Thymic stromal cells

Basic thymic structure

Thymus stromal cell heterogeneity is no doubt a direct reflection of the input of all three germ layers. The thymus is bounded by a connective tissue capsule. Between this and the underlying epithelium is a subcapsular space which contains some vasculature and is the site of entry of blood-borne precursors and macromolecules. It is thus very likely that the thymus is bathed in plasma constituents, which has very important implications for self tolerance. Fibroblast-and type 1 collagen-rich trabeculae branch off from the capsule and penetrate the cortex, terminating at the corticomedullary junction, thereby lobulating the thymus and providing a structural link between the subcapsule and the medulla. These trabeculae are well vascularized and innervated and contain predominantly fibroblasts and macrophages; there are also minor populations of adipocytes, eosinophils, neutrophils, plasma cells, mast cells and granulocytes. Towards the internal base of the trabeculae are the perivascular spaces which effectively constitute the blood-thymus barrier. These connective tissue-rich regions are actually external to the thymic parenchyma, being separated from it by type 1 epithelium (see below) and from the vascular endothelial cells by basal laminae. They contain multiple leukocytes, including neutrophils and mature T and B cells and even germinal centers. Although there is no formal barrier dividing the cortex from the medulla, both of these areas are readily definable by their desmosomal-linked epithelium which have been extensively classified ultrastructurally and phenotypically.

Epithelium

Subcapsular/subtrabecular/perivascular (type 1 epithelium) Immediately underlying the capsule and lining the internal face of the trabeculae and perivascular spaces is a continuous layer of flattened epithelium which rests on a basal lamina, effectively separating cells of the connective tissue compartment from the lymphoepithelial compartment. This is termed type 1 or lining epithelium. It completely encases the cortex at the interface with the trabeculae and the capsule, but it is discontinuous at the base of the trabeculae. This latter feature may have important implications for cellular migration from the vasculature into the thymus. Phenotypic similarity with neuroendocrine tissue suggests this epithelium has its neurogins from the neural crest. Many antigens expressed in the subcapsular epithelium are shared with the medullary epithelium, indicating a potential lineage relationship, but they also express region-specific molecules. This is best exemplified in the chicken, where the subcapsular type 1 epithelium is exclusively defined phenotypically. Furthermore there is ultrastructural and phenotypic heterogeneity within the type 1 epithelium. Being such a discrete region, its functional significance is difficult to investigate but its potential importance stems from the finding that there are

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Cortical Ep (heterogeneous)

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Figure 1 The thymic stromal architecture. (Reproduced by permission from Immunology Today 14: centrefold. The author is very grateful to D Godfrey for the provision of the figure.)

Capsula

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Cortical Ep (heterogeneous)

Capillary Macrophage

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Mcdulary Ep (heterogeneous)

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Figure 1 The thymic stromal architecture. (Reproduced by permission from Immunology Today 14: centrefold. The author is very grateful to D Godfrey for the provision of the figure.)

abnormalities in type 1 epithelium prior to the onset of autoimmune disease in genetically prone chickens. It is unlikely to involve abnormal selection to self peptides, however, because the type 1 epithelium is very poor, if not negative, in MHC class II expression. Given that this is the site of intrathymic precursor entry and subsequent localization and many of these are dividing, the working hypothesis for the role of the type 1 epithelium is that it attracts blood-borne precursor cells and induces their activation/proliferation, which may be subsequently coupled to TcR gene rearrangement and commitment to the T cell lineage.

Cortical epithelium Over 85% of thymocytes are localized within the cortex. The cortical epithelium consists of an interconnected network of stellate-shaped cells characterized by relatively small cell bodies and long cytoplasmic processes which surround the tightly packed thymocytes. There is a remarkable degree of elasticity in the cortical epithelium, as it is able to rapidly expand and contract in response to shifts in the thymocyte pool, essen tially keeping the desmosomal contacts intact. Although most monoclonal antibodies (mAbs) which react with cortical epithelium label the entire network, there is evidence at the ultrastructural level of heterogeneity. Types 2 and 3 epithelial cells are located in the outer cortex, have large nuclei, are metabolically active and often form thymic nurse cells (TNCs). Type 4 epithelial cells are "dark", electron dense with more oval/spindle-shaped nuclei and located in the deeper cortex/medullary regions.

Of the many structural features of the thymus, TNCs are arguably the most interesting. They are multicellular complexes consisting of a cortical epithelial cell (type 2 or 3) which has physically encompassed surrounding thymocytes varying in number from <5 to over 100, depending on the species. These T cells are not within the cytoplasm but rather encompassed within vacuoles formed by the extensive plasma membrane processes of the epithelial cell. Whether TNCs are fully enclosed structures in situ is debatable but the intimate association between the enclosed thymocytes and the epithelial plasma membrane is indicative of a major role in thymopoiesis. The epithelial cell expresses high levels of MHC class I and II. In fact these structures may be microcosms of T cell development because they contain CD3-CD4-CD8" precursors, CD3~ or CD31o CD4+CD8+ immature T cells and phenotypically and functionally mature CD3hi CD4+CD8" and CD3hi CD4~CD8+ cells. The level of mature T cells in TNCs is increased in TcR transgenic mice. Hence TNCs may be the primary site of positive selection, given the current dogma that this is the domain of cortical epithelium. Consistent with this is the presence in TNCs of acidic organelles necessary for antigen processing and presentation. There is also evidence of apoptosis in TNCs, which may reflect negative selection or death of T cells with a nonproductive TcR or one which fails to recognize self MHC-peptide complexes. Although there are relatively few TNCs per thymus, they may represent the most extreme form of dynamic opening and closing pockets of thymopoiesis in the cortex.

Medullary epithelium Unlike the stellate network of the cortex interconnected by long cytoplasmic processes, the medullary epithelium is more compact. The medulla is clearly distinguished from the cortex phenotypically, which may reflect different germ-layer origins, although the subcapsule and medulla share antigens. This relationship between the subcapsule (type 1 epithelium) and medulla is still unresolved, but since the former also lines the trabeculae, which extend into the corticomedullary junction, there is a continuous physical link between the outer and inner thymus. Hence precursor cells entering the thymus at either the subcapsule or corticomedullary junction will encounter the same epithelial cells. The profound phenotypic differences between the cortex and medulla no doubt reflect functional differences but these are still speculative. Medullary epithelium is not necessary for positive selection but it may contribute; it does have a role in tolerance at the level of inducing anergy rather than deletion. Given that T cell migration is thought to occur from the medulla, the epithelium in this region might provide the necessary differentiation signals. In addition to the interconnecting epithelium network in the medulla, there are also small clusters of swirling keratinized epithelial cells termed Hassall's corpuscles, particularly in the human, which typify the thymus. They are likely to be end-stage epithelium and may also be a site of removal of dying medullary thymocytes.

Origins of epithelium Although still unresolved, it is likely that both ectoderm and endoderm contribute to the thymic epithelium and mesoderm to the leukocyte component. Whether the cortex and medulla derive from separate precursors or are different maturation stages of a common lineage is also unresolved. Until recently, the dogma was that there is a putative epithelial stem cell expressing determinants of the cortex or medulla, which develops into either of these regions, depending on the inductive signals it receives. It now appears likely that both regions develop autonomously and their maturation and expansion, at least in part, are dependent on the T cells themselves - immature thymocytes regulating cortical epithelium and mature T cells of either a(3-or y8-TcR type inducing medullary epithelium expansion. This highlights an important symbiotic developmental relationship between the thymocytes and the stromal cells.

Nonepithelium

Despite its primary role as the central organ for inducing T lymphocyte development, but consistent with the need to present immature T cells with a broad spectrum of self antigens, there is a surprising heterogeneity in the stromal cell compartment.

While the epithelial cells constitute the specific microenvironment, they do not act alone but rather in concert with a heterogeneous group of nonepi-thelial stromal cells. Of these, the most important are the dendritic cells, which are the subject of separate entries in this encyclopedia. There are two types of dendritic cells in the thymus - those of so-called lymphoid origin and those of myeloid origin. Their relative intrathymic localization is not clearly known but, as a population, dendritic cells are spread throughout the thymus, with preferential localization at the corticomedullary junction where they are thought to be the major mediators of negative selection through their specialized ability to trap, process and present antigen. In addition, MHC-class II negative macrophages are also present throughout the thymus, including within the trabeculae; their major role is in the removal of the vast number of thymocytes which die in situ. One of the more unusual stromal cells in the thymus are myoid cells which are present in the medulla. These contain contractile proteins reminiscent of striated muscle. Their role is unknown but they may influence T cell migration, and the presence of acetylcholine receptors on them is no doubt important for establishing tolerance to neuroendocrine molecules. In thymic hyperplasia, these acetylcholine receptors may be involved in the pathogenesis of myasthenia gravis. Fibroblasts and reticular fibroblasts are a major part of the trabeculae but they also surround the vasculature and are present scattered throughout the cortex in particular.

While they clearly have a structural role in the thymus, undergoing activation following thymic damage, the fibroblasts also provide the differentiation-inducing ligand for CD44, which is expressed on intrathymic precursors.

Given the obvious importance of vascular endothelium cell adhesion molecules and/or their ligands in mediating leukocyte extravasation at sites of inflammation, and lymphocyte trafficking through high endothelial venules in lymph nodes, it is highly likely that they also play an important role in thymocyte migration. It is unclear precisely how and where precursors enter the thymus and mature cells emigrate, although it is likely to be complex because E-cadherin - a member of the cadherin superfamily of adhesion molecules - apparently mediates migration of prethymic but not intrathymic precursors.

How does the thymic stroma influence thymopoiesis?

Despite the obvious functional importance of the thymic stroma, precisely how it mediates T cell development is unclear. MHC-peptide complexes are essential for selection and while most cells can mediate negative selection, the current dogma is that cortical epithelium would appear to be unique in its ability to induce positive selection. There appear to be differences in MHC class II expression, peptide processing and antigen presentation properties between cortical and medullary epithelial cells, which may facilitate their preferential roles in positive selection and self tolerance, respectively. It is feasible, however, that medullary epithelium is potentially capable of inducing positive selection but, because of the structural restraints of the thymic architecture, do not contact the appropriate target thymocytes. The identity of the plasma membrane molecule(s) responsible for positive selection is one of the major unsolved questions of thymus function. While much of thymic function is organ autonomous, it is very much under the influence of neuroendocrine molecules.

Thymic epithelial cells produce many cytokines, particularly interleukin 7 (IL-7), which promotes precursor survival and TcR gene rearrangement, and transforming growth factor (B (TGFfJ), a product of subcapsular and cortical epithelial cells, which also regulates the differentiation of early thymocyte subsets. While cytokines obviously have important roles, no combination to date, alone or in conjunction with nonthymic stromal cells, has been able to recapitulate complete T cell differentiation from precursor through to mature T lymphocyte. Extracellular matrix proteins are important in cellular differentiation and function in many systems including the thymus. A wide variety of extracellular matrix constituents have been described in the thymus, such as laminin, fibronectin, merosin and type I and IV col-lagens, each localizing to discrete regions. These may influence thymopoiesis either directly through provision of ligands for specific T cell surface receptors or indirectly by trapping cytokines by acting as a reservoir. They may also facilitate thymocyte migration through the stromal milieu. Multiple cell adhesion receptor-counter-receptor pairs, such as LFA-1/ ICAM-1, -2, VLA-4/V CAM-1 and aE(37/E-cad-herin, have been proposed to be involved in thymic stromal cell-T cell interactions, and, while none appear to be imperative for thymopoiesis, their combined effects and coordinated expression patterns could be major constituents of the specific thymic microenvironment.

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