ARTICLE |
Correspondence to: Willem van Ewijk, Dept. of Molecular Cell Biology, Center for Electron Microscopy, Leiden University Medical Center, Wassenaarse weg 72, 2333 AL Leiden, The Netherlands. E-mail: vanewijk@lumc.nl
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Summary |
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Development of a mature T-cell repertoire in the thymus depends on lymphostromal interaction between thymocytes and stromal cells. To facilitate intercellular contact, the epithelium in the thymus has differentiated into a unique three-dimensionally (3D)-oriented network. Here we analyze factors influencing induction and maintenance of the 3D configuration of the epithelial network in fetal thymic lobes in vitro. We show that the 3D configuration of the thymic stroma depends on (a) the oxygen pressure in vitro and (b) permanent physical contact between stromal cells and developing thymocytes. This latter feature is demonstrated by incubation of fetal thymic lobes with deoxyguanosine (d-Guo), inducing a 2D-organized thymic stroma, with thymic cysts appearing. Reconstitution of d-Guo-treated lobes with a limited number of flow-sorted T-cell progenitors restores the 3D configuration of the thymic epithelium, but only at high oxygen pressure. This study underlines the plasticity of thymic epithelium and shows that the unique organization of the thymic epithelium is dependent on both oxygen and crosstalk signals derived from developing thymocytes. (J Histochem Cytochem 51:12251235, 2003)
Key Words: T-cell development, lymphostromal interaction, crosstalk, fetal thymic organ culture, thymic epithelium
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Introduction |
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THE THYMUS is concerned with the development of a broad repertoire of T-lymphocytes, reactive with foreign antigens but tolerant to self-determinants. During generation of this physiological T-cell repertoire, developing T-cells interact with various types of stromal cells located in different thymic microenvironments (
Epithelial cells form the basis of these microenvironments. Importantly, the organization and positioning of epithelial cells is unique for the thymus. Whereas in other organs epithelial cells are usually placed on a basement membrane to form two-dimensionally (2D)-organized sheets of cells which line off functionally different domains, epithelial cells in the thymus are organized in a sponge-like three-dimensionally (3D)-oriented fashion. This typical configuration facilitates migration and lymphostromal interaction during thymopoiesis (
The uniqueness of the thymic stroma is already established early in ontogeny in the region of the third pharyngeal pouch. In this region, expression of Hoxa3 and Foxn1 regulates early steps in the development of the thymic epithelium (
Signals derived from invading T-cell progenitors have been shown to contribute to the organization, positioning, and maintenance of the differentiated network configuration of the thymic epithelium, a phenomenon earlier designated as "thymic crosstalk" (
In the present study we analyze requirements for proper development of thymic microenvironments in vitro. First, we show that maintenance of the typical thymic architecture requires a high oxygen pressure in the culture system. Lowering the pO2 in the culture system results in reorganization of stromal cells and loss of one dimension in the tertiary organization of the thymic epithelium. Second, we show that maintenance of the tertiary organization of thymic microenvironments requires permanent physical contact between developing thymocytes and thymic epithelial cells. To demonstrate this latter feature, we have purified thymic epithelium using de-oxyguanosin (d-Guo). d-Guo treatment of fetal thymic lobes blocks lymphoid development and results in thymic lobes exclusively composed of stromal elements (
This study underlines the impressive plasticity of the thymic stroma and shows that the typical architecture of the thymic microenvironments depends on oxygen, together with continuous crosstalk signaling by developing thymocytes. Physiologically, both factors guarantee induction and maintenance of proper T-lymphocyte development.
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Materials and Methods |
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Mice
Time-mated pregnant C57Bl/6 (B6/Ly5.2) mice were obtained from SLC (Shizuoka, Japan), and B6Ly5.1 were maintained in the animal facility of the Kyoto University according to the institutional guidelines.
Organs and Cells
Fetal thymic lobes (TLs) of d14, d15 embryos (B6) as well as fetal livers (FLs) of d14 embryos (B6Ly5.1) were isolated using a stereomicroscope. FL and FT cell suspensions were prepared as described previously (
High Oxygen Submersion (HOS) and Low Oxygen Submersion (LOS) Organ Culture: Deoxyguanosine Treatment
FTs were obtained from d14 embryos, placed directly in wells of a 96-well V-bottom plate, and cultured for 5 or 9 days. To obtain hematopoietic cell-depleted FT lobes, thymuses were cultured on polycarbonate filters (pore size 8 µm) floating on culture medium containing 1.35 mM d-Guo for 6 days in humidified air containing 5% CO2. The lobes were washed and placed individually in wells of a 96-well U-bottom plate. To these lobes, 100 D14FL Lin- Sca-1lo progenitors were added using a fine capillary tube under direct microscopic visualization, followed by low-speed centrifugation of the plate for 5 min (experimental details in Table 1). All organ cultures that were to grow under HOS conditions were placed and sealed in plastic bags (Ohmi Oder Air Service; Hikone, Japan). The air in the bags was replaced with a gas mixture of 70% O2, 25% N2, and 5% CO2. These plates, as well as plates with FTs under LOS conditions (normal air containing 5% CO2), were placed in a 37C incubator. The cultures were maintained in RPMI 1640 complete medium (Life Technologies; Tokyo, Japan). Supplements were 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mg/ml sodium bicarbonate, 0.1 mM non-essential amino acid solution (Life Technologies), 5 x 105 mM 2-mercaptoethanol, 100 ng/ml streptomycin, and 100 U/ml penicillin. Medium was changed on day 5 for 9-day cultures and a new gas mixture was infused into each bag. For 5-day cultures the medium was not changed. After culture, all lobes were harvested and either processed for FACS analysis or frozen in OCT compound for immunohistochemistry.
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Monoclonal Antibodies and Flow Cytometric Analysis
After culture, cells were recovered and pooled in groups of three lobes, and were counted using trypan blue dye exclusion. Cells were incubated with antibodies and analyzed by flowcytometry (FACS Vantage; Becton Dickinson, Mountain View, CA) as previously described (ß (H57597; Caltag), FITCanti-TCR
(GL3; Caltag), FITCanti-CD4 (GK1.5; Caltag), FITCanti-CD3 (500A2; Caltag), PEanti-CD44 (IM7; PharMingen), anti-Ly5.1 (a201.7; kindly donated by Dr. Y. Saga, Banyu Seiyaku, Tokyo, Japan), and anti-CD25 (PC61.5.3; American Type Culture Collection, Rockville, MD). Anti-Ly5.1 and anti-CD25 were labeled with Cyanine5 (Cy5) using a labeling kit (Biological Detection Systems; Pittsburgh, PA). Anti-FcR
II/III (FcR; 2.4G2, PharMingen) was used to block nonspecific binding of monoclonal antibodies to the FcR.
Monoclonal Antibodies, Single-chain Antibodies, and Immunohistochemistry
From all different experimental groups, 6-µm frozen sections were prepared using a Leitz cryostat and collected on 0.1% gelatin-coated microscope slides. After a brief acetone fixation, sections were incubated with the following monoclonal antibodies: T-cells, anti-Thy1 (clone 59AD2.2), anti-CD3 (clone KT3), anti-CD4 (clone H129.19), anti-CD8 (clone 536.7); macrophages, F4/80, ER-HR3, ER-TR9 and MOMA1; cortical epithelium, ER-TR4; medullary epithelium, ER-TR5; fibroblasts, ER-TR7, anti-MHC class I (clone M1/42), anti-MHC class II (clone M5/114); dendritic cells, N418. After washing in PBS/Tween, the slides were incubated with rabbit anti-rat IgG conjugated to horseradish peroxidase (Dako; Glostrup, Denmark) as secondary reagent or horseradish peroxidase conjugated to rat anti-hamster IgG (Dako), to detect N418.
For staining with monoclonal single-chain antibodies (scFv), sections were fixed with 1% paraformaldehyde for 5 min followed by a brief wash in PBS/Tween. Slides were incubated with the cortical epithelial marker TB4-4 (
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Results |
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This study is based on the use of a fetal thymic organ culture system in which thymic lobes are completely submerged in tissue culture medium (
In the present study we cultured lobes at two different pO2 levels: (a) oxygen concentration at 70%, resulting in a local high oxygen pressure of 400 mmHg in the thymic lobes (high oxygen submersion culture; HOS), and (b) oxygen concentration at 20% resulting in a low pO2 of 175 mmHg (low oxygen submersion culture; LOS); (cf.
To study the influence of thymocytes on the development of the thymic epithelium, isolated T-cell progenitor cells were co-cultured with d-Guo-treated fetal TLs under either HOS or LOS conditions.
T-cell Development Occurs Normally at High pO2 But Is Arrested at the Triple Negative CD44+CD25- Stage at Low pO2
In day 14 fetal TLs, cultured under HOS conditions (group 1 in Table 1; and Fig 1, upper row) T-cell development proceeded normally. After 5 days of culture, the majority of the cells in the TLs were Thy1+ cells (Fig 1A), with both CD4+CD8+ (DP, double positive) cells developing, as well as SP (single positive) CD4+ and SP CD8+ thymocytes (Fig 1B). Both ß and
TCR-expressing T-cells were generated (Fig 1C; cf.
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In contrast, when TLs were placed under LOS conditions (group 2 in Table 1; and Fig 1, lower row), the cell recovery was much lower compared to the HOS cultures (see also legend to Fig 1). Although limited numbers of T-cells expressing CD4+CD8+ or CD4-CD8+ were still present (Fig 1E and Fig 1F), as well as some T-cells expressing ß TCR and
TCR (Fig 1G), most of the remaining thymocytes were arrested at the TN (CD3-CD4-CD8-) CD44+CD25- stage (Fig 1H). Incubation with propidium iodide indicated that the large majority of cells in TLs cultured under LOS conditions remained alive after 5 days of culture (data not shown).
3D-oriented Thymic Epithelium Develops in Thymic Lobes Only at High pO2
To study the architecture of thymic microenvironments under HOS and LOS conditions, d14 fetal TLs were placed in an organ culture under HOS (group 1 in Table 1) or LOS (group 2 in Table 1) conditions. After 5 days of culture, the lobes were frozen and prepared for immunohistochemistry. Microscopic analysis revealed two different areas in each TL cultured under HOS conditions (Fig 2). TB4-4, an scFv reactive with cortical epithelium, identified a peripheral area consisting of 3D-organized cortical epithelial cells and a central area with closely packed, undifferentiated epithelial cells (i.e., rounded cells with an euchromatic nucleus that lack long cytoplasm processes and show low cytokeratin expression; see Discussion) (Fig 2A, asterisk). An organized medulla was lacking but a few medullary epithelial cells, defined by the monoclonal antibody ER-TR5, were found scattered throughout the TL (Fig 2B). ER-TR7 identified the thymic capsule, septae, and individual fibroblasts in the center of the lobes (Fig 2D). Therefore, a well-localized medulla defined by groups of ER-TR5+ epithelial cells remains absent.
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In each lobe, T-cells were abundantly present (Fig 2C). Importantly, the large majority of T-cells co-localized with the fine reticular network of epithelial cells in the periphery of the TLs (compare Fig 2A and Fig 2C), indicating that a 3D organization of thymic epithelium is required for T-cell development.
When d14 fetal TLs were cultured under LOS conditions (group 2 in Table 1), cellularity remained low. In addition, cortical epithelial cells did not create their typical network architecture. Instead, closely packed cuboidal cortical epithelial cells were observed (Fig 2E). A few small, rounded medullary epithelial cells occurred spread throughout the lobes (Fig 2F). Fibroblasts, dendritic cells, and macrophages were present in TLs grown either under HOS or LOS conditions; they did not differ in frequency or in morphology (data not shown).
The 3D Architecture of Thymic Epithelium Is Lost After d-Guo Treatment
The finding that HOS conditions were required for the organization of a normal reticular network of epithelial cells raises the question of whether oxygen has a direct influence on the differentiation of epithelial cells alone. To investigate this possibility, endogenous thymocytes were removed by culturing the thymic lobes in the presence of d-Guo.
To determine the direct effect of d-Guo on the thymic stroma, d15 lobes were incubated with d-Guo for 6 days (group 3 in Table 1) and directly frozen in OCT compound. Frozen sections stained with scFv TB4-4 showed that cortical epithelial cells, instead of creating a network, now formed strands of cuboidal cells (Fig 3A). Similarly, medullary epithelial cells had retracted their cytoplasmic extensions (Fig 3B). Furthermore, thymic cysts developed under the influence of d-Guo, as shown in Fig 3A and Fig 3B. Staining with monoclonal antibody specific for the TCR-CD3 complex indicated that d-Guo treatment efficiently removed endogenous thymocytes (data not shown). Thus, d-Guo treatment of TLs not only causes depletion of endogenous thymocytes but also results in repositioning of both cortical and medullary epithelial cells.
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High pO2 Per Se Has No Influence on the 3D Architecture of Cortical Epithelial Cells
To study whether the pO2 directly influenced the differentiation of the thymic stroma we cultured d-Guo treated lobes for a period of 9 days at high pO2 or at low pO2 (HOS, c.q. LOS conditions; groups 4 and 5 in Table 1). As shown in Fig 3C, under high pO2 cysts remained present, the characteristic 3D organized epithelial network structure remained absent, and only closely packed, small cortical epithelial cells were observed, which at the periphery of the thymic lobe were oriented parallel to the surrounding basement membrane. These findings suggest that oxygen per se does not promote the differentiation and positioning of the epithelial network in the thymus.
Progenitor T-cells Induce Differentiation of the 3D Architecture of Thymic Epithelial Cells Only at High pO2
To study the potential of developing thymocytes to induce differentiation of the thymic epithelium, we co-cultured d-Guo-treated fetal TLs with T-cell progenitors under either high or low pO2 conditions.
Endogenous T cells were first removed by culturing d14 fetal TLs in the presence of d-Guo for 6 days. Subsequently, these d-Guo-treated lobes were in vitro reconstituted with 100 sorted Lin- c-kit+ Sca-1lo d14 fetal liver cells, which contain T lineage-committed progenitors (
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To study the development of the d-Guo-treated thymic stroma after reconstitution with T-cell progenitors, we stained cryostat sections of TLs with antibodies defining subsets of thymic epithelial cells and compared the organization of the thymic stroma with normal untreated fetal TLs (group 1 in Table 1). Comparison of Fig 5A and Fig 2A shows that the architecture of the thymic epithelium in both groups is quite similar. At the periphery of the reconstituted d-Guo-treated lobes, a well-differentiated MHC-expressing reticular network of cortical epithelial cells had developed (Fig 5A and Fig 5D), with thymocytes positioned in between processes of the epithelial cells (Fig 5C). In the middle of the lobes epithelial cells were present, but these cells are mainly undifferentiated cells, while thymocytes remained absent in this region. Compared to normal HOS cultures, ER-TR5+ medullary epithelial cells were present at increased frequency (Fig 5B), but these cells were not positioned in discrete medullae. They remained intermingled with TB4-4+ cortical epithelial cells (compare with Fig 5A). T-cell differentiation appeared normal, as demonstrated in Fig 4 and Fig 5C.
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By contrast, in d-Guo-treated fetal liver cell reconstituted lobes placed under LOS conditions, T-cells did not progress beyond the CD44+CD25- phenotype (Fig 4; and group 7 in Table 1). Moreover, in these lobes cortical epithelial cells were unable to form a 3D-oriented network and remained grouped together in strands of cuboidal epithelial cells (Fig 5E). Similarly, medullary epithelial cells did not expand and were unable to form medullae. Thymic cysts remained present in the lobes, as shown in Fig 5E. Thus, under low pO2, the 3D network of the thymic stroma was not induced. Together, these experiments clearly demonstrate an impressive plasticity of the thymic stroma and indicate that T-cell progenitors and a high pO2 are simultaneously required for the development and 3D orientation of thymic microenvironments.
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Discussion |
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Early steps in the development of the mouse thymus can be easily accessed by in vitro culture of d14-derived thymic lobes. Within a period of 6 days all thymocyte subsets differentiate normally, as under normal in vivo conditions (
In the cultured fetal thymus, thymocytes develop at the peripheral side of the lobes, in areas where epithelial cells are found in the thymus-characteristic network configuration. They do not develop in the central part of the lobe where clustered epithelial cells are located.
Such clustered epithelial cells have an undifferentiated morphological phenotype. Electron microscopic studies (van Ewijk et al. unpublished) have revealed absence of the characteristic long cytoplasmic processes. Moreover, bundles of tonofilaments are absent in these cells, corresponding to extremely low levels of cytokeratin expression, while incubation of sections with the antibody MTS 24 (
Organ culture of fetal thymic lobes requires oxygen. In the classical filter-based culture system, only a thin film of culture medium separates the developing thymocytes from the air in the incubator. In the submersion culture system, as we show here, thymopoiesis clearly depends on the pO2 in the culture system. At high pO2 the tertiary structure of thymic microenvironments is maintained. Lowering the pO2 induces a dramatic redistribution of epithelial cells, leading to the formation of thymic cysts lined with cells normally not occurring in the thymus.
This 3D to 2D conversion induced by hypoxia does not lead to cell death in the cultured thymic epithelium and is, in our opinion, not an in vitro artifact. First, thymic cysts also occur in vivo, even in normal mice ( T-cells. Finally, as shown by our present reconstitution experiments, the 2D nature can be remodeled back to 3D.
There is ample information in the literature on the requirement for oxygen during the development of the immune system. Interestingly, a recent in vivo study from
Redistribution of the thymic epithelium under hypoxic conditions appears to be an indirect phenomenon, primarily caused by lack of crosstalk signals derived from developing thymocytes. We do not feel that the reported impressive changes in the architecture of the thymic reticulum are caused by the mere physical absence of developing thymocytes, because other experimental models in which thymocytes are removed from the thymic microenvironment, such as hydrocortisone treatment (ß TCR-expressing thymocytes at the TN I (CD3-CD4-CD8-CD44+ CD25-) stage, while
T-cells develop normally. Therefore, crosstalk signals from early
ß TCR-expressing thymocytes appear to be of fundamental importance in development and maintenance of the thymic epithelial architecture. These in vitro findings are supported by our previously published in vivo work in which we showed that a block in early T-cell development, as occurs in CD3
transgenic mice, results in a similar 3D to 2D conversion of the epithelial phenotype (
transgenic mice with progenitor T-cells derived from RAGnull mice restores the 3D phenotype (
The question arises whether thymocytes at early stages in development induce this particular 3D configuration or whether these cells merely play a role in the maintenance of this architecture after other inductive events. In support of the latter notion, a recent study by
The molecular nature of thymic crosstalk signaling is now slowly emerging. Several different factors have been shown to influence the typical architecture of the thymic stroma. (a) Tumor necrosis factor (TNF) appears to be involved, since TCR ligation in developing T-cells induces upregulation of TNF in thymic stromal cells (
gene (c.f.
In summary, our observations indicate that in the absence of crosstalk signaling, thymic epithelial cells retract their typical cytoplasmic extensions and convert to a "simple" epithelium. This type of epithelium is found in the primitive thymic anlage and also in other endoderm-derived organs such as the respiratory tract, the gastrointestinal tract, thyroid, and parathyroid. In the presence of developing thymocytes, thymic epithelial cells differentiate into proper organized microenvironments. Maintenance of these microenvironments requires a high pO2 within the thymic lobes. The role of epithelial stem cells (
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Acknowledgments |
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Supported by the Netherlands Organization for Scientific Research (NWO), grant 90105273, and by Grants-in Aid for Scientific Research, Priority Area Research 12051219, and Special Coordination Funds from the Ministry of Education, Science, Sports and Culture, Japan.
We wish to thank Mr Tar van Os for photographic assistance.
Received for publication December 30, 2002; accepted April 2, 2003.
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