Activation of cortical and inhibited differentiation of medullary epithelial cells in the thymus of lymphotoxin-beta receptor-deficient mice: an ultrastructural study

Abstract

The reciprocal influences of thymic lymphocyte and nonlymphocyte populations, i.e. thymic cross-talk, are necessary for the proper maturation of thymocytes and the development/maintenance of thymic stromal microenvironments. Although the molecular influences exerted by thymic stromal cells on maturing thymocytes have been extensively studied, the identity of signalling molecules used by thymocytes to influence the thymic stromal cells is still largely unknown. Our study provides the first ultrastructural evidence that the functional lymphotoxin-beta receptor (LTβR) signalling pathway is engaged in the cross-talk between thymocytes and the thymic stromal cell population. We show that LTβR signalling is of the utmost significance for the preservation of the subcellular integrity of all thymic epithelial cells. In the absence of LTβR there is (1) hypertrophy and activation of cortical thymic epithelial cells, (2) the complete loss of fully differentiated medullary thymic epithelial cells, and (3) the inhibited differentiation of remaining medullary thymic epithelial cells with the appearance of prominent intercellular cysts in the thymic medulla.

Introduction

The thymus provides a specific inductive microenvironment for the proliferation and differentiation of precursor cells originating from the bone marrow into mature T lymphocytes. This process is stringently controlled. Only those lymphocytes that, on the one hand, acquire the capability to recognize the foreign antigens presented in the context of self-major histocompatibility complex (MHC) molecules and, on the other hand, become tolerant to self-antigens are allowed to exit the thymus and enter the pool of peripheral mature T lymphocytes. The former process is known as positive selection and is controlled by cortical thymic epithelial cells (cTEC; Jenkinson et al. 1992; Anderson et al. 1994). The latter process is called negative selection (recently often referred to as central tolerance) and occurs under the control of medullary thymic epithelial cells (mTEC) and mononuclear cells, whereby the autoreactive cells are removed by clonal deletion (Sprent & Kishimoto, 2002).

The thymic microenvironment is created by epithelial cells and cells of the mononuclear phagocyte system. Different types of these so-called stromal cells express or release a variety of active molecules (MHC molecules, adhesion molecules, chemokines, extracellular matrix components and hormones). Thymic epithelial cells show a great ultrastructural diversity. Electron microscopy enables an easy distinction between four cTEC and three mTEC types in man (van de Wijngaert et al. 1984) and rat (Milićević & Milićević, 1997). In this manner, several tissue niches are created which are suitable for different stages of thymocyte maturation to occur.

However, the interactions between stromal cells and maturing thymocytes are not unidirectional, but reciprocal. More specifically, not only do thymic stromal cells govern the process of thymocyte maturation within the thymus gland but, in turn, the thymocytes also exert a feedback influence on stromal cells controlling their differentiation, integrity and organization. These mutual influences are encompassed by the concept of thymic cellular cross-talk (Ritter & Boyd, 1993; van Ewijk et al. 1994). However, although the molecular influences exerted by thymic stromal cells on maturing thymocytes have been extensively studied (reviewed in Milićević & Milićević, 2004), the identity of signalling molecules used by thymocytes to influence the thymic stromal cells is still largely unknown. Only recently has it been shown that lymphotoxin beta (LTβ) is one such molecule, and that the functional LTβ/LTβ-receptor signalling pathway is of the utmost importance for the preservation of the structural and functional integrity of mTEC. In LTβ-receptor (LTβR)-deficient mice, a reduction in the number and aberrant maturation of mTEC has been shown by light microscopy, which is accompanied by the breakdown of negative selection, the release of autoreactive cells from the thymus and autoimmune manifestations (Boehm et al. 2003; Chin et al. 2003). Therefore we felt there was an urgent need to perform a detailed ultrastructural study and to investigate the subcellular organization of all epithelial cell types in the LTβR-deficient thymus. This would clarify the nature of epithelial aberrations in the LTβR-deficient thymus and further elucidate the causes of functional breakdown of thymic negative selection in these animals. Our study revealed a thorough ultrastructural alteration of all thymic epithelial cell types in LTβR-deficient animals.

Materials and methods

Animals

Normal C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Professor Klaus Pfeffer, Institute of Medical Microbiology, University of Düsseldorf, Germany, kindly provided LTβR-deficient mice. LTβR-deficient mice had been backcrossed to C57BL/6 mice at least six times and were genotyped as described previously (Futterer et al. 1998). Eight normal and eight LTβR-deficient animals of both sexes, 8–12 weeks old, were used in the study. The mice were housed and bred under specific pathogen-free conditions in the animal facility of the University of Lübeck, Germany. They were given standard laboratory chow and had free access to tap water. Permission to perform these animal experiments was issued by the Ministry of Nature and Environment (V 252-72241.122-1 (24-3/02)).

Tissue preparation for light and electron microscopy

To obtain a general overview, the pieces of thymic tissue were prepared for light microscopy. The pieces were fixed in neutral buffered formaldehyde or Bouin's solution, routinely processed for paraffin wax sectioning (3–5 µm) and stained with hematoxylin-eosin. The tissue quickly frozen in liquid nitrogen was used for cryostat sectioning (12 µm) and immunohistochemical demonstration of MHC class II. The biotinylated hamster anti-mouse monoclonal antibody (clone 25-9-17, BD Biosciences, Heidelberg, Germany) was used. In brief, cryostat sections were air-dried at room temperature for 2 h and fixed in a 1 : 1 (v/v) mixture of methanol-acetone for 10 min at –20 °C. Sections were incubated with primary antibody for 1 h and reaction was revealed by incubation with extravidine-peroxidase (Dako, Glostrup, Denmark) for 30 min. The tissue prepared for electron microscopy (see below) was used to produce semithin sections (1 µm), which were routinely stained with toluidine blue.

For electron microscopy the pieces of thymic tissue were quickly immersed in glutaraldehyde and cut into 1-mm³ cubes. Thereafter the tissue was fixed for 2 h in a solution of 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH = 7.4) at 4°C. 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 °C. 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). Ultrathin sections were routinely contrasted with uranyl acetate and lead citrate. The material was examined with a Zeiss EM-109 electron microscope (Carl Zeiss, Oberkochen, Germany).

Results

Light microscopy

We found that the thymus of normal C57BL/6 mice shows a well-developed cortex and medulla. The boundary between them was clear. The cortex was densely populated with small thymocytes. The regions subjacent to subcapsular and paraseptal connective tissue were found to be occupied by one to two rows of large, blastoid thymocytes. The cortical epithelial cells were delicate, with slender cell processes, which often ran in parallel, perpendicularly oriented to the medulla, and forming fine, small meshes in the thymic cortex (Fig. 1A). The medulla was less densely populated with thymocytes and formed a compact, clearly delineated area, within the thymic tissue (Fig. 1A). The thymus of LTβR-deficient mice appears largely unchanged, as already noted in earlier studies of these animals (Ettinger et al. 1998; Fütterer et al. 1998). More careful inspection, however, revealed the structural alterations in both the thymic cortex and medulla. The cortical subcapsular/paraseptal regions, populated with large lymphoblastoid thymocytes, were expanded, comprising more cell layers than in the normal thymus, at the expense of the deep cortex, which was reduced in size. The cortical epithelial network was coarser than in the normal thymus (Fig. 1B). The cortical epithelial cells were enlarged, with bulky and thickened prolongations, which were irregularly oriented, and formed the distended meshes harbouring more thymocytes than in the normal mice (Fig. 1B). The intensity of MHC class II immunostaining was comparable to that observed in the normal thymus (compare Fig. 1A with 1B). The medulla was broken down into numerous smaller islands and reduced in size (Fig. 1B). In this region, the cystic structures were frequently observed: sometimes small and circular, often elongated and channel-shaped (Fig. 1C). The fibrosis of the thymic medulla was also prominent, whereby the thickened connective tissue fibers were often observed (Fig. 1C).

Fig. 1.

The organization of cortical and medullary epithelial compartments is altered in the LTβR-deficient thymus. In the normal thymus the cortical epithelial cells are delicate, with slender prolongations, often perpendicularly oriented to the medulla, forming fine, small meshes in the thymic cortex. The medulla forms a compact and clearly delineated area (A; ×100). In the LTβR-deficient thymus the cortical epithelial cells are enlarged, with bulky, thickened, irregularly oriented prolongations, which form the distended meshes in the thymic cortex. The medulla is broken down in smaller islands and reduced in size (B; ×100). In the medulla of the LTβR-deficient thymus the cystic structures and thickened connective tissue fibers are frequently observed (C; ×100). C, cortex; M, medulla. Immunohistochemical demonstration of MHC class II (A,B); hematoxylin-eosin staining (C).

Electron microscopy

Normal thymus

Four epithelial cell types were readily discernible in the cortex. ‘Subcapsular epithelial cells’ (type 1) were positioned against the capsule, septa and perivascular connective tissue, and always had a basal lamina. These cells were found to be irregular in shape and showed cellular prolongations that interconnected them with the adjacent epithelial cells by means of desmosomes. The nucleus was found to be mostly euchromatic and the cytoplasm had a very active appearance: the Golgi complex was well developed; smooth and coated vesicles, multivesicular bodies and elongated profiles of rough endoplasmic reticulum were abundant, whereas only a moderate number of mitochondria and polyribosomes were seen. Fine bundles of keratin tonofilaments were positioned around the nucleus and throughout the cytoplasm (Fig. 2A). ‘Pale epithelial cells’ (type 2) were usually positioned in the outer regions of the thymic cortex and showed the low electron density of the nucleus and cytoplasm. They were found to be stellate in shape, with several, delicate cytoplasmic prolongations. The nucleus was very large, oval, and markedly euchromatic, with patent nucleolus. The cytoplasm was not abundant, but reflected a high cell activity: multiple Golgi complexes, cisternae of rough endoplasmic reticulum, numerous smooth and coated vesicles were seen. The bundles of cytokeratin tonofilaments in the perinuclear cytoplasm and cytoplasmic prolongations were usually delicate and sparse (Fig. 2B). ‘Intermediate epithelial cells’ (type 3) were located in deeper regions of the thymic cortex and showed higher electron density of the nucleus and cytoplasm in comparison with the ‘pale epithelial cells’. The nucleus of these cells was often polygonal in shape and showed a characteristic pattern of chromatin organization: smaller or larger clumps of heterochromatin were evenly dispersed throughout the nucleus. The nucleolus was prominent and in an eccentric position. These cells were rich in cytoplasm and had massive extensions containing abundant organelles, especially secretory vacuoles. The bundles of tonofilaments were localized within the cytoplasmic prolongations (Fig. 2C). ‘Dark epithelial cells’ (type 4) were positioned in the deep cortical regions and at the cortico-medullary boundary. Characteristically, these cells show a very high electron density of the nucleus and cytoplasm. The large clumps of heterochromatin were scattered all over the nucleus. The nucleolus was very prominent. The cytoplasm was sparse, but with plenty of organelles: Golgi complexes, large secretory vacuoles, multilamellar bodies and lipid droplets were prominent. The cytoplasm was found to give off very long and delicate cytoplasmic extensions, which in distant dilatations contain numerous organelles, the same as those in the perinuclear cytoplasm. Often the secretory vacuoles and lipid droplets discharge their contents into the intercellular space. The keratin bundles were ample and massive (Fig. 2D).

Fig. 2.

Subcellular organization of cortical epithelial cell types in the normal thymus. ‘Subcapsular epithelial cells’ (type 1) are positioned against the connective tissue, and always have a basal lamina (arrowheads). These cells are irregular in shape and have delicate cellular prolongations (arrows). The nucleus (N) is euchromatic and the cytoplasm has a very active appearance with fine bundles of keratin tonofilaments (A; ×7000). ‘Pale epithelial cells’ (type 2) show the low electron density of the nucleus and cytoplasm. They are stellate in shape with delicate cytoplasmic prolongations. The nucleus (N) is very large, oval, markedly euchromatic, with patent nucleolus. The cytoplasm is not abundant, but reflects a high cell activity. The cytokeratin tonofilaments (arrow) are usually delicate and sparse (B; ×7000). ‘Intermediate epithelial cells’ (type 3) show higher electron density of the nucleus and cytoplasm. The nucleus is polygonal in shape and shows a characteristic pattern of chromatin organization with prominent nucleolus (Nu). Cytoplasm is abundant with bundles of tonofilaments (arrows) and numerous secretory vacuoles (v), and shows massive extensions (C; ×12 000). ‘Dark epithelial cells’ (type 4) show a very high electron density of the nucleus and cytoplasm. The large clumps of heterochromatin are scattered all over the nucleus (N). The cytoplasm is sparse, but with plenty of organelles: Golgi complexes (arrows), large secretory vacuoles, multilamellar bodies (arrowhead) and lipid droplets. The cytoplasmic prolongations are extensive and packed with organelles (D; ×7000). Contrasted with uranyl acetate and lead citrate.

Three types of epithelial cells may be distinguished in the thymic medulla. ‘Undifferentiated epithelial cells’ (type 5) are most often located at the cortico-medullary boundary. These cells were found to be rounded in shape, with short, delicate cytoplasmic extensions, and in general had an immature, blastoid appearance: the nucleus being extremely euchromatic and the nucleolus very large. In the cytoplasm the polyribosomes prevailed, whereas other organelles were sparse and small (Fig. 3A). The ‘large medullary epithelial cells’ (type 6) had very abundant cytoplasm and several inconspicuous cytoplasmic prolongations. Their cytoplasm displayed the prominent signs of intense metabolic and secretory activity: in addition to numerous transport vesicles, dilated profiles of rough endoplasmic reticulum and large Golgi fields, clusters of secretory vacuoles were prominent. The vacuoles were usually smaller and acquired a grape-like form (Fig. 3B); but sometimes they were larger, whereby the vacuoles became compressed (Fig. 3C) or presented a solitary cyst-like structure (Fig. 3D). ‘Spindle-shaped epithelial cells’ (type 7) were small, often arranged in groups and connected to each other by large desmosomes. The cytoplasm was sparse, with scanty organelles and well-developed bundles of cytokeratin (Fig. 3E). Hassall bodies were small, and were usually composed of just two to three epithelial cells in various stages of activity. Sometimes the cells had a quiescent appearance with prominent cytoplasmic keratin bundles. Sometimes the signs of secretory activity prevailed: the dilatations of rough endoplasmic reticulum and abundance of polyribosomes stood out (not shown).

Fig. 3.

Subcellular organization of medullary epithelial cell types in the normal thymus. ‘Undifferentiated epithelial cells’ (type 5) are rounded in shape, with very delicate cytoplasmic extensions (arrow). The nucleus (N) is extremely euchromatic, the nucleolus is large and in the cytoplasm the polyribosomes prevail, whereas other organelles are sparse and small (A; ×4000). ‘Large medullary epithelial cells’ (type 6) have very abundant cytoplasm and inconspicuous prolongations. The cytoplasm displays signs of intense metabolic and secretory activity: numerous transport vesicles, dilated profiles of rough endoplasmic reticulum, large Golgi fields (arrowheads) and clusters of secretory vacuoles (v), which are usually small and have a grape-like form (B; ×7000). Sometimes the vacuoles (v) are larger and compressed (C; ×12 000) or appear as a solitary cyst-like (Cy) structure (D; ×12 000). ‘Spindle-shaped epithelial cells’ (type 7) are small, the cytoplasm is sparse, with massive bundles (arrows) of cytokeratin (E; ×20 000). N, nucleus; Contrasted with uranyl acetate and lead citrate.

LTβR-deficient thymus

In the LTβR-deficient thymus it was found that all cTEC show signs of hypertrophy and activation. Ultrastructural hallmarks of activation encompass all components of the cell. Briefly, the cell in general becomes enlarged, the nucleus becomes euchromatic with patent or multiple nucleoli, and the organelles more abundant with indications of increased biosynthetic activity (extensive, dilated profiles of rough endoplasmic reticulum, multiple enlarged Golgi complexes, prominently accumulated or markedly discharged secretory products) and more developed cellular prolongations (Rhodin, 1974; Ghadially, 1988). According to this, ‘subcapsular’ and ‘pale cells’ (types 1 and 2) show signs of activation. They are markedly increased in size and morphologically become similar, except for the characteristic localization of the former. Therefore they will be described jointly. We observed that the nucleus is large, oval and extremely euchromatic with one to two patent nucleoli. The amount of perinuclear cytoplasm is increased and the cellular prolongations are massively enlarged. The cytoplasm is markedly electron-lucent, and the cytokeratin bundles are very delicate, but prominent, and more abundant than in normal cells. Numerous polyribosomes, scanty profiles of granular endoplasmic reticulum, as well as multivesicular bodies and coated vesicles were seen. However, the Golgi complexes were very well developed, and numerous large secretory vacuoles partly filled with flocculent material could be observed (Fig. 4A,B). ‘Subcapsular cells’ sometimes formed the overlapping layers (Fig. 4A). These cells were found to be surrounded by an increased number of blastoid thymocytes, setting them back from the basal lamina. The bulky cytoplasmic prolongations of ‘pale epithelial cells’ encompass and clearly segregate the large groups of thymocytes, which is suggestive of emperipoiesis. The ‘intermediate epithelial cells’ (type 3) showed a reduced amount of cytoplasm, and the secretory vacuoles were less numerous than in the normal cells. However, numerous dilated profiles of granular endoplasmic reticulum were seen. The nucleus retained the characteristic polygonal shape, with very prominent nucleolus and dilated perinuclear cistern. Some cells were observed to be binuclear (Fig. 4C). The nucleus of the ‘dark epithelial cells’ (type 4) showed a giant nucleolus. The number of vacuoles with multilamellar bodies and lipid droplets was markedly increased. The Golgi zones were enlarged and very prominent. The cytoplasmic prolongations were extremely enlarged and encompassed small groups of thymocytes (Fig. 4D). Occasionally, even individual thymocytes were almost completely enveloped by prolongations of these cells. Distant portions of their processes were broadened and packed with organelles in the same manner as the perinuclear cytoplasm (Fig. 4D).

Fig. 4.

Activation of cortical epithelial cells in the LTβR-deficient thymus. ‘Subcapsular epithelial cells’ (type 1) form the overlapping layers against the capsular connective tissue (Ca) and basal lamina (arrowheads). The nucleus is large, outstandingly euchromatic with markedly developed nucleolus (Nu). Cytoplasm and cellular prolongations are massively enlarged with increased amount of organelles, secretory vacuoles (v) and keratin filaments (arrow) (A; ×7000). ‘Pale epithelial cells’ (type 2) have the bulky cytoplasmic prolongations and increased amount of cytoplasm with abundance of organelles, secretory vacuoles (v), numerous well-developed Golgi complexes (arrowheads) and delicate keratin filaments. Nucleus (N) is euchromatic (B; ×12 000). ‘Intermediate epithelial cells’ (type 3) have a reduced amount of cytoplasm and secretory vacuoles, but show dilated profiles of granular endoplasmic reticulum (arrow). Some cells are binuclear with a very prominent nucleolus (Nu) (C; ×7000). The nucleus of ‘dark epithelial cells’ (type 4) shows a giant nucleolus (Nu). The number of multilamellar bodies (arrowheads) is markedly increased and Golgi zones are very prominent (arrows). Cytoplasmic prolongations are enlarged and packed with organelles (D; ×7000). Contrasted with uranyl acetate and lead citrate.

The population of mTEC was completely different. Very significantly, the fully differentiated ‘large medullary cells’ (type 6) were completely absent and could not be found, although the residual medullary regions were largely composed of clustered epithelial cells with few or no thymocytes at all (Fig. 5A). These groups were mostly composed of special epithelial cells that could not be seen in the thymic medulla of normal mice. Some of them were large and rounded with very abundant cytoplasm, and with a large, rounded and very euchromatic nucleus with a prominent nucleolus. The cytoplasm showed signs of high activity: polyribosomes and mitochondria were abundant, and Golgi complexes numerous and well developed. Smooth and coated vesicles, as well as endocytic vacuoles, were abundant, whereas cytokeratin bundles were sparse and delicate (Fig. 5B). Some mTEC were elongated or polygonal in shape, similar to ‘spindle-shaped cells’ (type 7) of the normal thymus, but often these cells also showed the ultrastructural features described above (Fig. 5C). The prominent features of these cells were exceedingly well developed interdigitations with similar neighbouring cells (Fig. 5B,C). Small amounts of homogeneous, electron-dense material in the form of intercellular cysts could be seen between such cells (Fig. 5A,C). Similar mTEC formed the walls of the large cystic structures that were frequently observed in the medulla of LTβR-deficient thymus. These cells had a somewhat less active appearance: the nucleus was more heterochromatic and polygonal in shape, but the nucleolus was often very prominent. The cytoplasm was found to be more electron-dense and to show numerous mitochondria, but the other organelles were less developed. The hallmarks of these cells were exceedingly well developed lateral interdigitations and prominent junctional complexes with neighbouring cells (Fig. 6A). Lymphocytes transmigrating between the epithelial cells were often encountered. Delicate microvilli decorated the cell surface facing the lumen of the cyst. Fibroblasts with collagen fibrils were sometimes seen underneath a well-developed basal lamina (Fig. 6A). Various cells were found to be present within the lumen of larger cysts.

Fig. 5.

Inhibited differentiation of medullary epithelial cells in the LTβR-deficient thymus. The residual medullary island is largely composed of clustered epithelial cells (Ep). Some of them are large and rounded and some are more fusiform in shape. Prominent interdigitations (arrows) and cysts (arrowhead) are seen between the cells (A; ×4000). The nucleus (N) of oval cells is very euchromatic and the abundant cytoplasm shows signs of high activity with well-developed Golgi complexes (small arrowheads) and delicate cytokeratin bundles (large arrowhead). The interdigitations (arrow) with neighbouring cells are very prominent and exceedingly developed (B; ×7000). Polygonal and fusiform epithelial cells (Ep) show prominent interdigitations (arrowheads) and enclose an intercellular cyst (Cy) filled with electron-dense material (C; ×7000). Contrasted with uranyl acetate and lead citrate.

Fig. 6.

Aberrant differentiation of medullary epithelial cells in the LTβR-deficient thymus. An epithelial cell (Ep) within the wall of a large intercellular cyst has a less active appearance. These cells have exceedingly developed lateral interdigitations (arrows) with neighbouring cells. A lymphocyte (Ly) transmigrates between the epithelial cells. Underneath the epithelial cell the basal lamina (black arrowheads) and a fibroblast (Fi) with collagen fibrils (white arrowheads) are seen (A; ×7000). A rounded immature epithelial cell with inconspicuous prolongations, similar to normal ‘undifferentiated epithelial cell’ (type 5) is preserved. The nucleus (N) is also rounded and very euchromatic. Nucleolus is large, and organelles are scanty with a small Golgi zone (white arrowhead) and very fine keratin bundles (black arrowhead) (B; ×7000). Contrasted with uranyl acetate and lead citrate.

Only one mTEC type that has a counterpart in the normal thymus was found to remain preserved. Single cells having an immature appearance, similar to ‘undifferentiated epithelial cells’, could be seen: very large and rounded, with extremely inconspicuous prolongations. The nucleus was also rounded and very euchromatic, with a large nucleolus, and the organelles were scanty. Due to their blastoid appearance, these cells may be difficult to recognize, but delicate and very fine keratin bundles denote their epithelial nature (Fig. 6B).

The typical Hassall bodies could not be seen. Sometimes, mTEC were concentrically arranged, but without signs of secretory activity.

The morphological changes that occur in the thymus of lymphotoxin-beta receptor-deficient mice are summarized in Table 1.

Table 1.

Summary of the morphological changes in cortical and medullary epithelial cells that occur in lymphotoxin-beta receptor-deficient thymus compared with normal mice

Cell type	Features	Interpretation
Cortex
Type 1 (subcapsular)	Enlarged, increased biosynthetic activity	Increased production of chemotactic factors
Type 2 (pale)	Enlarged, increased biosynthetic activity and emperipoiesis, enhanced and disoriented cell prolongations	Disturbed positive selection and flow of thymocytes towards medulla
Type 3 (intermediate)	Increased biosynthetic activity	Increased production and/or release of hormones
Type 4 (dark)	Increased biosynthetic activity, enhanced cell prolongations	Increased glycoprotein production, disturbed flow of thymocytes towards medulla
Medulla
Type 5 (undifferentiated)	Preserved ultrastructure	Correspond to normal
Type 6 (large medullary)	Loss	Inhibited differentiation, disturbed negative selection
Type 7 (spindle-shaped)	Altered, form small intercellular cysts	Inhibited differentiation
Cyst lining type	Increased number of large intercellular cysts	Inhibited differentiation
Hassall body type	Loss	Inhibited differentiation

Discussion

Our study is the first to show at the subcellular level that the LTβR is engaged in the cross-talk between thymic stromal and thymocyte populations. We were able to show that the functional LTβR signalling pathway is necessary for the appropriate maintenance of all TEC types: in LTβR-deficient thymus the ultrastructural signs of cTEC hypertrophy and activation are seen, as well as the loss and inhibited differentiation of mTEC, along with an impaired development of thymic medulla.

Earlier studies of the thymus in mutant mice deficient in LTβR, or its downstream signalling molecules, focused on the structural and functional alterations of mTEC (Burkly et al. 1995; Naspetti et al. 1997; Boehm et al. 2003; Chin et al. 2003; Kajiura et al, 2004; Kinoshita et al. 2006; Zhang et al. 2006). Here we demonstrate that the cortical epithelium is also markedly altered in the LTβR-deficient thymus, which shows that the defects are more extensive than initially thought and do not encompass only the medullary epithelium (Boehm et al. 2003; Chin et al. 2003). We believe that similar alterations of cTEC may be expected in mutant mice lacking various signalling molecules downstream of LTβR. The generation and maintenance of cTEC also depends on interaction with developing thymocytes (Goverman et al. 1997; Klug et al. 1998; Anderson & Jenkinson, 2001). Thus the observed alterations probably reflect the disruption of cellular cross-talk between the cTEC and thymocytes at the corresponding developmental stage, i.e. CD4⁻CD8⁻ double-negative and CD4⁺CD8⁺ double-positive cells that normally reside in the vicinity of type 1 and 2 cTEC (Milićević & Milićević, 2004). Indeed, in chemokine receptor CCR7-deficient mice CD4⁻CD8⁻ double-negative thymocytes fail to localize in the subcapsular region of the thymic cortex and accumulate at the border of the cortex and medulla (Misslitz et al. 2004). In this manner, the contact with corresponding type 1 cTEC is precluded, which could induce the alterations of these cells. Double-positive CD4⁺CD8⁺ thymocytes regulate the differentiation of early thymocyte progenitors and γδ T cells via LTβR (Silva-Santos et al. 2005) and in a similar manner could also influence the physiology of cTEC (this study). There is some controversy about the functional role of type 4 ‘dark’ cTEC, i.e. whether they represent the active or rather the dying cells. Notably, in many other organs there are ‘light’ and ‘dark’ varieties of cells, as for example in gustatory corpuscles, where the latter represent the fully active cells (Rhodin, 1974). Interestingly, similar ultrastructural changes indicative of cell activation are seen in ‘dark’ cTEC when thymocyte maturation is blocked by the application of cyclosporin A to the animals (Milićević & Milićević, 1997, 2004), which also confirms that ‘dark’ cTEC in LTβR-deficient thymus represent a highly functional, rather than a dying cell type.

Our data demonstrate the loss and inhibited differentiation of all types of mTEC in LTβR-deficient mice. Very notably, all variants of highly differentiated type 6 ‘large medullary epithelial cells’ (Milićević & Milićević, 1997; this work) corresponding to Ulex europaeusagglutinin-1 (UEA-1)-positive mTEC, which contain the intracellular vacuoles (Farr & Anderson, 1985), disappear from the LTβR-deficient thymus. This finding is in full agreement with the light microscopic finding of UEA-1-positive mTEC reduction in the LTβR-deficient thymus (Boehm et al. 2003). The functional significance of intracellular cysts in ‘large medullary epithelial cells’ is still unknown. Our results suggest that these structures may be related to the negative selection involving this type of mTEC. Moreover, the disappearance of UEA-1-positive mTEC was registered in animals deficient in various molecules positioned downstream in the LTβR signalling pathway: IκB kinase Kinoshita et al. 2006), NF-κB-inducing kinase (NIK; Kajiura et al. 2004), nuclear factor-κB2 (NF-κB2; Zhang et al. 2006) and RelB (Burkly et al. 1995; Naspetti et al. 1997). Interestingly, however, no ultrastructural studies of mTEC in these mutant mice are yet available. In addition, the typical Hassall corpuscles also disappear from the LTβR-deficient thymic medulla, which is a further sign of impaired mTEC maturation.

In LTβR-deficient thymus the elongated or polygonal mTEC are seen. They are reminiscent of mature ‘spindle-shaped epithelial cells’ (type 7) in the normal thymus, but their ultrastructural features are notably different. The signs of increased activity, prominent interdigitations and small intercellular cysts are present. Therefore these cells appear structurally similar to the mTEC that form the large intercellular cystic structures in LTβR-deficient medulla. Although the presence of such structures has been occasionally reported in normal thymus of various species (Oksanen, 1977; Chan, 1986; Khosla & Ovalle, 1986; Atoji et al. 1999), large intercellular cysts are much less frequent in the normal mouse thymus (Farr et al. 2002; Dooley et al. 2005; Gillard et al. 2007). Thus the prominent presence of cysts in LTβR-deficient thymus may also be regarded as an indication of arrested maturation and differentiation of mTEC. This view is confirmed by a very recent study of AutoImmune REgulator (AIRE)-deficient thymus (Gillard et al. 2007). AIRE transcription factor is positioned downstream of and controlled by LTβR (Chin et al. 2003). In addition to phenotypic aberrations, an increased prevalence of cystic epithelial structures was observed and this was interpreted as another indication that mTEC differentiation has been affected in these mutant mice (Gillard et al. 2007).

The only recognizable type of mTEC which remains preserved in LTβR-deficient thymus corresponds to ‘undifferentiated epithelial cells’ (type 5) in the normal thymus.

Our study does not provide an analysis of mTEC at various developmental stages. Thus our observations may reflect either an inhibition of differentiation or a failure of maintenance of mTEC types in LTβR-deficient thymus. Without further work, it is impossible to distinguish whether both or either of these possibilities is the direct result of the lack of LTβR signalling in mTEC. However, it is significant that at embryonic day (E) 11.5, prior to the formation of the three-dimensional epithelial network, the thymic primordium is composed of a two-dimensional cell bilayer. This forms the cysts with the innermost TEC layer showing prominent claudin-positive intercellular junctions (Hamazaki et al. 2007; Holländer, 2007). This situation is very similar to the cystic structures observed in the medulla of LTβR-deficient thymus. Moreover, these claudin-positive precursor-TECs give rise exclusively to various mTEC including an AIRE-positive subset (Hamazaki et al. 2007; Holländer, 2007). With these data in mind, we favour the latter possibility and propose that our ultrastructural observations reflect the inhibition of differentiation of mTEC in LTβR-deficient thymus, i.e. that signalling through LTβR is necessary for differentiation of mTEC from their embryonic form (as also suggested by Boehm et al. 2003).

We speculate that – just as in secondary lymphoid organ development (Mebius, 2003) – LTβR plays a crucial role in the development and organization of thymic medulla. The initial LTβ-mediated signalling through LTβR on mTEC is necessary for the induction of chemokines, which further attract the mature thymocytes (Kwan & Killeen, 2004; Ueno et al. 2004). In this manner a positive feedback loop is formed – as for example during the formation of lymphoid follicles and FDC maturation (Ansel et al. 2000) – which enables the development of the thymic medulla and maturation of mTEC. Indeed, the structure of the thymic medulla is prominently disturbed in chemokine CCL19- and CCL21-deficient plt/plt mice; the medullary areas in mutant thymus are smaller and more numerous than in normal mice (Misslitz et al. 2004; Ueno et al. 2004), which precisely corresponds to the morphological defects observed in LTβR-deficient animals (this work).

Our results are therefore pertinent to thymic physiology, especially to the negative selection. In recent years it has been shown that mature thymic mTEC promiscuously express a large variety of genes and synthesize highly tissue-specific self-antigens (Derbinski et al. 2001, 2005). This mechanism provides the basis for negative selection in the thymic medulla (Liston et al. 2003). The expression of self-genes by mTEC is controlled by AIRE transcription factor, and the mutations in AIRE gene are accompanied by defects in negative selection, with a consequent appearance of autoimmune diseases (Liston et al. 2003). Recently it was shown that AIRE expression and negative selection depend on the functional LTβR signalling pathway (Boehm et al. 2003; Chin et al. 2003). If the LTβR axis is disrupted, AIRE expression and negative selection become defective (Boehm et al. 2003; Chin et al. 2003; Villasenor et al. 2005). We confirm at the ultrastructural level that LTβR signalling is necessary for differentiation of mTEC. These data, together with the recent data of Farr's group (Gillard et al. 2007), suggest that LTβR signalling via AIRE induces the structural and functional maturation of mTEC, enabling them to effect the negative selection. Interestingly, thymic metallophilic macrophages, which also seem to be involved in this process, are lacking in the thymus of LTβR-deficient mice as well (Milićević et al. 2006).

In conclusion, LTβR signalling axis plays a critical role in homeostasis of all types of TEC. However, the deficiency of LTβR shows the opposing effects on thymic cortical and medullary epithelial populations resulting in activation/hypertrophy of the former and inhibited differentiation/loss of the latter.