothesis: *Hoxa3* may regulate *Foxn1*, which, in turn, regulates *Pax1* expression in the thymic primordium, in a *Foxn1*-dependent manner, or *Hoxa3* and *Foxn1* induce *Pax1* expression in the third pharyngeal pouch and in early thymus primordium.

It has also been shown that *Foxn1* regulates the expression of *CCL25* and *Dll4* (**Figure 2**). These genes play a pivotal role in the thymocyte development, since *CCL25* regulates the colonization of the fetal thymus (97), while the Notch ligand *Dll4* is involved in the commitment of hematopoietic progenitors to the T cell lineage (30). In both early fetal TEC and in the post-natal thymus, *Dll4* expression is directly related to the *Foxn1* expression (71). Furthermore, these molecules are absent in the *Foxn1* null thymus, even though there is evidence indicating that their expression may occur in a *Foxn1*-independent manner in TECs (98, 99). Eventually, in a recent report it has been shown that *Foxn1* is upstream of dll4a and ccl25a expression in *medaka fish*, thus confirming the relationship with this transcription factor (100).

## **THE HUMAN NUDE/SCID PHENOTYPE: A MODEL OF THYMIC MICROENVIRONMENT DISRUPTION AND FAILURE OF THE T CELL DEVELOPMENT**

The Nude/severe combined immunodeficiency (SCID) phenotype represents the prototype of thymic architecture disruption due to alterations of the *FOXN1*, which is the master regulator of TE-lineage specification (71).

In humans, as in mice and rats, mutations in the "nude" *Foxn1* gene induce the hairless phenotype, associated with a rudimentary thymus gland (T cell related primary immunodeficiency). The human Nude/SCID phenotype (MIM 601705; Pignata Guarino Syndrome) was first identified in 1996, after more than 30 years from the initial mouse description, in two sisters originated from a small community with a high grade of inbreeding, who showed congenital alopecia of the scalp, eyebrows, and eyelashes, nail dystrophy, and a severe T cell immunodeficiency, inherited as an autosomal recessive disorder (154). This phenotype was associated with a C792T transition in the *FOXN1* gene, which resulted in the nonsense mutation R255X in the exon 4 (formerly exon 5), with a complete absence of a functional protein similar to the previously described rat and mouse *Foxn1* mutations (101–103).

and functional T cells.

In the absence of *Foxn1* expression, thymic development is halted at a rudimentary stage. As a consequence, in the affected patients the thymic lobe is still present but intra-thymic lymphopoiesis is completely blocked (63, 104) leading to severe primary T cell immunodeficiency (105–107) and to death in early childhood from severe infections (105, 108–112, 154). *Foxn1* is also involved in morphogenesis and maintenance of the 3D thymic micro-structure, which is necessary for a fully functional thymus (113, 114). In fact, evidence is available that in an *in vitro* 2D culture system consisting of a monolayer of mouse bone marrow stromal OP9 cells it is possible to generate mature T cells, only if these cells are transduced with the Notch ligand Deltalike 1 (OP9-DL1) (115, 116), whose pathway exerts a pivotal and necessary role in promoting the induction of T cell-lineage commitment (117–119). Of note, in all these co-culture systems, the stromal cells are enforced to overexpress Notch ligands, and their expression by TECs seems to be maintained only in a 3D thymus structure (120). In human Nude/SCID, the T cell defect is

Eya1 are also required in neural crest cells (NCCs). In the phase 2, Foxn1

characterized by the absence of proliferative response to the common mitogens and a severe blockage of the T cell differentiation (154). Recent studies revealed the presence of some circulating T cells of non-maternal origin in patients carrying alterations of *FOXN1* gene. These cells have been shown to be predominantly double-negative αβT cells (CD3+CD4−CD8−, DN) and to exhibit a regulatory like T cell phenotype (FoxP3+). This finding raised important issues regarding the site of differentiation of these cells. One hypothesis is the persistence of a thymic rudiment, which allows a partial T cell development (109). Alternatively, a T cell differentiation, even though partial and ineffective to result in a productive immunity, could occur at an extra-thymic site. In both pre-natal and post-natal life, the TCRBV spectratype repertoire in Nude/SCID patients is oligoclonal, thus confirming the immaturity of the process and, at the same time, that developmental events do take place at some extent (111, 112).

For many years, the human counterpart of the nude mouse phenotype has been erroneously considered the DiGeorge syndrome, which occurs spontaneously and is mainly characterized by thymic hypo/aplasia and a mild T cell defect. However, several lines of evidence argue against the analogy between these two disorders. In fact, the DiGeorge syndrome is often associated with neonatal tetany and major anomalies of great vessels. These defects are due to malformation of the parathyroid and heart, derived from a major embryologic defect in the third and fourth pharyngeal pouch from which the thymus primordium emerges. In addition, in this syndrome hairlessness is missing and gross abnormalities of skin annexa are not found. Children with DiGeorge syndrome may also have lymphopenia, with a mild reduction of T cells, that are however usually responsive to common mitogens.

In Nude/SCID patients, skin is tighter than usual and is characterized by basal hyperplasia and dysmaturity. Alopecia is primitive in nature, in that it can be observed at birth and persists after bone marrow transplantation, thus ruling out the acquired nature of the disorder. In keeping with this, in athymic mice, completely lacking body hair, restoration of the thymus did not lead to hair growth, indicating a direct participation of FOXN1 to hair follicle development (87). The most frequent phenotypic alteration affecting the nails is koilonychia ("spoon nail"), characterized by a concave surface and raised edges of the nail plate, associated with significant thinning of the plate itself; canaliform dystrophy and a transverse groove of the nail plate (Beau line) may also be observed (121). However, the most specific phenotypic alteration is leukonychia, characterized by a typical arciform pattern resembling a half-moon and involving the proximal part of the nail plate. These alterations of digits and nails were also reported in a few strains of nude mice. Of note, nail dystrophy has also been observed in heterozygous subjects carrying *FOXN1* alterations (121). *FOXN1* is known to be selectively expressed in the nail matrix, where the nail plate originates, thus confirming that this transcription factor is involved in the maturation process of nails and suggesting nail dystrophy as an indicative sign of heterozygosity for this molecular alteration (121).

Autoptical study of a fetus homozygous for R255X mutation revealed multiple-site neural tube defects, including anencephaly and spina bifida. This finding may help explaining the high rate of mortality *in utero* observed in the population where the first patients were identified (105). Intriguingly, the other forms of SCID become clinically evident only during the postnatal life, when the protection of the newborn transferred from the mother immune system declines. This observation, suggests that other causes different from immunodeficiency, are responsible for the high rate of mortality *in utero* and led to consider the Nude/SCID mutation and anencephaly causally related. Of note, in a recent study, the mouse Foxn1 gene was found to be expressed also in epithelial cells of the developing choroids plexus, a structure filling the lateral, third and fourth ventricles of the embryonic brain (105). Moreover abnormality in the development of corpus callosum were also found in another FOXN1 mutated fetus even in the absence of anencephaly, indicating that the transcription factor may play a role as a co-factor in the brain ontogeny (105).

Altogether these findings suggest that FOXN1 may also be implicated as co-factor in the development of vital systems required for a proper fetus development, thus explaining the mortality in the first trimester in fetuses carrying the genetic alterations, which is not justified by the SCID *per se*.

## **FOXN1 MUTATION PREVENTS THE PRE-NATAL T CELL DEVELOPMENT IN HUMANS**

It is now clear that FOXN1 acts as a transcription factor implicated in the differentiation of thymic and skin epithelial cells, even though many of its molecular targets still remain to be discovered. Most of the knowledge so far available has been achieved in humans in the post-natal life, while little is known about FOXN1 role during the pre-natal life.

Of note, other FOX family members, including *Foxq1* and *Foxm1b*, are important during embryogenesis, being involved in a variety of biological processes (122). Approximately 50% of *Foxq1*−/− murine embryos die *in utero*, thus suggesting the requirement of this gene during embryogenesis (123). Similarly, *Foxm1b* is important during liver regeneration (124).

Studies on thymus organogenesis revealed that *Foxn1* is expressed in all TECs during fetal stages. Of note, *Foxn1*−/− mice showed undifferentiated TECs responsible for a blockage of thymopoiesis and severe immunodeficiency (125). Recently, the identification of a human *FOXN1*−/− fetus gave the unique opportunity to study in humans the T cell development *in utero*, in the absence of a functional thymus. Vigliano et al. documented a total blockage of the CD4+ T cell maturation and a severe impairment of CD8+ cells, with an apparent bias toward TCRγδ+ cells (112). In this case in the congenital absence of the thymus was due to R255X missense mutation in the *FOXN1* gene. In particular, it has been reported that in the absence of FOXN1 a few not functional CD8+ cells, mostly bearing TCRγδ in the absence of CD3, presumably of extra-thymic origin could develop in both humans and mice (126–128). Further analysis of the fetal RNA, performed to evaluate the variable-domain β-chain (Vβ) families' usage among T lymphocytes, revealed that the generation of TCR diversity occurred at some extent in the *FOXN1*−/− fetus, but was abnormal. Thus, these data provided a further evidence of the crucial role for FOXN1 in the early pre-natal stages of T cell development and not in the B and NK-cell differentiation, these populations being normally present in the Nude/SCID fetus (112). A similar impairment of the T cell differentiation with a selective blockage of CD4 differentiation but not of CD8, was detected in murine models characterized by the absence of the nuclear high-mobility group (HMG) box protein TOX (107).

The identification of a limited number of CD8+ cells bearing the TCRγδ suggests that this cell population may develop at extra-thymic sites in a *FOXN1*-independent manner, even though they are unable to sustain a productive immune response into the periphery. Indeed, evidence exists indicating that T cells may also differentiate at extra-thymic sites, as intestine and liver (129–133). Of note, the majority of thymus-derived T lymphocytes bears the αβ chains of TCR and a few of them express the γδ heterodimer (134), while the T cell pool developed outside the thymus is characterized by a higher proportion of TCRγδ+ T cells expressing the CD8αα homodimer, instead of the CD8αβ (135, 136). Moreover, also DN T cells (CD3+CD4–CD8– ) and lymphocytes expressing CD7 and CD2 in the absence of CD3 (CD2+CD3–CD7+) are generally considered of extra-thymic origin (135–137).

In spite of the well documented knowledge on the role of the primary lymphoid organ to foster T cell development, some still unsolved issues in human athymic conditions indicate that an indepth information of the overall process is still to be achieved and, in particular, the involvement of different tissues in T cell ontogeny must be definitively clarified. Since FOXN1 is selectively expressed in the thymus and skin, one possibility to explain the presence of the few non-functional CD8+TCRγδ+ cells in Nude/SCID fetus is that skin epithelial cells could play a partial role in T cell ontogeny, as already shown in *in vitro* models (138, 139).

## **THYMUS TRANSPLANTATION: A PROMISING TREATMENT TO ATHYMIC DISORDERS**

*Forkhead-box n1* deficiency is a very rare immunodeficiency with unfortunately poor chance of curative treatments. Recently, thymus transplantation has emerged as a promising treatment for children affected with congenital athymia (140–143), as that observed in complete DiGeorge anomaly and in *FOXN1* deficiency. Conceptually, the thymus transplant seems to be in principle the more appropriate therapeutic strategy, taking into account that bone marrow transplantation performed in one child with *FOXN1* deficiency, failed to induce a long-term sustained immune reconstitution. In particular, in this patient no reconstitution of the naïve T cell pool was observed (144).

Thymus transplantation has been first used in children affected with complete DiGeorge anomaly, with excellent clinical and immunologic results (141). In order to achieve immune reconstitution, cultured post-natal allogeneic thymus tissue slices were transplanted into the quadriceps muscles of the athymic host (145). The migration of host bone marrow stem cells to the donor graft allow them to develop into naive T cells, which then emigrate out of the engrafted thymic tissue into the peripheral blood. Thymopoiesis is observed in biopsies of the transplanted thymus within 2 months of transplantation (140) and naive T cells are detected in the peripheral blood approximately 3–5 months after transplantation (146, 147). Taking advantage from this previous experience, a few years ago an allogeneic thymus transplantation has been used for the first time in two unrelated infants with Nude/SCID phenotype due to a deficiency of the transcription factor FOXN1 (111). The clinical phenotype of the two subjects was characterized by the absence of naïve T cells, total alopecia, nail dystrophy, and severe infections, as disseminated *Bacillus* Calmette–Guérin in subject 1 and severe respiratory infections in subject 2. Molecular analysis, performed to confirm the clinical suspect of the Nude/SCID phenotype, revealed the presence of a homozygous R255X mutation in the *FOXN1* gene in subject 1, the same of that previously described (107), and a homozygous R320W novel missense mutation in the subject 2. Moreover, subject 1 showed, like a small percentage of complete DiGeorge patients, referred as atypical complete DiGeorge, circulating oligoclonal T cells of non-maternal origins, which were predominantly double-negative T cells, and a T cell proliferative response to PHA within the normal range. Because of that, before thymus transplantation subject 1 have required immunosuppression regimen to prevent graft rejection. Differently,immunosuppression was not used for the subject 2, who had, like typical complete DiGeorge patients, very few T cells (141, 146).

Results obtained with thymus transplantation were encouraging in both FOXN1-deficient subjects, and led to a full T and B cell reconstitution and functional rescue. Indeed, both subjects developed naïve T cells, diverse TCR repertoires and an *in vitro* proliferative T cell responses against different antigens. Eventually they reached normal serum Ig levels with generation of protective antibody specific titers. Of note, HLA matching for class I and II did not seem to interfere with T cell counts after thymus transplantation, being subject 2 transplanted without any HLA matches. However, CD8+ T cell number, although apparently functional, was disproportionally low compared to CD4+ T cells (111).  Possible explanations are that the phenomenon is related to the HLA mismatch between host hematopoietic precursors and allograft thymic epithelia or to alterations in the thymic graft due to transplantation procedures.

Functionality of the thymic allograft has been assessed for the first time through signal joint (sj) and DβJβ T cell receptor rearrangement excision circle (TREC) analyses (109). The sj/βTREC represents a ratio between early and late products of TCR rearrangements, which directly correlate with thymic output and provide an indirect measurement of thymocyte divisionrate (149–151). The sj/βTREC ratio quantification, conducted in subject 1 with R255X mutation, was very low during the peri-transplant period and comparable to those observed in healthy children at 2.5 years post-transplant. Of note, 4 years posttransplantation a decrease of sj/βTREC ratio associated with a reduction in sjTREC levels and in the number of naïve cells were found, suggesting the decline in thymic allograft output (109). This decline might be due to the reduced longevity of the thymus allograft or to peripheral homeostasis of the T cell pool maintenance following its replenishment. Overall, the thymus transplantation seems to be a promising curative strategy for subjects with athymia due to *FOXN1* deficiency or complete DiGeorge syndrome in the perspective of long-term clinical benefit.

## **CONCLUSION**

The integrity of the thymic epithelial architecture allows the growth, the differentiation, and TCR repertoire selection of immature T cells, thus originating fully mature and functional T cells. Of note, the failure to generate or to maintain the proper 3D thymic architecture leads to severe immunodeficiency or autoimmunity. The unique function of the thymus in the establishment/maintenance of the T cell pool is related not only to the peculiar 3D structure, but also to the specialized functions of the thymic stroma. Indeed, lympho-stromal interactions within the multicellular thymic microenvironment play a crucial role in the regulation of the T cell development. Moreover, these interactions are based on a bilateral crosstalk between stromal cells and traveling thymocytes, which, in turn, are able to provide important signals for the TECs differentiation.

Thymus organogenesis and T cell development are sophisticated biological processes, which require the activation of a wide panel of genes. There is evidence that the master regulator of the thymus development is the *Foxn1* gene, since it is required at multiple intermediate stages of the TE-lineage specification either in the fetal and adult thymus, through the direct or indirect regulation of genes involved in the thymus development and function. These genes include *Pax1*, *Hoxa3*, *CCL25*, *Dll4*, *p63*.

Studies on the animal and human model of the Nude/SCID phenotype have provided an enormous contribution in identifying the crucial role of *Foxn1* to drive the thymus development, even though many issues regarding the transcriptional regulation of the TECs specification and homeostasis still remain to be solved. The development *in vitro* of cellular models of TEC lineage

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 17 April 2013; accepted: 25 June 2013; published online: 12 July 2013. Citation: Romano R, Palamaro L, Fusco A, Giardino G, Gallo V, Del Vecchio L and Pignata C (2013) FOXN1: a master regulator gene of thymic epithelial development program. Front. Immunol. 4:187. doi: 10.3389/fimmu.2013.00187*

*This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2013 Romano, Palamaro, Fusco, Giardino, Gallo, Del Vecchio and Pignata. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Which model better fits the role of Aire in the establishment of self-tolerance: the transcription model or the maturation model?

## **Mitsuru Matsumoto\*,Yumiko Nishikawa, Hitoshi Nishijima, Junko Morimoto, Minoru Matsumoto and Yasuhiro Mouri**

Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, Tokushima, Japan

### **Edited by:**

Claudio Pignata, Federico II University, Italy

**Reviewed by:**

Nikolai Petrovsky, Flinders Medical Centre, Australia Claudio Pignata, Federico II University, Italy

### **\*Correspondence:**

Mitsuru Matsumoto, Division of Molecular Immunology, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto, Tokushima 770-8503, Japan e-mail<EMAIL_ADDRESS>The discovery of Aire-dependent transcriptional control of many tissue-restricted selfantigen (TRA) genes in thymic epithelial cells in the medulla (medullary thymic epithelial cells, mTECs) has raised the intriguing question of how the single Aire gene can influence the transcription of such a large number of TRA genes within mTECs. From a mechanistic viewpoint, there are two possible models to explain the function of Aire in this action. In the first model, TRAs are considered to be the direct target genes of Aire's transcriptional activity. In this scenario, the lack of Aire protein within cells would result in the defective TRA gene expression, while the maturation program of mTECs would be unaffected in principle.The second model hypothesizes that Aire is necessary for the maturation program of mTECs. In this case, we assume that the mTEC compartment does not mature normally in the absence of Aire. If acquisition of the properties of TRA gene expression depends on the maturation status of mTECs, a defect of such an Aire-dependent maturation program in Aire-deficient mTECs can also result in impairedTRA gene expression. In this brief review, we will focus on these two contrasting models for the roles of Aire in controlling the expression of TRAs within mTECs.

**Keywords: autoimmunity, thymic epithelial cell, self-antigen, gene transcription, cell differentiation**

The current prevailing view regarding the role of Aire in selftolerance is that it is involved in the transcriptional control of many tissue-restricted self-antigen (TRA) genes in medullary thymic epithelial cells (mTECs) (1). In other words, TRAs are considered to be the direct target genes of Aire's transcriptional activity (the transcription model) (2). This view was first suggested in a paper reporting that Aire-deficient mTECs showed dramatically lower expression of TRAs than wild-type mTECs (3). Since this landmark report, the transcription model has prompted many studies of Aire in an attempt to clarify how the single *Aire* gene can influence the transcription of such a large number of TRAs within mTECs (4–7). Unfortunately, to obtain a mechanistic insight into this interesting phenomenon, it has been necessary to employ cultured cells transfected with the *Aire* gene, because the fraction of naturally Aire-expressing mTECs in the thymic stroma is too small to work with. However, no appropriate cultured cell lines that could be used reliably in place of Aire-expressing mTECs *in vivo* have been available. Nonetheless, overexpression of Aire in cultured cells resulted in increased transcription of many TRAs, in accordance with the transcription model. However, it is important to pay more attention to the uniqueness of bona fide mTECs *in vivo*, which show characteristics very different from those of cultured cell lines; although several cell lines derived from the thymic stroma are available for both humans and mice, none of them show typical characteristics of mTECs such as high expression of FoxN1, MHC class II, and CD80, even though they are positive for cytokeratin and epithelial cell markers (e.g., keratin 5, EpCAM, and lectin UEA-1 binding). Most importantly, none of these cell lines express a reliable level of Aire at the protein level. However, large aspects of Aire's action on TRA gene expression in the transcription model were deduced on the basis of the effects of lack of Aire expression in mTECs *in vivo* (i.e., the phenotypes of Aire-deficient mice) and the opposite effects of Aire overexpression in mTEC-"like" cells *in vitro* (i.e., in transfection studies). The reality is that there is still a fairly wide gap between these two experimental settings that needs to be bridged.

In comparison with the remarkable changes noted in the expression profiles of TRA genes in Aire-deficient mTECs, morphological alterations in the medullary components from Airedeficient mice were not initially appreciated until Farr's paper had appeared (8). This was the main reason why insufficient attention was paid initially to another proposed explanation for the reduced TRA gene expression in Aire-deficient mTECs: the maturation model (2). However, fairly recent detailed studies of Aire-deficient thymi have revealed several important aspects of the Aire-dependent differentiation programs of mTECs, such as increased numbers of mTECs with a globular cell shape (8, 9), contrasted with reduced numbers of terminally differentiated mTECs expressing involucrin, the latter being associated with reduced numbers of Hassall's corpuscles (9, 10). Although not fully investigated, increased percentages of mTECs expressing high levels of CD80 (CD80high) is another suggested aspect of the Airedependent mTEC differentiation program (11–13). In this regard, it is noteworthy that the Aire-dependent mTEC differentiation

program can be linked to the control of TRA gene expression, in which the role of Aire may be perceived from a different viewpoint. Given that acquisition of the properties of TRA gene expression depends on the maturation status of mTECs (14, 15), any defect in such an Aire-dependent maturation program could also account for defects of TRA gene expression in Aire-deficient mTECs. In such a case, TRA genes would not have to be the direct target of Aire. Instead, Aire-deficient mTECs would have defective TRA gene expression, because they are not fully differentiated to stage(s) where other undetermined transcriptional means for TRA gene expression beyond Aire become available and/or active (**Figure 1**). Having said so, the exact point in the differentiation process at which Aire-deficient mTECs are prevented from differentiating further still remains unclear. Investigation of this issue has been hampered by the current lack of suitable markers for the mTEC differentiation program: so far, CD80 and MHC class II remain the few that are available. Precise elucidation of the target gene(s) relevant to the progression of mTEC differentiation controlled by Aire is an essential task to support the maturation model.

Currently, there is no firm evidence to support either model for Aire's role in the control of TRA gene expression. Therefore, there is a need to develop better *in vivo* experimental systems for investigating this issue. In this connection, the results obtained by examining *Aire* gene expression under control of the rat insulin promoter using transgenic mice merit attention (16). It was found that alterations of the transcriptome did not mirror those created by abrogation of Aire within mTECs. This may not be surprising, but it is nevertheless important: if cell types differ, the effects of functional gain or loss of a transcription factor, such as Aire, can in turn differ markedly. This is especially important in the case of Aire, since mTECs are unique in showing promiscuous gene expression and a heterogeneous composition (17, 18). Thus, it cannot be over-emphasized that the roles of Aire need to be studied using *in vivo* models, and not *in vitro* systems using mTEC-surrogate cells.

Regardless of the models employed, there are several important issues related to the molecular regulation of TRAs within mTECs in the context of Aire. Obviously, the transcriptional control of TRAs is different from the regulation seen in their authentic tissues, as exemplified by differences in the transcriptional start sites of individual TRA genes (19). Although the fact that transcriptional hierarchies driving the development of the pancreas and transcription of the *insulin 2* gene are not maintained in mTECs (19, 20) might be more consistent with the transcription model, it does not contradict the maturation model in explaining why Aire-deficient mTECs show impaired *insulin 2* gene expression; the maturation model does not require the transcriptional hierarchies driving the development of authentic tissues. Instead, the maturation model has its own mTEC developmental process in which expression of particular TRAs is acquired at specific time points during the differentiation program. For example, expression of Aire-dependent TRA genes, such as *insulin 2* and *SAP1*, can be accomplished in terminally differentiated mTECs that have fully matured with the help of Aire protein. Lack of Aire in mTECs results in premature termination of differentiation, although Airedeficient mTECs can still develop and pass a certain maturation stage. These Aire-less mTECs, which are rather mature (CD80high)

**FIGURE 1 | Schematic representation of the roles of Aire in TRA gene expression associated with the mTEC differentiation program**. Aire-expressing mTECs develop from their progenitor via an immature stage (CD80lowMHC-2low) where only limited numbers of TRAs (e.g., 1 and 2 ) are expressed. Aire is expressed at the mature stage (CD80highMHC-2high), and the transcription model (upper) suggests that Aire directly activates many target TRA genes within mature mTECs (so-called Aire-dependent TRAs; 3 – 6 ). Consequently, lack of Aire results in defective expression of Aire-dependent TRA genes. In contrast, the maturation model suggests that Aire plays an important role in the maturation program of mTECs, and that expression of Aire-dependent TRA genes, such as 3 – 6 , can be accomplished in terminally differentiated mTECs showing a dendritic to fibroblastic morphology that have fully matured with the help of Aire. According to this scenario, lack of Aire causes a halt in mTEC maturation at a stage where promiscuous gene expression in the cells is still not possible (marked as "Aire-KO mTECs"); lack of Aire in mTECs results in premature termination of differentiation, and these CD80high Aire-less mTECs have a more globular cell shape and lack the transcriptional machinery for Aire-dependent TRA genes (lower). Because Aire-independent TRA genes, such as 1 and 2 , can be expressed before the mature stage, lack of Aire has little impact on their expression.

but not fully competent for TRA expression, have a more globular cell shape and lack transcriptional machinery and/or activity for Aire-dependent TRA genes (9) (**Figure 1**). In this scenario, Aireindependent TRA genes, such as*CRP* and*GAD67*, can be normally expressed even in Aire-deficient mTECs, because these TRAs can be expressed before the terminal differentiation stage(s), and consequently the lack of Aire has little impact on their expression. It is still unknown why some (Aire-independent) TRAs are expressed from the immature stage(s), whereas (Aire-dependent) others are expressed only after they become fully mature. For this reason, it would be important to clarify the exact timing of Aire expression during the course of mTEC differentiation (21). Nevertheless, promiscuous gene expression seems to be accomplished in terminally differentiated mTECs that have matured in the presence of Aire protein (the maturation model). Alternatively, reduced TRA gene expression could represent failure of heterogeneity in terms of TRA gene expression due to a halt in differentiation at a premature stage before heterogeneity of individual mTECs has occurred. In contrast, the transcription model may explain why some TRA genes are Aire-dependent and others are not, as outlined in the following. Aire-PHD1 binding with H3K4me0 is an interesting finding, and a model has been proposed suggesting that Aire's

PHD1 acts as a chromatin reader, searching for genes showing low expression (5, 22); Aire preferentially binds with weakly expressed genes harboring the silent chromatin signature of H3K4me0, and may help to up-regulate TRA genes whose expression levels would otherwise remain low (23). In this model, TRAs showing relatively high expression would do not require the help of Aire, and their expression would be Aire-independent.

One caveat of the transcription model is that expression of Aire and Aire-dependent genes does not always overlap at the singlecell level (19, 24, 25). If Aire were controlling the expression of TRAs in any way by means of direct transcriptional control, we would expect to see both Aire and Aire-dependent TRAs within the same individual cell, although one could argue that the timing of expression of Aire and Aire-target genes might not always be the same within any given analytical snapshot time frame.

Involucrin is an interesting TRA in that it is sometimes used as an example of an Aire-dependent TRA that follows the transcription model: when cultured cell lines were introduced with an Aire-expressing plasmid, transcription of the endogenous *involucrin* gene was up-regulated possibly due to the transcriptional activity of Aire (26). However, at the same time, involucrin is also used as a marker of mTEC maturation status. Similarly to its expression in the epidermis of the skin, involucrin is expressed by mature epithelial cells in the thymic medulla; it is expressed most strongly in Hassall's corpuscles, which seem to be the product of terminally differentiated mTECs, and Aire-deficient thymi show reduced numbers of involucrin-expressing mTECs, even in Hassall's corpuscles (9, 10). These findings are consistent with data favoring the maturation model derived from *in vivo* systems suggesting that Aire is required for promotion of the mTEC maturation program. Thus, there is a need to understand the types of effects that can be expected according to the experimental systems employed.

Regarding the role of Aire in the mTEC maturation program, an important issue that needs to be carefully investigated is whether or not Aire has proapoptotic activity. Introduction of Aire into cultured cells has been reported to result in apoptosis (11). Accordingly, increased MHC class IIhigh/CD80high mTECs seen in Aire-deficient mice was considered to explain the lack of Aire-mediated proapoptotic activity, because loss of Aire did not result in augmented proliferation of mTECs (11). Given that Aire plays an important role in the induction of a wide variety of TRAs,

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concomitant induction of apoptosis in Aire-expressing mTECs by Aire itself might be an effective way to promote cross-presentation, thereby facilitating negative selection (1). Once again, however, this attractive hypothesis needs to be investigated in more depth using *in vivo* experimental systems.

Finally, there is a need to discuss the implications of these two different models for the mechanisms underlying the defect of negative selection in Aire-deficient animals. In principle, the transcription model restricts the failure of negative selection in Aire-deficient mice to reduced expression of TRA gene products. In contrast, the maturation model suggests that Aire may affect the thymic microenvironment more globally than through simple control of TRA expression levels. Consequently, the maturation model allows for the possibility that regulation of TRA gene expression may not be the major defect of Aire-deficient mTECs responsible for impaired negative selection. Instead, other alterations in the function of mTECs lacking Aire might equally account for the defective negative selection in Aire-deficient mice. These changes could include processing and/or presentation of self-antigens within the mTECs (27), the process of thymocyte maturation (28), the process by which mature thymocytes are attracted to their proper location for negative selection by production of chemokines from mTECs (27, 29), control of cross-presentation through alteration of the relationship between BM-APCs and mTECs (30), and the balance between negative selection and regulatory T cell production (31). Furthermore, modification of microRNA-regulated TRA gene expression by Aire might represent another dimension in this field that warrants further investigation (32). All of the above issues may be largely clarified once the target genes of Aire have been determined using *in vivo* models. Thus, our current understanding of the fundamental function of Aire still seems to be in its infancy, and the proposal and evaluation of different models would doubtless lead to further advances in this fascinating field of research.

## **ACKNOWLEDGMENTS**

This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science and from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and CREST, the Japan Science Technology Agency.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 30 May 2013; accepted: 09 July 2013; published online: 22 July 2013. Citation: Matsumoto M, Nishikawa Y, Nishijima H, Morimoto J, Matsumoto M and Mouri Y (2013) Which model better fits the role of Aire in the establishment of self-tolerance: the transcription model or the maturation model? Front. Immunol. 4:210. doi: 10.3389/fimmu.2013.00210 This article was submitted to Frontiers in T Cell Biology, a specialty of Frontiers in Immunology.*

*Copyright © 2013 Matsumoto, Nishikawa, Nishijima, Morimoto, Matsumoto and Mouri. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.*

## Regulations of gene expression in medullary thymic epithelial cells required for preventing the onset of autoimmune diseases

#### **Taishin Akiyama1\*, Miho Shinzawa1 , Junwen Qin1,2 and Nobuko Akiyama1**

1 Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan

2 Key Laboratory for Regenerative Medicine, Department of Developmental and Regenerative Biology, Ministry of Education and International Base of Collaboration for Science and Technology, the Ministry of Science and Technology and Guangdong Province, Jinan University, Guangzhou, China

## **Edited by:**

Claudio Pignata, Federico II University, Italy

## **Reviewed by:**

Anna Villa, National Council of Research, Italy Petteri Arstila, University of Helsinki, Finland

## **\*Correspondence:**

Taishin Akiyama, Division of Cellular and Molecular Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan e-mail<EMAIL_ADDRESS>
Elimination of potential self-reactiveT cells in the thymus is crucial for preventing the onset of autoimmune diseases. Epithelial cell subsets localized in thymic medulla [medullary thymic epithelial cells (mTECs)] contribute to this process by supplying a wide range of self-antigens that are otherwise expressed in a tissue-specific manner (TSAs). Expression of some TSAs in mTECs is controlled by the autoimmune regulator (AIRE) protein, of which dysfunctional mutations are the causative factor of autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED). In addition to the elimination of self-reactive T cells, recent studies indicated roles of mTECs in the development of Foxp3-positive regulatory T cells, which suppress autoimmunity and excess immune reactions in peripheral tissues.TheTNF family cytokines, RANK ligand, CD40 ligand, and lymphotoxin were found to promote the differentiation of AIRE- andTSA-expressing mTECs. Furthermore, activation of NF-κB is essential for mTEC differentiation. In this mini-review, we focus on molecular mechanisms that regulate induction of AIRE and TSA expression and discuss possible contributions of these mechanisms to prevent the onset of autoimmune diseases.

**Keywords: medullary thymic epithelial cells, autoimmune disease, NF-**κ**B,TNF receptor family, gene expression**

## **INTRODUCTION**

The thymus contributes to self-tolerance of T cells by eliminating potentially self-reactive T cells and generating immunosuppressive T cells, which are essential for preventing the onset of autoimmune disease. Epithelial cells localized in the thymic medulla [medullary thymic epithelial cells (mTECs)] are non-hematopoietic in origin and play non-redundant roles in the elimination of self-reactive T cells (1–4). Recent studies have revealed that mTECs also contribute to the selection and survival of immunosuppressive Foxp3-positive regulatory T cells (Tregs) (5–8).

Medullary thymic epithelial cells express several functional molecules required for the selection of self-tolerant T cells and Tregs (3). Mature types of mTECs express MHC molecules and co-stimulatory molecules essential for antigen presentation to developing T cells. In addition, mTECs secrete several types of chemokines (e.g., CCL19, CCL21, and CCL22) that attract T cells or dendritic cells in the medulla (2, 9). Moreover, a recent study has shown that the expression of CD70 in mTECs enhances the development and survival of Tregs via an interaction with its receptor, CD27, which is expressed on thymic T cells (5). A key feature of mTECs is their ability to express hundreds of self-antigens that are normally expressed in a tissue-specific manner (TSAs) (4, 10). TSAs are processed and directly presented by mTECs or indirectly presented by thymic DCs receiving TSAs from mTECs (4, 7, 11–13). T cells that recognize TSAs with high avidity undergo apoptosis (so-called negative selection) or survive as regulatory T cells (4, 14). Many studies have suggested significant roles of mTEC-dependent self-tolerance in preventing the onset of some autoimmune diseases in humans. Expression of some TSAs requires a nuclear protein autoimmune regulator (AIRE), the dysfunctional mutations of which are responsible for an inherited human autoimmune disease, autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED) (15, 16). Whereas the expression of AIRE mRNA is detected in different cell types, AIRE expression at the protein level is remarkably high in mTECs (17). A previous study using AIRE-deficient mice provided evidence that autoimmunity, provoked by dysfunction of AIRE, is thymic stroma-dependent (18). In addition to APECED, recent studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the *AIRE* gene are associated with rheumatoid arthritis (19, 20). In addition to mutations in the *AIRE* gene, reduced expression of the muscle acetyl choline receptor (*CHRNA1*) in mTECs was shown to be associated with the onset of myasthenia gravis (21). Moreover, impairment of the mTEC-dependent tolerance might explain the relationship between myocarditis and autoimmunity (22). These findings also imply that the onsets of various human autoimmune diseases could be related to dysregulation of mTEC-dependent tolerance. Interestingly, in addition to relationships with autoimmune diseases, recent studies have uncovered roles for mTEC-dependent T-cell tolerance in tumor tolerance (8, 23, 24).

Because expression of AIRE and TSAs is characteristic of mTEC, mTECs should harbor specific mechanisms to direct AIRE and TSA expression. Expression of TSAs appears to be correlated with the differentiation of mTECs. In this mini-review, we specially focus on molecular mechanisms regulating the expression of AIRE and TSAs and the process of mTEC differentiation.

## **DEVELOPMENT OF mTECs**

Thymic epithelial cells are classified into mTECs and cortical thymic epithelial cells (cTECs) (2). Several lines of evidence indicate the existence of a bi-potent TEC progenitor capable of differentiating into mTECs and cTECs in the fetal and adult thymus (25–29). The bi-potent TEC progenitor seems to give rise to each progenitor of mTECs and cTECs in the next stage (30, 31). Recent studies revealed that mTECs differentiate from progenitors expressing cTEC-markers (32, 33). These data imply that mechanisms determining the mTEC commitment suppress the cTEC-driving program. However, master molecules that decide the fate of the bi-potent TEC progenitor expressing cTEC-markers to the mTEC lineage have not been determined yet.

Currently, mTECs are classified based on the expression of MHC II, CD80, AIRE, and involucrin (**Figure 1**). mTECs (typically defined as CD45− EpCAM+ Ly51− and UEA-1+ by flow cytometric analysis) in adult mice are divided into two subpopulations, according to the expression levels of MHC II and CD80 (34). mTECs expressing high levels of MHC II and CD80 (mTEChi) express a more diverse set of TSAs than mTECs expressing lower levels of MHC II and CD80 (mTEClo) do (35). Moreover, precursor-product relationship analysis has suggested that the mTEClo fraction can differentiate into mTEChi (36, 37). Therefore, the mTEChi fraction would be the more mature type of mTEC than mTEClo .

The mTEChi fraction is further separated on the basis of AIRE expression (36, 38). Because previous studies have indicated that the AIRE-expressing mTECshi (AIRE+ mTEChi) are postmitotic and susceptible to apoptosis (36), AIRE+ mTECshi are postulated to be the more differentiated cell types than AIRE-negative mTECshi. mTECs expressing involucrin, a marker of terminally differentiated keratinocytes, are considered to be terminally differentiated mTECs that may be derived from AIRE+mTEChi (39, 40).

## **REGULATION OF AIRE mRNA EXPRESSION**

Molecular mechanisms regulating the expression of AIRE, which are likely critical for preventing autoimmunity, remain unclear. In the fetal thymus, expression of AIRE starts at embryonic day 14.5 (41). Consistently, mature mTECs emerge around this embryonic day (42). Thus, AIRE expression seems to be closely linked to mTEC differentiation. However, because mTEChi is separated into AIRE+ and AIREfractions, the mTEC differentiation mechanism might be necessary but is not entirely sufficient for AIRE expression.

A study using a luciferase reporter assay identified a plausible minimal promoter region of the *AIRE* gene (43). This region contains binding sequences for Sp1, AP-1, NF-Y, and ETS family of transcription factors. Indeed, luciferase reporter analysis suggested regulation of the *AIRE* gene promoter by ETS family proteins (44). However, *in vivo* genetic studies are necessary to prove that these sequence-specific transcription factors are critical for the regulation of AIRE expression.

high levels of MHC II and CD80, and a more diverse set of tissue-specific antigens (TSAs). MHC II-high and CD80-high mature mTECs are further separated into AIRE-positive and AIRE-negative subpopulations. AIRE-positive mature mTECs are postmitotic and undergo apoptosis or otherwise differentiate into involucrin-positive mTECs.

The promoter region of AIRE contains a high ratio of CpG sites (43). These CpG sites are hypermethylated in established cell lines defective in the AIRE expression. A subsequent study showed that these CpG sites are hypomethylated in isolated mTECs compared to thymocytes (45). These findings suggest that DNA demethylation might be prerequisite for AIRE expression. However, interestingly, hypomethylation was also observed in cTECs and thymoma with defective AIRE expression (45). Hence, DNA hypomethylation appears to be required but not sufficient for inducing AIRE expression.

Overall, AIRE expression seems to be regulated by combinations of chromatin modification and sequence-specific transcription factors. However, precise mechanisms and regulatory molecules remain to be determined.

## **REGULATION OF TSA mRNA EXPRESSION**

TSA expression appears to be regulated by complicated mechanisms. Single-cell PCR analyses revealed a stochastic nature of TSA expression in mTECs (38, 46). Each TSA is expressed in a subset of mTECs (38, 46). The frequency of mTECs expressing a particular TSA was different, depending on the TSA (38, 46). Interestingly, various combinations of TSAs are expressed in individual mTECs (38, 46). These studies suggest that regulatory mechanisms of TSA expression in mTECs are different from those used in inherent tissues.

Several studies suggest that TSA expressions are epigenetically controlled. A comprehensive mRNA expression study revealed that TSA gene loci tend to co-localize in chromosomal clusters (35, 47). Moreover, genomic imprinting of the *Igf2* gene, a TSA, was lost in mTECs (35), implicating the involvement of a DNA demethylation mechanism in TSA expression. Interestingly, another imprinted gene, *Cdkn1c*, was not affected. These data imply the existence of mTEC-specific mechanisms for demethylation of DNA.

Control of TSA gene expression by AIRE has been intensively studied (48–50). Several studies have revealed a function of AIRE as a transcription factor that directly promotes TSA expression (51, 52). Furthermore, AIRE binds to hypomethylated Histone 3 Lys 4 (H3K4) through its plant homology domain (53, 54). This finding suggests that AIRE modifies the chromatin structure in the TSA genes. AIRE also binds to DNA-PK (55–57), which functions in the repair of DNA-double strand breakage. A study using an mTEC cell line suggested that interactions of AIRE with H3K4 and DNA-PK are critical in recruiting AIRE to TSA gene loci and promoting TSA expression (57). Additionally, it was reported that AIRE interacts with P-TEFb, a component of the super elongation complex (58). It is generally accepted that transcription elongation, via the release of "paused" RNA polymerase II, is critical for the regulation of many genes (58, 59). AIRE may recruit P-TEFb to the TSA gene locus and promote elongation of the arrested TSA transcripts by releasing RNA polymerase II from the proximal promoter (60). Recent comprehensive analysis of mRNA transcripts in mTECs supports this mechanism (61). In addition to the TSA expression, the AIRE-dependent expression of some microR-NAs (miRNAs) was recently revealed (62, 63). Consistently, genetic studies revealed important roles played by miRNA expressions in functions and maintenance of mTECs (63–65).

Compared to the mechanisms underlying Aire-dependent TSA expression, molecular mechanisms underlying Aire-independent TSA expression are less understood. As described above, whereas epigenetic regulations of TSA genes would be critical, mechanisms underlying epigenetic changes specific for mature mTECs remain unclear. Moreover, unidentified transcription factors may be involved in the promotion of Aire-independent TSA expressions.

## **EXTRACELLULAR SIGNALING TO PROMOTE DIFFERENTIATION OF mTECs EXPRESSING AIRE AND TSAs**

Differentiation of TECs is well known to be correlated to differentiation of T cells in the thymus (so-called thymic cross-talk) (3). mTEC maturation was reported to be abolished in severe combined immunodeficiency (SCID) patients (66). This finding supports the idea that failure of the thymic cross-talk results in the onset of autoimmune manifestation through inhibition of mTEC function. Interestingly, a recent study showed that administration of anti-CD3ε antibody ameliorated autoimmunity in leaky SCID model mice possibly through improvement of the thymic cross-talk (67).

Molecular basis of the thymic cross-talk in mTEC development has been reported. Several lines of evidence revealed that TNF family cytokines expressed in thymocytes and other cells of hematopoietic origin (2) and their receptors expressed in mTEC are critical for the thymic cross-talk. Briefly, signaling of TNF receptor family members, RANK, CD40, and lymphotoxin-β receptor (LtβR), play essential roles in the development of mTECs expressing Aire and TSAs. This topic has been summarized in a recent review (1).

## **DOWNSTREAM OF TNF RECEPTOR FAMILY SIGNALING**

TNF receptor family signaling induces the activation of NF-κB and MAPK pathways (68). To date, the involvement of the MAPK pathway in the development of mTEC remains to be addressed. However, several lines of evidence have indicated that the NF-κB family plays a critical role in the development of mTECs expressing AIRE and TSAs.

NF-κB members are sequestered in the cytoplasm in an inactive state by the binding of the inhibitory protein IκB in resting cells (69–71). Ligations of receptors induce phosphorylation and subsequent degradation of IκB proteins, thereby leading to nuclear localization of NF-κB to activate transcription. Two distinct NFκB activation pathways, the classical pathway and the non-classical pathway, are currently known (70–72) (**Figure 2**). The classical pathway is required in inflammatory responses and lymphocyte activation (71). On the other hand, the non-classical pathway mainly promotes development and architecture formation of lymphoid organs, including the thymus. In the non-classical pathway, receptor ligation induces accumulation of the NF-κB-inducing kinase (NIK), which is normally degraded by the ubiquitindependent proteasome in resting cells. Subsequently, accumulated NIK phosphorylates and activates IKKα, which induces partial degradation of p100 to p52. p100 preferentially binds to and sequesters RelB in the cytoplasm, and the partial degradation of p100 to p52 induces translocation of RelB and p52 as a heterodimer into the nucleus.

The requirement for NF-κB activation in the development of mTEC was initially identified by the analysis of RelB-deficient mice (73, 74). RelB-deficient mice showed severe reduction in medulla size, accompanied by a lack of UEA-1-positive mTECs. Consistently, the expression of AIRE was abolished in the RelBdeficient thymus (6, 41, 75). As expected, RelB-deficient mice showed severe autoimmune diseases. A recent study demonstrated that autoimmunity of RelB mice was due to the defect in thymic stroma function (6). Mice carrying a dysfunctional mutation, NIK (*aly/aly*), also showed a similar defect in mTEC development and autoimmune phenotypes (76–78). Whereas IKKα-deficient mice die shortly after birth, neonatal IKKα-deficient mice and transplantation of IKKα-deficient thymic stroma indicates a requirement of IKKα in the development of mTECs (79, 80). mTEC development in p100-deficient mice is partially defective (81, 82), but this appears to be due to a partial rescue of p100 function by p105 (or its processed product, p50) because the double deficiencies of p100 and p105 resulted in severe defects in mTEC development, similar to the RelB- and NIK-mutant mice (83). Overall, these results support the idea that activation of the non-classical NF-κB pathway is essential for the development of mTECs.

TRAF6 is a signal transducer that mediates signaling from TNF receptor family members (84, 85). TRAF6-deficient mice exhibit severe autoimmune disease (86, 87). Additionally, recent studies suggest possible associations between SNPs of the *TRAF6* gene with rheumatoid arthritis and systemic lupus erythematosus in humans (88, 89). Previous studies showed that TRAF6 promotes the development of mTECs expressing AIRE and TSAs, thereby suppressing autoimmunity (86). Moreover, RANK-mediated differentiation of mTECs requires TRAF6 in *in vitro* organ culture

of fetal thymic stroma (90). Notably, TRAF6 is a signal transducer that mediates the activation of the classical NF-κB pathway but not the non-classical NF-κB pathway (84, 85). Thus, these data imply a role for TRAF6-mediated activation of the classical NF-κB pathway in mTEC differentiation.

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	-

In addition to the above findings, a scaffold protein, Sin (also called Efs), was proposed to be expressed downstream of TNF receptor family signaling. Sin-deficient mice showed reduced numbers of mTECs and thymic stroma-dependent autoimmunity (91). In addition to the role of Sin in FGF-mediated proliferation signaling (91), a recent study suggested that Sin might regulate the non-classical NF-κB pathway activated by RANKL signaling (92). Because the SH3 domain and phosphorylation of tyrosine residues of Sin might be critical for its function (93, 94), these studies also imply unrecognized roles of Src-type tyrosine kinases in mTEC development.

## **CONCLUDING REMARKS**

Whereas significant roles for NF-κB in signal activation of mTEC differentiation and subsequent expression of AIRE and TSAs are indisputable, molecular events connecting these signaling pathways to induction of AIRE and TSA remain unclear. It was reported that LtβR signaling induces the expression of AIRE in an mTEC line in the presence of a DNA methylation inhibitor (95). However, it is still unclear whether NF-κB binds to the promoter of the *AIRE* gene. Moreover, a wide variety of TSA expression would not be explained only by NF-κB-dependent transcriptional activation because NF-κB family members are generally known to be sequence-specific transcription factors. Thus, the link between NF-κB activation and expression of AIRE and TSAs remains largely enigmatic.

In addition, differentiation stages regulated by these signaling molecules and their mechanisms need to be clarified. mTECs have different properties in each developmental stage, with regard to TSA expression, AIRE expression, and DNA methylation status. Therefore, it is important to clarify types of mTECs in which each TNF receptor family signal functions. Overall, more studies are needed to understand the molecular and cellular mechanisms regulating the development of mTECs with the final aim to develop novel therapeutic strategies preventing autoimmune diseases caused by defective thymic functions.

## **ACKNOWLEDGMENTS**

This work was supported by a grant from the Japanese Society for the Promotion of Science (Taishin Akiyama).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 07 June 2013; paper pending published: 24 June 2013; accepted: 09 August 2013; published online: 26 August 2013.*

*Citation: Akiyama T, Shinzawa M, Qin J and Akiyama N (2013) Regulations of gene expression in medullary thymic epithelial cells required for preventing the onset of autoimmune diseases. Front. Immunol. 4:249. doi: 10.3389/fimmu.2013.00249*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2013 Akiyama, Shinzawa, Qin and Akiyama. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## **T. Petteri Arstila1\* and Hanna Jarva1,2**

1 Department of Bacteriology and Immunology, Immunobiology Research Program, Haartman Institute, University of Helsinki, Helsinki, Finland 2 HUSLAB, Helsinki University Hospital Laboratory, Helsinki, Finland

## **Edited by:**

Claudio Pignata, Federico II "University", Italy

### **Reviewed by:**

Patrick Jerome Bertolino, Centenary Institute, Australia Taishin Akiyama, University of Tokyo, Japan

### **\*Correspondence:**

T. Petteri Arstila, Department of Bacteriology and Immunology, Immunobiology Research Program, Haartman Institute, University of Helsinki, P.O. Box 21, Helsinki FIN-00014, Finland e-mail<EMAIL_ADDRESS>
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 regulatoryT 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.

**Keywords: APECED, AIRE,T cells, autoimmunity, thymus**

## **INTRODUCTION**

Monogenic diseases, although rare, provide a unique possibility to obtain information in the human system on the significance and function of the molecules affected by the mutations. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), also known as autoimmune polyendocrine syndrome type 1 (APS-1), is one such natural knockout phenotype, and has provided important information on T cell selection and pathogenesis of organ-specific autoimmunity (1, 2). It is a recessively inherited human autoimmune disease, caused by lossof-function mutations in the Autoimmune Regulator (AIRE) gene (3, 4). It is enriched in certain populations, most notably the Finns (prevalence 1:25 000), Sardinians (1:14 000), and Iranian Jews (1:9 000) (5). The pathognomonic triad consists of chronic candidiasis, hypoparathyroidism, and Addison's disease, with several other endocrine and non-endocrine manifestations affecting a smaller fraction of the patients.

Since the discovery of the underlying genetic defect in 1997, the pathogenesis of this rare polyendocrine syndrome has been the focus of considerable interest. In particular, studies in Airedeficient mice have shown that AIRE plays an important role in T cell development and negative selection in the thymus, thus elucidating general pathways of thymic development (2). However, the murine phenotype differs in several key points from the human disease, not least in the absence of all the defining triad components mentioned above (6). It is clear that to understand how AIRE works in the human immune system, the human disease mechanisms have to be studied on their own terms, and not only as an extension of the murine phenotype. Such an approach has already proved successful, for example by revealing the role of anti-cytokine antibodies in causing the increased susceptibility to *Candida* infections (7). Here, we review the main features of human APECED, both clinical and immunological. Although several important questions remain open, we also attempt to provide an explanation of the pathogenetic mechanisms, looked at from the human viewpoint.

## **CLINICAL FEATURES**

The classic triad of APECED consists of Addison's disease, hypoparathyroidism, and chronic mucocutaneous candidiasis

**Abbreviations:** AIRE, autoimmune regulator; ALAT, alanine amino transferase; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; APS-1, autoimmune polyendocrine syndrome type 1; CMC, chronic mucocutaneous candidiasis; GAD, glutamic acid decarboxylase; LKM, liver-kidney microsomal; mTEC, medullary thymic epithelial cells; RTE, recent thymic emigrant; TRA, tissue-restricted antigen; Treg, regulatory T cell.

(CMC), two of which are required for the diagnosis. The autoimmunity in APECED is organ-specific but with many components. There is no gender bias. Virtually all patients have more than two disease components and up to 10 components have been reported. On the average patients have four components (8). First symptoms occur on average at the age of five (range 0.2–18 years) (9). The most common endocrinopathies, hypoparathyroidism, and Addison's disease are usually diagnosed at the age of 3–5 years and 11–15 years, respectively. New components appear through life (9).

Chronic mucocutaneous candidiasis is usually the first sign of APECED and in Finnish patients its prevalence is 100%. Candidiasis is the most common component of APECED except in Iranian Jews in whom it is rarely described (10). The severity of the symptoms varies from redness and soreness of the corners of the mouth to inflamed mucosal surfaces in the whole oral cavity. Candidiasis increases the risk of oral carcinoma by causing chronic inflammation (1).

Hypoparathyroidism is the most common autoimmune component in APECED and APECED should be considered in the differential diagnosis of every patient with hypoparathyroidism of unknown cause. Addison's disease is the second most common autoimmune component and its prevalence was 78% in a large patient series (9). It most often presents with both mineralo- and glucocorticoid deficiency.  Ovarian insufficiency is actually the third most common autoimmune component, affecting approximately 65% of APECED women and starting in early adulthood (9). It can present with primary amenorrhea with a complete failure of or arrested pubertal development. About half of the cases develop premature menopause (9). Testicular failure has a maximum prevalence of 25% in men and starts usually at an older age (11).

About one third of APECED patients develop hypothyroidism, usually after puberty (9). Thyroid autoantibodies are found commonly but clinical disease is not always present. The prevalence of type I diabetes mellitus among APECED patients varies between populations. In a large Finnish patient series, the prevalence was about 30% (8). Gastrointestinal symptoms, such as chronic diarrhea, constipation, hepatitis, and gastritis are common. Autoimmune gastritis with pernicious anemia is present in approximately 30% of patients by middle age (9). In severe forms, patients develop chronic atrophic gastritis and pernicious anemia (9). About 20% of patients develop autoimmune hepatitis with variable severity (8).

Ectodermal manifestations include enamel hypoplasia of the teeth, alopecia, nail dystrophies, vitiligo, and ocular keratopathy (8, 9). Keratitis occurs in 25% of the patients and can lead to vision loss. Keratitis can be an early and even the first manifestation of the disease. Alopecia and vitiligo affect up to 30–40% of the patients by middle age (5, 12).

## **DIAGNOSIS AND AUTOANTIBODY FINDINGS**

The diagnosis of APECED is based on the clinical features, detection of autoantibodies, and genetic analysis. Two of the most common disease components are required for APECED diagnosis. Candidiasis is usually the first symptom, while

hypoparathyroidism and Addison's disease are the most common endocrinopathies. Since the gene test is available, the diagnosis is confirmed with the identification of the mutation. The type of mutation, however, does not predict the disease course. Due to the rarity of APECED, there is often delay of years before the diagnosis is set (13).

An important feature in the diagnostics is the existence of IgG autoantibodies. Their possible pathogenetic role is mostly unknown. Many autoantibodies in APECED are targeted against intracellular enzymes (14). It is possible that the detected autoantibodies are not pathogenetic but, instead, are a marker of the ongoing T cell activity at the target tissue (14). The presence of autoantibodies correlates with the disease components but autoantibodies can also precede the onset of the target organ failure (15). Some autoantibodies are closely associated with the corresponding disease manifestation, while others are found only in a subset of patients with the particular manifestation. For example, autoantibodies against calcium-sensing receptor are found in almost all APECED patients with hypoparathyroidism, whereas anti-NALP5 antibodies, also linked to hypoparathyroidism, are much less common (16, 17). Also, autoantibodies found in APECED patients may be different from those found in patients with an isolated autoimmune disease. A case in point is GAD (glutamic acid decarboxylase), an important autoantigen in type I diabetes but rarely targeted in APECED patients with diabetes as a disease component (18).

Once the initial diagnosis is made, APECED patients must be evaluated regularly and tested for autoantibodies, since new disease components appear through life (19). Suggestions for laboratory testing in the follow-up of APECED patients are presented in **Table 1**.

The recently discovered neutralizing antibodies against type I interferons are found in 100% APECED patients (20, 21). Antiinterferon antibodies are present before symptom development at high titers. In addition to APECED, type I interferon autoantibodies have been found only in patients with thymoma, but with lower prevalence and titers (20, 21). Thus, measurement of neutralizing

**Table 1 | Suggested tests in suspected APECED and for the follow-up of APECED patients.**

In diagnosed APECED patients the aim is to screen for new disease components for early diagnosis.

antibodies against the type I interferons α and ω is a sensitive diagnostic test.

Chronic mucocutaneous candidiasis correlates with autoantibodies against Th17 class cytokines, both in APECED patients and CMC patients without APECED (22–24). The autoantibodies are neutralizing and have been found against IL-22 (91% of patients), IL-17F (75%), and IL-17A (41%) (22). Testing for these antibodies is not commonly used in the diagnostic evaluation, since the abovementioned type I interferon antibodies are more prevalent and specific.

NACHT leucine-rich-repeat protein 5 (NALP5) is a protein expressed in the cytoplasm of parathyroid chief cells. Forty-nine percent of APECED patients with hypoparathyroidism are positive for NALP5 antibodies (16). These antibodies are not found in APECED patients without hypoparathyroidism or in non-APECED patients with hypoparathyroidism. NALP5 antibodies thus represent APECED-specific autoantibodies.

Adrenocortical autoantibodies can be detected by indirect immunofluorescence assays. These antibodies recognize 21 hydroxylase, 17-hydroxylase, and side-chain cleavage enzymes, which are involved in the steroid hormone synthesis (11). Ovarian insufficiency correlates with the presence of autoantibodies against side-chain cleavage enzyme (11). Steroid cell autoantibodies can be screened for by indirect immunofluorescence assays with ovarian and testicular tissues. APECED patients with ovarian insufficiency also have elevated FSH and LH levels and low estrogen levels (1).

Fifty percent of APECED patients with hepatic involvement have liver-kidney microsomal (LKM) antibodies (25). The cytochrome target antigen is CYP1A2. Autoantibodies against CYP1A2 are highly specific (100%) but their sensitivity is only 50% (12). Plasma alanine amino transferase (ALAT) are a good marker for developing autoimmune hepatitis in APECED (1). Smooth muscle cell antibodies, which are a marker for autoimmune hepatitis, are not commonly present in APECED-associated hepatitis (25).

In APECED,autoantibodies against IA-2 (tyrosine phosphataselike protein IA-2) correlate with the development of type I diabetes. However, the sensitivity is low (11). GAD antibodies are relatively common (33%) in APECED patients but their presence does not correlate with diabetes, in contrast to non-APECED patients (11, 18).

Gastrointestinal symptoms, for example constipation and diarrhea are common among APECED patients. Hypocalcemia due to hypoparathyroidism can cause diarrhea. Exocrine pancreatic failure occurs in a few percent of patients and can present with malabsorption and steatorrhea (9). Plasma calcium levels and exocrine pancreatic function should be assayed in APECED patients with diarrhea.

## **GENETICS OF APECED**

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is caused by loss-of-function mutations in the *AIRE* gene. The human *AIRE* gene is found in the q22 region of chromosome 22, and shares a 71% sequence homology with its murine counterpart Aire (3, 4, 26). At full length, AIRE encodes a 58 kDa protein of 545 amino acids, although two other, shorter splice variants of unknown significance have been described (4, 27). AIRE contains a N-terminal caspase-recruitment domain and SAND domain that together regulate AIRE's multimerization, two plant homeodomains (PHDs), and a proline-rich region (**Figure 1**) (28, 29). The N-terminus also contains a nuclear localization signal, while C-terminus is important for transcriptional activation. PHDs are zinc fingers involved in protein-protein interaction, and PHD1 has been shown to mediate the binding of AIRE to non-methylated histone H3 (30–34). Together, these sequence and structural features suggest a role in the regulation of gene transcription, but despite some data suggesting otherwise (35), it is unlikely that AIRE is a transcription factor that directly binds DNA.

Today, more than 60 APECED mutations have been identified. The mutations are found throughout the coding region in AIRE and include nonsense mutations causing premature stop codons, frameshifts caused by deletions and missense mutations (36). The most common ones are the mutations R257X (c.769C > T) in exon 6 and a 13-base pair deletion (c.967–979del) in exon 8 (37, 38). APECED is inherited in an autosomal recessive manner and heterozygosity does not cause APECED. However, there is one Italian family where a dominantly inherited mutation in the AIRE gene (G228W) causes APECED (39).

In general, the type of mutation in APECED does not correlate with the clinical features (40). Phenotype can vary even between siblings carrying the same mutation (9). However, the R257X mutation carriers have more commonly candidiasis than patients with other mutations (40). Also, Iranian Jews APECED patients have a unique mutation (Y85C, c.374A > G) and do not develop keratopathy or candidiasis (10).

## **AIRE's EXPRESSION AND FUNCTION**

A few years after the discovery of the genetic defect responsible for APECED, experiments with knockout mouse models linked AIRE to the transcription of tissue-restricted antigens (TRAs) in thymus. The highest expression of AIRE is found in medullary thymic epithelial cells (mTECs) (41), which are capable of expressing a diverse set of genes normally restricted to certain tissues (42, 43). This phenomenon has been denoted ectopic transcription and is likely to be important for the deletion of autoreactive thymocytes. In the absence of Aire a subset of thymic TRAs was

**FIGURE 1 | The structure of AIRE protein**. CARD: caspase recruitment domain; SAND: human Sp100, Aire1, NucP41/P75, and Drosophila DEARF1 domain; PHD: plant homeodomain; PRR: proline-rich region. CARD and SAND are associated with AIRE multimerization and nuclear localization, while PHD1 mediates binding to non-methylated histone H3. Mutations disrupting AIRE's function have been found in all domains except PRR, and also at the extreme C-terminus. Areas where recessive mutations have been found are shown below the schematic structure as horizontal lines, while the location of the dominant mutation is shown as a vertical line.

down-regulated, suggesting that Aire functioned as a regulator of the ectopic transcription of some TRAs in mTECs (44). Another study using a model antigen with Aire-regulated promoter provided evidence of increased development of autoreactive T cells and subsequent autoimmunity when Aire was knocked out, at least in that particular transgenic system (45). Later studies have confirmed but also complicated the link between TRA transcription and Aire (2).

It has been difficult to establish how AIRE facilitates TRA transcription, given the promiscuous nature of its function and the diverse and context-dependent set of genes affected by AIRE's absence. Several studies have reported that AIRE can bind DNA, suggesting that it might function as a transcription factor (35, 46). Others, however, have challenged the significance of direct DNA binding by AIRE (32, 47, 48). Moreover, the genes regulated by AIRE lack shared promoter elements (47), and it is thus difficult to see how AIRE could by direct DNA binding regulate such a diverse set of genes.

A detailed analysis of AIRE's molecular partners suggests a less direct role for AIRE in gene transcription. A common feature of AIRE-regulated genes is that they are associated with a stalled RNA polymerase II (RNAPII), which stops full-length transcription (34, 49, 50). AIRE then seems to allow further elongation of the target genes. A recent attempt to formulate a model suggests that AIRE first binds to non-methylated histone H3 (29). It then recruits positive transcription elongation factor b (P-TEFb) to phosphorylate RNAPII, reactivating the stalled polymerase and gene transcription. Although this would allow for a broad range of genes to be activated, a major problem with this model is to explain how AIRE's control on TRAs is maintained even when the TRAs are expressed as transgenes, outside of their normal epigenetic environment.

A competing explanation is that AIRE is not directly involved in TRA transcription, but rather regulates mTEC maturation and death, thus indirectly affecting also mTEC-expressed TRAs. In support of this possibility are numerous studies showing that Airedeficiency results in fundamental changes in the mTEC population (51–54). The expression of AIRE is a late event in the lifespan of mTECs (55), and it has been suggested to be a terminal differentiation factor for mTECs. Thus, in the absence of AIRE, the altered developmental pathway of mTECs might lead to a decrease in the TRA-expressing stages, either because of abnormal cell death or diverted or arrested maturation. However, although the mTEC differentiation model is compatible with most reported effects of AIRE-deficiency, the molecular mechanisms by which AIRE regulates mTEC biology are largely unknown.

The strengths and weaknesses of scenarios involving regulation of gene transcription versus regulation of mTEC homeostasis have been more fully summarized in a recent review (47). At the moment the available data do not provide unequivocal support to either of the two main models, and it should also be noted that practically all the data come from studies on the murine system or cell lines. Nevertheless, we would argue that the human phenotype is difficult to reconcile with models invoking regulation of TRA transcription as the main function of AIRE. We discuss these aspects in more detail below, in the chapter on the pathogenesis of APECED.

A further complication is AIRE's expression in peripheral tissues, a phenomenon of largely unknown significance (56). Early studies showed AIRE mRNA expression in a wide range of tissues, but not all of these studies were confirmed on protein level. In the periphery AIRE is expressed at significantly lower levels than in the mTECs, making reliable detection with mostly polyclonal antibodies difficult, and some studies have questioned the presence of any extrathymic AIRE (57). Nevertheless, a wide agreement exists for the expression of AIRE in lymphoid tissues, with perhaps dendritic cells as the main AIRE+ population (58, 59). The peripheral AIRE+ cells can also express TRAs, although the genes are only partly overlapping with the thymic set (60).

## **IMMUNOLOGICAL ABNORMALITIES**

The most obvious immunopathological finding in APECED patients is the diverse set of autoantibodies, mostly against tissues affected by the disease. The autoantibodies are often directed against enzymes involved in hormone synthesis, but since these enzymes are intracellular, the organ-specific autoantibodies rarely have pathogenetic significance. Of special interest are the neutralizing autoantibodies to cytokines, since they are likely to have pathogenetic effects. These autoantibodies are found in practically all APECED patients, while extremely rare in healthy people, and can block interferon-induced gene expression both *in vivo* and *in vitro* (21, 61). The more recently described neutralizing autoantibodies against the Th17 cytokines IL-17A, IL-17F, and IL-22 are linked to the defective antifungal defense and CMC (22, 24).