Ultrastructure of medullary thymic epithelial cells of ... - Nature

Immunology and Cell Biology (2010) 88, 50–56 & 2010 Australasian Society for Immunology Inc. All rights reserved 0818-9641/10 $32.00 www.nature.com/icb

ORIGINAL ARTICLE

Ultrastructure of medullary thymic epithelial cells of autoimmune regulator (Aire)-deficient mice Zˇivana Milicévic´1, Novica M Milicévic´1, Martti Laan2, Pa¨rt Peterson2, Kai Kisand2, Hamish S Scott3 and Ju¨rgen Westermann4 The significance of the autoimmune regulator (Aire) transcription regulator in establishing central tolerance has recently been elucidated in great detail. Still, the role of Aire in medullary thymic epithelial cell (mTEC) physiology is not fully understood. To shed more light on this issue, we studied the ultrastructure of mTECs in Aire-deficient thymus. We show that all types of mTECs show ultrastructural signs of activation and increased intracellular traffic, which suggests that in the absence of Aire their physiology is impaired. Type 6 ‘large’ mTECs are fully developed in Aire-deficient mice and more frequent than in the normal thymus. The frequency of type 5 ‘undifferentiated’ mTECs is also increased. Collectively, our results suggest that the role of Aire in the physiology of mTECs could be more profound and not restricted only to the presentation of self-tissue-restricted antigens and/or apoptosis of end-stage fully mature cell types. Immunology and Cell Biology (2010) 88, 50–56; doi:10.1038/icb.2009.55; published online 1 September 2009 Keywords: thymus; medullary epithelial cells; ultrastructure; Aire; mice

The thymocytes selected for export from the thymus into the peripheral lymphoid organs are at the same time self-major histocompatibility complex (MHC)-restricted and self-tolerant. This is achieved through a stringent process of selection. During positive selection, which takes place in the thymic cortex under the control of cortical thymic epithelial cells, the thymocytes become self-MHCrestricted,1 whereas during negative selection that predominantly occurs in the thymic medulla, autoreactive thymocytes are removed by clonal deletion.2 In recent years, the physiological basis of negative selection has been much elucidated. It has been shown that medullary thymic epithelial cells (mTECs) are capable of expressing a wide variety of genes and synthesizing self-tissue-restricted antigens (TRAs) under the control of the autoimmune regulator (Aire) transcription regulator.3,4 The ectopic Aire-controlled expression of TRAs in the thymic medulla enables the clonal deletion of autoreactive thymocytes.5,6 However, additional roles for Aire in mTEC physiology have been proposed. According to the model of ‘terminal differentiation,’ Aire expression occurs in highly differentiated mTECs and drives a broad display of TRAs.7 In contrast, the concept of ‘developmental restriction’ suggests that Aire expression occurs in immature mTECs and fosters their differentiation into progressively restricted cell fates showing the TRAs limited by the chosen destiny.8 Very recently, this concept has been reformulated,9 which supersedes the one by the same group;8 now, it is proposed that Aire deficiency leads to an expansion

of end-stage terminally differentiated mTECs. Furthermore, it has been shown that Aire is involved in the control of the mTEC life cycle inducing their apoptotic cell death.10 Considering that ultrastructural studies depicting all subcellular elements provide veritable and complete information on the functional state and differentiation stage of a given cell type,11,12 we felt it was imperative to investigate all mTEC types in Aire-deficient mice to shed more light on the role of Aire in mTEC physiology. Our study provides new insight into the functional role of Aire showing that in Aire-deficient thymus all mTEC types show ultrastructural signs of activation and increased intracellular traffic suggestive of profound alterations in cell physiology. In addition, we show that the most mature ‘large’ mTECs are fully developed in Airedeficient mice. Finally, the least mature ‘undifferentiated’ mTECs appear to be increased in frequency as well. RESULTS Light microscopy The thymic medulla of normal C57BL/6 mice is well developed, neatly organized and uniformly populated with thymocytes. In semi-thin sections, the mTECs can be easily distinguished and only some of them possess the characteristic varieties of intracellular vacuoles (Figure 1), as described earlier in the rat13 and mouse.14 A majority of mTECs do not present the intracellular vacuoles (Figure 1).

1Faculty of Medicine, Institute of Histology and Embryology, University of Beograd, Beograd, Serbia; 2Department of Molecular Pathology, Institute of General and Molecular Pathology, Tartu University, Tartu, Estonia; 3Division of Molecular Pathology, Institute of Medical and Veterinary Science and The Hanson Institute, and School of Medicine, University of Adelaide, Adelaide, South Australia, Australia and 4Center for Structural and Cell Biology in Medicine, Institute of Anatomy, University Lu¨beck, Lu¨beck, Germany Correspondence: Dr NM Milicévic´, Faculty of Medicine, Institute of Histology and Embryology, Visˇegradska 26, 11000 Belgrade, Serbia. E-mail: [email protected] Received 6 April 2009; revised 8 July 2009; accepted 9 July 2009; published online 1 September 2009

Ultrastructure of Aire-deficient thymus Zˇ Milicévic´ et al 51

Table 1 Frequency of different medullary epithelial cell types in the normal and Aire-deficient thymus Normal

Aire-deficient

Type 5 ‘undifferentiated’

+

+++

Type 6 ‘large’ Type 7 ‘spindle-shaped’

+ ++

+++ ++

0

+

‘Signet-ring cells’

Abbreviation: Aire, autoimmune regulator. 0 ¼ absent; + ¼ singular cell; ++ ¼ groups with two cells; +++ ¼ groups with three or more cells.

intracytoplasmic vacuoles, even in those creating Hassall’s corpuscles (Figure 2). Some mTECs are enlarged, rounded and filled with vacuoles to such an extent that these cells attain the appearance of signet-ring cells (Figure 2, inset). Thus, owing to the increased amount of vacuoles, only some mTECs can be related to normal mTEC types. Figure 1 Normal thymus. The thymic medulla is well populated with lymphocytes. All variants of ‘large’ medullary thymic epithelial cells are easily recognizable: having numerous, smaller (white arrows), fewer, larger, compressed vacuoles (black arrow) or a single, large vacuole (arrowhead). Toluidine blue. Bar, 10.0 mm.

Figure 2 Autoimmune regulator-deficient thymus. The thymic medulla seems less populated with thymocytes, and medullary thymic epithelial cells (mTECs) show an untidy organization. Most of the mTECs are enlarged and the majority of them show the presence of vacuoles to a varying degree. However, only a rare one (black arrow) can be related to the variant of normal ‘large’ mTECs indicated by a black arrow in Figure 1, but showing more vacuoles. Hassall’s corpuscle is well developed (arrowhead). Inset: mTEC is enlarged, rounded, filled with vacuoles, attaining the signet-ring cell appearance. Toluidine blue. Bar, 10.0 mm.

The thymic medulla of Aire-deficient mice is smaller, besides being clumsily organized and unevenly populated with thymocytes. mTECs in the Aire-deficient thymus form large groups without thymocytes more often than in the normal thymus (Figure 2). The majority of mTECs are much enlarged, rounded and without notable cytoplasmic prolongations. They show an extremely euchromatic nucleus with multiple prominent nucleoli. Very notable is the presence of numerous

Electron microscopy Normal thymus. Similarly, as in the normal human15 and rat thymus,13 three types of epithelial cells may be distinguished using ultrastructural criteria in the thymic medulla of normal mice.14 Type 5 ‘undifferentiated’ mTECs are infrequently observed as singular cells (Table 1). They are rounded in shape, with short, delicate cytoplasmic extensions, and characteristically show an immature, blastoid appearance; the nucleus is extremely euchromatic with the patent nucleolus. In the cytoplasm, the polyribosomes prevail, whereas other organelles are sparse and small. Cytokeratin bundles are very delicate, too (Figure 3). These cells are most often located at the corticomedullary boundary. Type 6 ‘large’ mTECs have very abundant cytoplasms and several inconspicuous cytoplasmic prolongations. These cells have a strictly polarized subcellular organization. The hallmark of these cells is the presence of vacuoles, which are clustered in one area of the cytoplasm in the vicinity of the nucleus. The vacuoles are usually smaller and acquire a grape-like form, occasionally showing delicate internal microvillous projections (Figure 4). Sometimes, the vacuoles are fewer in number, but larger and compressed (Figure 5). Rarely, a solitary cyst-like structure is observed, sometimes with internal cilia (not shown, see Figure 1). The vacuoles are surrounded by larger cytokeratin bundles, with smaller bundles projecting between them. In the neighboring cytoplasm, numerous transport vesicles, sometimes fusing with the vacuoles, as well as large Golgi complexes are observed (Figures 4 and 5). In the cytoplasmic region opposite to that occupied by the vacuoles, profiles of the rough endoplasmic reticulum are observed (Figure 4). The surface membrane shows prominent intercellular junctional complexes (Figure 4). Type 7 ‘spindle-shaped’ mTECs are small, often arranged in groups (Table 1), and connected to each other by large desmosomes and interdigitations. The cytoplasm is sparse, with scanty organelles and thick bundles of cytokeratin (Figure 6). Hassall’s corpuscles are small, usually composed of just a few epithelial cells, often with a quiescent appearance and prominent cytoplasmic keratin bundles. Aire-deficient thymus. At the ultrastructural level, three major observations can be made. First, all types of mTECs can be readily identified, including the most mature ‘large’ phenotype. Second, all mTEC types show the various morphological signs of increased activity, with hypertrophied cellular compartments involved in exocytosis and endocytosis, whereby some very large, rounded mTECs are filled with giant vacuoles to such an extent that they attain the Immunology and Cell Biology


Figure 3 Normal thymus. Type 5 ‘undifferentiated’ medullary thymic epithelial cell is rounded in shape, with a very euchromatic nucleus (N). The cytoplasm is scanty and shows very delicate cytoplasmic extensions (arrow). In the cytoplasm, the polyribosomes prevail, whereas other organelles, including the keratin filaments (arrowheads), are sparse and small. Uranyl acetate and lead citrate. Bar, 0.8 mm.

Figure 4 Normal thymus. Type 6 ‘large’ medullary thymic epithelial cell has a very abundant cytoplasm and inconspicuous prolongations. It shows signs of intense metabolic/secretory activity and has a strictly polarized subcellular organization. In one area of the cytoplasm, in the vicinity of the very euchromatic nucleus (N), a characteristic grape-like cluster of smaller vacuoles (v) is located. In the neighboring cytoplasm, numerous transport vesicles (black arrowhead), sometimes fusing with the vacuoles, as well as a large Golgi complex (black arrow) are seen. In the opposite region of the cytoplasm, profiles of the rough endoplasmic reticulum are observed (white arrowheads). The surface membrane shows prominent intercellular junctional complexes (white arrow). Uranyl acetate and lead citrate. Bar, 1.0 mm.

appearance of signet-ring cells. Third, the frequency of the least mature ‘undifferentiated’ mTECs is also increased. As briefly mentioned, type 5 ‘undifferentiated’ mTECs are more numerous and, in contrast to the normal thymus, can be seen in groups even in the medulla (Figure 7, Table 1). They retain their immature appearance: the nucleus is very euchromatic and large; the polyribosomes are numerous and the cytokeratin bundles remain very delicate. These cells, however, are enlarged and the cytoplasm is more abundant. Signs of increased activity are observed: the Golgi complexes are notable and surrounded by numerous vesicles, whereas the Immunology and Cell Biology

Figure 5 Normal thymus. Type 6 ‘large’ medullary thymic epithelial cell shows vacuoles (v) that are fewer in number, but larger and compressed, strictly positioned in the vicinity of the euchromatic nucleus (N). The vacuoles are surrounded by larger cytokeratin bundles (small arrow), with smaller bundles projecting between them. In the neighboring cytoplasm, numerous transport vesicles (black arrowhead), as well as a large Golgi complex (large arrow), are seen. In the distant cytoplasmic region, numerous mitochondria and profiles of the rough endoplasmic reticulum (white arrowhead) are observed. Uranyl acetate and lead citrate. Bar, 0.8 mm.

Figure 6 Normal thymus. Type 7 ‘spindle-shaped’ medullary thymic epithelial cell is small. The cytoplasm is sparse, with scanty organelles and thick bundles of cytokeratin (arrows) around the euchromatic nucleus (N), which has a prominent nucleolus. The interdigitations with surrounding cells are prominent. Uranyl acetate and lead citrate. Bar, 0.8 mm.

perinuclear envelope forms the large dilatations continuous with the dilated cisternae of the rough endoplasmic reticulum (Figure 7). Type 6 ‘large’ mTECs are also seen often in groups (Figure 8, Table 1). The cells are enlarged, polygonal in shape with an extremely euchromatic nucleus and very prominent nucleoli. The cytoplasm is occupied by hypertrophied organelles, but the ‘large’ mTECs retain a characteristically polarized organization (Figures 8–11). The accumulation of vacuoles dominates in the perinuclear region. They are greatly increased in number and show signs of hyperactivity: many have a tortuous shape and present intravacuolar microvillous projections that sometimes acquire a giant size (Figure 9); coalescence with transport vesicles is observed much more often than in normal cells (Figures 9 and 10). They are surrounded by a myriad of transport vesicles and keratin bundles of normal appearance (Figures 9–11). In the vicinity


Figure 7 Autoimmune regulator-deficient thymus. A group of type 5 ‘undifferentiated’ medullary thymic epithelial cells (Ep). The cells are enlarged and the cytoplasm is more abundant. They show an immature appearance: the nucleus is very euchromatic; the polyribosomes are numerous and the cytokeratin bundles are very delicate (arrowheads). They also show signs of increased activity: the cisternae of rough endoplasmic reticulum (small arrows) are dilated and continuous with the perinuclear envelope, which also forms the large dilatations (large arrows). Uranyl acetate and lead citrate. Bar, 1.0 mm.

Figure 8 Autoimmune regulator-deficient thymus. A group of type 6 ‘large’ medullary thymic epithelial cells (Ep). The cells are very large with a very euchromatic nucleus and a very prominent nucleolus. The cytoplasm is very abundant and polarized with an increased number of compressed and tortuous vacuoles. The Golgi complexes are numerous and very enlarged (large arrows). Opposite to the vacuoles, dilatations of the rough endoplasmic reticulum (arrowheads) and endocytic compartments (small arrows) are seen. Uranyl acetate and lead citrate. Bar, 2.5 mm.

of vacuolar accumulations, hypertrophied Golgi complexes are observed, accompanied by a large number of vesicles (Figures 8–11). Mitochondria are enlarged and more numerous. In the opposite region of the cytoplasm, the granular endoplasmic reticulum, sometimes showing large dilatations, and very developed compressed membrane profiles associated with lipid droplets are encountered (Figure 10). Large endosomal compartments also appear surrounded by a wealth of vesicles (Figures 8 and 11). The interdigitations of the surface cell membrane with the neighboring cells are also more prominent than in normal cells (Figure 8). Type 7 ‘spindle-shaped’ mTECs can be readily recognized because of their characteristic shape

and massive keratin bundles. However, these cells also acquire a more active appearance: the cytoplasm is more abundant; the nucleus is more euchromatic with numerous nuclei; the number of large mitochondria is increased; dilated cisternae of the granular endoplasmic reticulum and active Golgi complexes with numerous vesicles and numerous polyribosomes are present in the cytoplasm (Figure 12). Finally, mTECs with special subcellular features that do not fit into the classification of normal mTECs are also seen (Table 1). They have a very euchromatic nucleus and abundant cytoplasm, which is almost filled up with giant multilamellar and multivesicular bodies lending them a signet-ring cell appearance. Between these giant multilamellar and multivesicular Immunology and Cell Biology


Figure 9 Autoimmune regulator-deficient thymus. Cytoplasm of a type 6 ‘large’ medullary thymic epithelial cell with the characteristic vacuoles showing increased activity: giant, multiple internal microvillous projections (small black arrows), tubular vesicle-like structures topped with coated vesicles (white arrows), as well as very numerous fusions with numerous coated vesicles. Large numbers of transport vesicles in the surrounding cytoplasm and dilated saccules of Golgi complexes (large black arrows) are seen. N, nucleus; Uranyl acetate and lead citrate. Bar, 0.4 mm.

Figure 11 Autoimmune regulator-deficient thymus. The cytoplasm of a type 6 ‘large’ medullary thymic epithelial cell shows a strict polarization. In the vicinity of the nucleus (N), a large accumulation of characteristic vacuoles with numerous enlarged Golgi complexes (arrows) is seen. In other parts of the cytoplasm, the enlarged endocytic compartments (arrowheads), accompanied by a huge number of transport vesicles of varying size, are observed. Uranyl acetate and lead citrate. Bar, 1.0 mm.

Figure 10 Autoimmune regulator-deficient thymus. Type 6 ‘large’ medullary thymic epithelial cell with greatly increased numbers of characteristic vacuoles, some fusing with multiple coated vesicles (v), surrounded by a myriad of transport vesicles. The dilatations of a rough endoplasmic reticulum (black arrowheads) and prominent proliferation of condensed membrane profiles (white arrows), as well as Golgi complexes (arrows) and mitochondria, are seen. N, nucleus; Uranyl acetate and lead citrate. Bar, 1.0 mm.

Figure 12 Autoimmune regulator-deficient thymus. Type 7 ‘spindle-shaped’ medullary thymic epithelial cell retains a characteristic shape and prominent keratin bundles (small arrows). However, the cytoplasm is more abundant, the nucleus (N) is more euchromatic with numerous nuclei, the number of large mitochondria is increased, the cisternae of granular endoplasmic reticulum (arrowheads) are dilated and Golgi complexes (large arrow) are well developed. Uranyl acetate and lead citrate. Bar, 0.8 mm.

bodies, all grades of smaller bodies are seen. The presence of keratin bundles confirms the epithelial nature of these cells (Figure 13). Hassall’s corpuscles are regularly observed, composed of mTECs that also show the increased number of vesicles and cytoplasmic vacuoles (Figure 2).

Therefore, in our study, we used the ultrastructural approach— this enables the precise identification of cell types and provides a thorough insight into the physiological aspects (including stage of differentiation) of mTECs.13,14 Indeed, using ultrastructural criteria, we show that in the Aire-deficient thymus: (i) all types of mTECs show signs of activation and increased intracellular traffic, (ii) the most mature ‘large’ mTECs are present and, in fact, even more numerous than in the normal thymus and (iii) the frequency of the least mature ‘undifferentiated’ mTECs is increased as well. The increased frequency and changed morphology of all organelles involved in the intracellular traffic, exocytosis and endocytosis (transport vesicles, Golgi complexes, large vacuoles and endosomal compartments) in all types of mTECs, but especially in the most mature

DISCUSSION In earlier studies on the role of Aire in mTEC function and differentiation, a light microscopic approach was used8–10,16 with a limited number of previously established criteria for a phenotypic classification of thymic epithelial cells (TECs).17–19 In contrast, ultrastructural studies on the Aire-deficient thymus encompassing all morphological features of these cells are lacking in the literature. Immunology and Cell Biology


Figure 13 Autoimmune regulator-deficient thymus. Medullary thymic epithelial cell with the signet-ring cell appearance. Most notable is the giant multivesicular vacuole (V) with numerous small vesicles (small arrowheads) at the inner aspect of the vacuolar membrane and endosomes of varying size fusing with the coated vesicles (large arrowheads) in the directly neighboring cytoplasm. Numerous lamellar bodies (asterisks), as well as light (L) and dark (D) multivesicular bodies, are also seen. The nucleus (N) is euchromatic. The presence of keratin bundles (arrow) confirms the epithelial nature of the cell. Uranyl acetate and lead citrate. Bar, 1.0 mm.

‘large’ mTECs in the Aire-deficient thymus, strongly suggest the uncoupling of negative feedback signaling with subsequent hypertrophy of affected/involved cellular components. Specifically, the communication between TECs and maturing thymocytes is not unidirectional, but the thymocytes at a certain stage of development regulate the proper development of the corresponding TEC type, which is referred to as thymic cross talk.20 Very recent studies point out that the expansion and development of mature Aire-positive mTECs depend on the receptor activator of nuclear factor-kappaB ligand (RANKL) and CD40L produced by positively selected CD4+ autoreactive thymocytes.21,22 Thus, the following scenario may be proposed: (i) the autoreactive mature thymocytes stimulate the mTECs,21,22 (ii) but, owing to the absence of Aire, the mTECs cannot express the TRAs, which hampers the apoptosis of autoreactive thymocytes5,6 and (iii) the surviving autoreactive thymocytes provide for the sustained stimulation of mTECs with a consequent increase in frequency of all organelles involved in intracellular traffic until some mTECs acquire the appearance of signet-ring cells. Such ‘giant multivesicular bodies’ showing positive vacuolar staining for immunoglobulin and T-cell antigens (both of which are normally found on the cell membrane) have been reported in ultrastructurally similar forms of B and T signet-ring cell lymphomas and also have been interpreted as a defect in plasma membrane recycling.23–26 Using the ultrastructural classification of mTECs,13,14 we show that the most mature ‘large’ mTECs are present in the Aire-deficient thymus and even more numerous than in the normal thymus. This fact implies that Aire is not directly involved in the control of mTEC differentiation, as in its absence the process proceeds unimpaired producing numerous mTECs of the most mature ‘large’ phenotype. In comparison with the normal thymus, the mature mTECs of ‘large’ phenotype are frequently localized in groups. This is consistent with the light microscopic findings showing that the minor population of ‘globular,’ fully differentiated mTECs present in the Aire-sufficient thymus is significantly expanded in the Aire-deficient thymic medulla.9,16 At this place, the advantages of ultrastructural studies become evident. Our study clearly shows that three mTEC types in

the Aire-deficient thymic medulla may actually correspond to the ‘globular’ mTECs observed with increased frequency in light microscopy:9,16 type 5 ‘undifferentiated’ mTECs, type 6 ‘large’ and mTECs with special subcellular features that do not fit into the classification of normal mTECs. All these mTEC types are increased in number, enlarged and rounded, which may appear as ‘globular’ in light microscopy. Finally, the increased frequency of mature ‘large’ mTECs in the Aire-deficient thymus also confirms that normally Aire is involved in the induction of their apoptotic cell death.10 Lastly, the increased frequency of the least mature ‘undifferentiated’ mTECs may also be a result of sustained stimulation by autoreactive thymocytes in the absence of Aire. If so, the role of Aire in the control of mTEC numbers could be more profound and not restricted to apoptosis of end-stage, fully mature mTECs.10 Possibly, the Aire activity could influence the general number of mTECs by reducing the frequency of autoreactive thymocytes and, in this manner, indirectly suppressing the proliferation of immature mTECs. In conclusion, our findings collectively show that Aire deficiency affects all mTEC types inducing a general change in the thymic medulla. METHODS Mice Autoimmune regulator-deficient mice of both sexes (8–10 weeks of age) were used in the study. Mice deficient in the Aire gene were generated at The Walter and Eliza Hall Institute (Melbourne, Australia) by a homologous recombination of targeting vectors in mouse C57BL/6 embryonic stem cells. Insertion of the Aire-targeting vector disrupted exon 8 and brought the LacZ reporter gene under the control of the endogenous Aire promoter, creating an Aire–LacZ fusion. A phosphoglycerate kinase neomycin cassette was used to select positive recombination events and was later removed using the flanking LoxP sites and Cre recombinase.27 Wild-type C57BL/6 mice (8–10 weeks of age) were used as controls. Wild-type and Aire-knockout mice were bred and maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. Permission to carry out these animal experiments was issued by the Estonian State Committee for Licensing of Animal Experiments.

Light and electron microscopy For light microscopy, the thymic tissue was fixed in neutral buffered formaldehyde or Bouin’s solution, routinely processed for paraffin wax sectioning (3– 5 mm) and stained with hematoxylin–eosin. For electron microscopy, pieces of thymic tissue were quickly immersed in glutaraldehyde and cut into 1-mm3 cubes, which were fixed for 2 h in a solution of 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH¼7.4) at 4 1C. Then, the tissue was thoroughly washed three times in 0.1 M sodium cacodylate buffer and post-fixed in 1% OsO4 in 0.1 M sodium cacodylate buffer for 2 h at 4 1C. After the repeated washing, the tissue was dehydrated in graded alcohols and embedded in Epon 812. The blocks were cut on an LKB Ultramicrotome III (LKB, Bromma, Sweden). The semi-thin sections (1 mm) were stained with toluidine blue, whereas the ultrathin sections were routinely contrasted with uranyl acetate and lead citrate. The material was examined with a JEOL JEM-1011 electron microscope (JEOL, Tokyo, Japan). The frequency of different mTEC types was estimated using the following approach. First, a certain mTEC type was identified. Then, we investigated whether it appeared as a singular cell or in a group of identical cells. Thereafter, the frequency of this mTEC type was described using the objective scale based on the pattern of mTEC organization (0¼absent; +¼singular cell; ++¼small groups with two identical cells; +++¼larger groups with three or more identical cells) in the normal and Aire-deficient thymus.

ACKNOWLEDGEMENTS Thanks are due to Maire Pihlap, Gudrun Knebel and Ljubomir Ognjanovic´ for expert technical assistance. This work was supported by the Ministry for Immunology and Cell Biology

Ultrastructure of Aire-deficient thymus Zˇ Milicévic´ et al 56 Science and Technological Development of Republic of Serbia (grant no. 145016) and is a part of the Institutional Academic Cooperation between Beograd and Lu¨beck (project DEU/1033146), which is financially supported by the Alexander von Humboldt-Foundation, Bonn, Germany. Martti Laan was supported by the Estonian Science Foundation (grant no. 7559).

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