Normal Structure,Function and Histology of the Thymus

Abstract

The thymus, a primary lymphoid organ and the initial site for development of T cell immunological function, is morphologically similar across species. It is actually an epithelial organ in which its epithelial cells provide a framework containing T cells as well as smaller numbers of other lymphoid cells. A symbiotic interaction exists between the thymic microinvironment and developing T cells, and the specificity of T cell release into the systemic circulation is under thymic control. The thymic cortex in a young animal is heavily populated by developing T cells along with a smaller proportion of associated epithelial cells. Larger, more mature T cells are found in the medulla where epithelial and other cell types are more abundant. Understanding normal morphological features of the thymus and their perturbations provides a cornerstone to assessing immune system function.

Embrology/Development

The rodent thymus develops from the endoderm of the 3rd and 4th pharangeal pouches and surrounding mesenchyme. (Dijkstra and Sminia, 1990) The pharangeal pouch connects with the pharynx via the thymopharangeal duct, remnants of which may be incorporated into the developing thymus giving rise to epithelial cystic structures (Figures 1 and 2). As development progresses, the thymus along with the thyroid and parathyroid, sharing the same pharangeal pouch origin, migrate caudally. They separate around day 15 when the thymus migrates into the thorax. Embryonic thymic remnants can give rise to ectopic thymic tissue in the neck (Figure 3), thyroid (Figure 4) and parathyroid glands (Suster and Rosai, 1992). Once migration is complete the epithelial cells organize into a loose meshwork separated by the developing vasculature. Following the rapid population with lymphocyte precursors from developing hematopoetic tissues (gestational day 11–12 in the mouse), the thymus becomes a lymphoepithelial organ. Because of their common pharyngeal pouch origin, ectopic thyroid (Figure 5) and parathyroid (Figure 6) can occasionally be found in the thymus.

The thymus is the first of the lymphoid organs to be formed and grows considerably immediately after birth in response to postnatal antigen stimulation and the demand for large numbers of mature T cells. Genetic factors also influence the age of onset, rate and magnitude of thymus dependant immunological function. In rats and mice, the thymus reaches maximal size by sexual maturity and then gradually involutes.

Fixation and Processing of the Thymus

Thymic weights are usually measured prior to fixation and following removal of adjacent fat and connective tissue. The thymus of aged, immunosuppressed, or immunodefficient animals may be difficult to locate. In this case, adipose and connective tissues from the anterior mediastinum containing thymic tissue should be collected and fixed without weighing (Kuper et al., 1995).

In routine studies, the thymus is normally fixed in 10% buffered formalin. The majority of thymic T cells can be shown to express CD3 on formalin fixed, paraffin-embedded tissue. Similarly, B cells can be demonstrated in formalin fixed tissue with antibodies to CD45R. However, if specific phenotyping of T cells is required, use of antigen retrieval methods and use of frozen sections may be required (Ward et al., 2006). Some authors prefer that the tissue should be snap-frozen, since phenotyping involves the use of antibodies against cell surface antigens such as CD4 and CD8, which are present only in small amounts and which are lost with conventional fixation (Schuurman et al., 1994). Ethanol fixation is preferred for the demonstration of keratin expression. Special fixation methods used for various procedures in immunotoxicity studies are discussed by Kuper et al. (1995).

Following fixation, the thymus is trimmed transversely through the middle of the organ, to include both lobes and then embedded. (Figure 7).

Normal Anatomy

The mammalian thymus is located in the pericardial mediastinum, anterior to the major vessels of the heart, and ventral to the base of the heart and aortic arch, with variable extension of one or both lobes into the cervical region in the rat (Haley, 2003). In the guinea pig, it is located in the neck region (Dijkstra and Sminia, 1990). The thymus consists of two distinct lobes connected by a connective tissue isthmus. A thin connective tissue capsule surrounds each lobe and, in most species, gives rise to septae, that partially subdivide the thymus into interconnecting lobules of variable size and orientation (Figure 8A, B). There is no sublobulation in the mouse (Figure 8C, D). Evidence for a functional cervical thymus in mice has been reported (Terszowski et al., 2006).

Blood, Lymphatic and Nerve Supply

The thymic arteries follow the course of the interlobular connective tissue septae and enter the organ substance at the corticomedullary junction. The corticomedullary arterioles ramify into capillaries that extend into the cortex and medulla. In the cortex they form a complex of capillary arcades which together with perivascular lymphocytes, macrophages and peripheral reticular epithelial cells form the blood-thymus barrier (Banks, 1993). Capillaries in the cortex are rarely fenestrated (Kuper et al., 2002). This restricts access of circulating antigenic molecules to developing cortical lymphocytes. By contrast, medullary capillaries are fenestrated and freely permeable to circulating antigens. Blood drains into the postcapillary venules, and finally returns to the corticomedullary junction in medullary veins (Banks, 1993). The structural organization of the blood and lymphatic vessels comprising the thymic microvasculature of the mouse is reviewed by Kato (1997).

Prothymocytes are thought to enter the thymic stroma through the large venules at the corticomedullary junction, and re-enter the circulation through the vascular lining of post-capilliary venules. These perivascular areas contain accumulations of phenotypically mature T cells that express the marker Mel 14, a receptor involved in the migration of lymphocytes through the lymphoid vasculature (van Ewijk et al., 1988). Efferent lymphatics drain into an adjacent pair of lymph nodes. The thymus has no afferent lymphatics. The rodent thymus has an abundant noradrenergic innervation but some cholinergic innervation has also been demonstrated (De Waal et al., 1997). Nerves follow the vasculature, within the capsule and septae and the highest concentration of nerve fibres is in the corticomedullary junction.

Histology

Of the various lymphoid tissues, the thymus is histologically most consistent across species (Haley, 2003). It is unique among the lymphoid organs in being an epithelial organ. The epithelial cells form an open framework containing predominantly T lymphocytes, smaller populations of B lymphocytes and plasma cells and scattered populations of other cells such as neuroendocrine cells. It is divided into a morphologically distinct cortex and medulla separated by a vascular corticomedullary zone.

Epithelial Stroma

The bulk of the supporting framework in the thymus is composed of the network of epithelial-reticular cells (Banks, 1993). Epithelial cells in the subcapsular region of the thymus form a layer 1 or 2 cells deep. In the outer cortex and ensheathing blood vessels, epithelial cells are thin and sheet-like, but elsewhere they assume a stellate appearance.

Epithelial cells are divided into distinct populations that differ in antigenic expression, ultrastructural characteristics (DeWaal et al., 1997), and their capacity to synthesize the thymic hormones thymulin, thymosin, thymopoetin and thymic humoral factor. Immunohistochemically, epithelial cells can be divided into four distinct subtypes: subcapsular cortical, inner cortical, medullary and Hassalls corpuscles. A decrease in the number of different antigenic epithelial populations occurs with age in mice. Main antigenic determinants of human thymic epithelium are listed in Table 1. More extensive and detailed lists of immunohistochemical markers are provided by Greaves (2000); Kuper et al. (1995); and Suster and Rosai (1992).

Capsule

A thin connective tissue capsule surrounds each lobe and, in most species, gives rise to septae, that partially subdivide the thymus into interconnecting lobules of variable size and orientation. There is no distinct sublobulation in the mouse. The capsule is composed of an outer and inner layer of collagen and reticular fibres between which are occasional clusters of lymphocytes. This inner layer invaginates to form the septae. Fine trabeculae also extend from the capsule or septae into the centre of the lobules. With reticulin staining a small amount of connective tissue can be demonstrated in the capsule, trabeculae and perivascular regions, but unlike other lymphoid organs, there is little supporting connective tissue. The bulk of the supporting framework in the thymus is composed of the network of epithelial reticular cells.

Epithelium-free areas (or “holes”) are compartments lacking stromal elements, which have been identified in the sub-capsular area, extending deep into the cortex (Bruijntjes et al., 1993; Elmore, 2006).

Cortex

Histologically, the darkly staining cortex contains densely packed, small, immature lymphocytes, which overshadow the sparse epithelial cell population (Figure 9), and a transient bone-marrow derived population of predominantly phagocytic macrophages. Large, mitotically active lymphoblasts, may be found in the subcapsular cortex. These cells have a round to oval nucleus with one or two prominent nuclei and relatively abundant, strongly basophilic cytoplasm. A gradient of small, less mitotically active cells occurs from the outer cortex to the corticomedullary junction. In the subcapsular region and deep cortex large numbers of rapidly dividing lymphocytes are short-lived and undergo apoptosis therefore, prominent cortical apoptotic bodies are a normal background finding. Apoptotic bodies are phagocytosed by macrophages and are visible within their cytoplasm resulting in the name “tingible-body macrophages” (Figure 10).

Corticomedullary Junction

The corticomedullary junction is characterized by plentiful blood vessels (predominantly arterioles) with scant perivascular connective tissue and mature and immature T lymphocytes. Dendritic cells and variable numbers of perivascular B-lymphocytes and plasma cells occur in the corticomedullary zone. B-cells and plasma cells increase with increasing age of the animal.

Medulla

The medulla, is continuous between adjacent lobules (Haley, 2003) and can form small buds that reach deep into the cortex that in some places reach the capsule (Figure 11). The medulla is paler staining, less densely cellular than the cortex, and contains more mature T-cells, prominent epithelial cells (Figure 12), Hassalls corpuscles, admixed macrophages, dendritic cells (non-phagocytic, bone marrow-derived cells), B lymphocytes and rarely myoid cells. The medullary T-lymphocytes are larger, paler-staining and have more cytoplasm than cortical lymphocytes.

Hassall’s corpuscles, are rare in rodent species when compared with humans and primates. In the mouse they can be very small and not readily visualized without immunostaining. Hassalls corpuscles react strongly with antibodies to high molecular weight keratin (AE2), a marker of terminal epithelial maturation, but are unreactive to other immunohistochemical markers for thymic epithelium. Component cells are polygonal with a large nucleus, and pale, occasionally granular cytoplasm with variable cystic degeneration and dystrophic calcification. In rats they can form whorls of flattened cells surrounding cell debris or concentrically arranged keratin (Figure 13). In mice these structures are less well defined and do not form keratin in their centers.

Myoid cells are rare in the thymic medulla, of rodents and humans (Suster and Rosai, 1992). They have ultrastructural and immunohistochemical features of striated muscle; staining positively for desmin and myosin. Their histogenisis is uncertain but some studies have demonstrated shared epitopes with thymic epithelial cells.

Neuroendocrine cells occur in low numbers. Their physiological function is not understood but they can give rise to carcinoid tumors. Mast cells and eosinophils are also variably present.

The Corticomedullary Ratio

The ratio between the cortex and medulla is typically determined subjectively from a low to medium magnification of the thymus and is frequently part of an enhanced histopathological evaluation of the thymus (Elmore, 2006). Since this ratio can vary depending upon the orientation of the thymus at trimming, it is recommended that a standardized trimming procedure be followed (see Figure 7). Tryphonas et al. (2004) provided morphometric measurements of histological sections of the thymus in control Sprague–Dawley rats. By their method, the average cortico-medullary ratio was determined to be 4.4 to 4.7 (30 and 90 day-old males) and 3.9 to 6.3 (30-and 60 day-old females).

Electron Microscopy

On electron microscopy, cortical epithelial cells have elongated cytoplasmic processes connected by desmosomes to adacent cells. Processes are covered in a basal lamina. Medullary epithelial cells are more voluminous and oval-shaped with short cytoplasmic extensions and many secretory organelles. DeWaal et al. (1997) discuss the ultrastructural details of the thymic component cells. Kato (1997) provides ultrastructural details of the mouse thymic microvascular system.

Normal Function

The thymus is a primary lymphoid organ, viz. bone marrow derived progenitor cells undergo differentiation/maturation, within the thymic microenvironment, to form the functional T cell repertoire. For an excellent overview of the functional aspects of the immune system one should consult: Abbas (2005), Brown et al. (2002), Tizard (2004), and Lebish et al. (1986).

Prothymocytes migrate from the bone marrow, enter the thymus via the vasculature at the corticomedullary junction, and undergo 4 stages of maturation, as they pass from the sub-capsular zone through the cortex to the medulla and finally enter the circulation as mature peripheral T cells. During intrathymic migration, developing T cells (thymocytes) proliferate and differentiate, resulting in changes in cell size and expression of differentiation antigens and interleukin receptors (van Ewijk et al., 1988). Phenotypic change is accompanied by T cell receptor (TCR) gene rearrangement: necessary for expression of surface T cell receptors and the generation of T cell competence. T cell receptor gene rearrangement is reviewed by Perryman (2004).

Transitional stages of thymocyte maturation can be characterized on the basis of the immunological phenotype of the cells as judged by the expression of the T cell receptor (TCR)-CD3 complex and its coreceptors CD4 and CD8 (differentiation antigens) (Kuper et al., 2002). Thymocytes differentiate from an immature double negative (CD4−/CD8−) phenotype, to an intermediate double positive phenotype (CD4+/CD8+), found on the majority of cortical thymocytes. CD4+/CD8+ thymocytes express a diverse repertoire of TCR specificities. Successful engagement of TCR with major histocompatibility complex (MHC) class I molecules, leads to differentiation into precursors of cytotoxic/suppressor T cells (CD4−/CD8+ phenotype). Engagement of TCR with major histocompatibility complex (MHC) class II molecules leads to differentiation into precursors of helper T cells (CD4+/CD8− phenotype). These single positive, mature phenotypes are located predominantly in the medulla. (De Waal et al., 1997).

The thymus also controls the specificity of T cells entering the circulation by means of positive and negative selection. Positive selection involves major histocompatibility complex (MHC) restriction, in which there is clonal expansion only of those T cells capable of recognizing antigen in the context of host MHC 1 and 2. The thymic nurse cells, express MHC class I and II and play a major role in positive selection. In negative selection, developing T cells, which bear receptors to self-antigens (autoreacive cells) undergo apoptosis, thereby causing clonal deletion of potentially harmful cells.

Thus, there are many more cortical lymphocytes than those entering the medulla, and eventually released by thymus into the general circulation. Apoptotic bodies and tingible body macrophages are therefore a normal histological feature of the cortex, particularly in young animals. The vast majority of apoptotic cells in the thymus is thought to be a reflection of failure to undergo positive selection. (De Waal et al., 1997)

Epithelium-Free Areas (or “Holes”)

These areas contain occasional phagocytic macrophages and abundant lymphocytes, (predominantly CD4+/CD8+) with a high rate of proliferation (Figure 14). They are thought to offer a separate intrathymic pathway for T-lymphocytes, whereby immature lymphocytes can move between the cortex, corticomedullary zone and medulla without coming into contact with the stromal elements concerned with selection (Bruijntjes et al., 1993). A summary of thymic function by region is presented in Table 2.

The cortical and medullary epithelial cells, macrophages and reticular/dendritic cells all play a role in development and differentiation of the thymocytes. The epithelium is involved in positive and negative selection. All epithelial cells express MHC class I products, but MHC class II antigens are differentially expressed in the cortical and medullary epithelium. In the subcapsular region, specialized epithelial cells known as thymic nurse cells engulf and form strong cell-cell membrane interactions with large numbers of developing lymphocytes (Brelinska and Warchol, 1997). These nurse cells are important in early T cell differentiation. Sub-capsular and medullary epithelial cells produce the thymic hormones. Thymosin is involved in late intrathymic T cell maturation. Thymopoietin, thymic humoral factor, and serum thymic factor are important in enhancing lymphocyte responsiveness (Banks, 1993). Dendritic cells are non-phagocytic, bone marrow-derived cells, which are responsible for antigen presentation to T cells (via class 1or 2 MHC) and are especially important in antigen presentation to naïve T cells. Dendritic cells are more efficient than epithelial cells in mediating negative selection.

Genetically Modified Animals

A symbiotic interaction exists between the thymic microenvironment and developing T cells. Various mouse models have been developed to demonstrate the role of thymic microenvironment in positive and negative selection of T cells and the potential influence of T cells on the development of thymic microenvironments (van Ewijk, 1991; De Waal et al., 1997).

SCID Mouse

Severe Combined Immunodefficiency Syndrome (SCID) is a genetic disorder characterized by failure of B and T lymphocyte differentiation. An important aspect of T lymphocyte differentiation requires rearrangement and expression of genes encoding T lymphocyte antigen-specific receptors (and B lymphocyte surface immunoglobulin receptors). Failure to properly complete these gene rearrangement events results in the elimination of lymphocyte precursors and near absence of mature, functional T and B lymphocytes. In mice the enzyme DNA-dependent protein kinase (DNA-PK) is required for gene rearrangement. Lack of this enzyme occurs as a spontaneous autosomal recessive mutation in C.B-17 BALB/c mice. Animals homozygous for the SCID mutation are severely deficient in functional B and T lymphocytes.

Since T lymphocyte development is necessary for the maintenance of thymic stromal integrity, and the development of a distinct cortex and medulla, the thymus is difficult to identify in these animals. Van Ewijk (1991) demonstrated that the thymic microenvironment was also abnormal in SCID mice. Stromal elements in these mice have a cortical phenotype and low numbers of medullary epithelial cells are scattered throughout the entire thymus. Histologically, the thymus consists of small dysplastic lobules, in which there is a marked paucity of lymphocytes (Figure 15). Hassall’s corpuscles may be evident. In some mice, known as “leaky SCIDS,” T cell differentiation may be partially restored over time and in these animals there is patchy re-organisation of the medullary epithelium.

In children, SCID is caused by mutation of the Recombinase-activating genes 1 and 2 (RAG1, and RAG2), which may impare DNA synthesis in the lymphocytes. Mutant mice developed to express RAG1 and RAG2 mutant genes develop non-T, non-B SCID similar to that observed in children with RAG-1 or RAG-2 deficiencies.

Athymic/Nude Mouse

In the homozygous nude (nu) mouse, there is failure of haircoat development and dysgenisis of the thymus, which does not develop beyond a rudimentary stage of epithelial delineated canaliculi, lacking in lymphoid cells (Hansen, 1978).