Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.     

The expression of keratin k10 in the basal layer of the epidermis inhibits cell proliferation and prevents skin tumorigenesis

Forced expression of K10, a keratin normally expressed in postmitotic, terminally differentiating epidermal keratinocytes, inhibits the progression of the cell cycle in cultured cells (Paramio, J. M., Casanova, M. Ll., Segrelles, C., Mittnacht, S., Lane, E. B., and Jorcano, J. L. (1999) Mol. Cell. Biol. 19, 3086-3094). This process requires a functional retinoblastoma (pRb) gene product and is mediated by K10-induced inhibition of Akt and PKCzeta, two signaling intermediates belonging to the phosphoinositide (PI) 3-kinase signal transduction pathway (Paramio, J. M., Segrelles, C., Ruiz, S., and Jorcano, J. L. (2001) Mol. Cell. Biol. 21, 7449-7459). Extending earlier in vitro studies to the in vivo situation, this work analyzes the alterations found in transgenic mice that ectopically express K10 in the proliferative basal cells of the epidermis. Increased expression of K10 led to a hypoplastic and hyperkeratotic epidermis due to a dramatic decrease in skin keratinocyte proliferation in association with the inhibition of Akt and PKCzeta activities. The inhibition of cell proliferation and Akt and PKCzeta activities was also observed although to a minor extent in low hK10-expressing mice. These animals displayed no overt epidermal phenotype nor overexpression of K10. In these non-phenotypic mice, ectopic K10 expression also resulted in decreased skin tumorigenesis. Collectively, these data demonstrate that keratin K10 in vivo functions include the control of epithelial proliferation in skin epidermis.     

BMP signaling is required for normal thymus development

The microenvironment of the thymus fosters the generation of a diverse and self-tolerant T cell repertoire from a pool of essentially random specificities. Epithelial as well as mesenchymal cells contribute to the thymic stroma, but little is known about the factors that allow for communication between the two cells types that shape the thymic microenvironment. In this study, we investigated the role of bone morphogenetic protein (BMP) signaling in thymus development. Transgenic expression of the BMP antagonist Noggin in thymic epithelial cells under the control of a Foxn1 promoter in the mouse leads to dysplastic thymic lobes of drastically reduced size that are ectopically located in the neck at the level of the hyoid bone. Interestingly, the small number of thymocytes in these thymic lobes develops with normal kinetics and shows a wild-type phenotype. Organ initiation of the embryonic thymic anlage in these Noggin transgenic mice occurs as in wild-type mice, but the tight temporal and spatial regulation of BMP4 expression is abrogated in subsequent differentiation stages. We show that transgenic Noggin blocks BMP signaling in epithelial as well as mesenchymal cells of the thymic anlage. Our data demonstrate that BMP signaling is crucial for thymus development and that it is the thymic stroma rather than developing thymocytes that depends on BMP signals.     

Severe abnormalities in the oral mucosa induced by suprabasal expression of epidermal keratin K10 in transgenic mice

Previous studies have demonstrated that keratin K10 plays an important role in mediating cell signaling processes, since the ectopic expression of this keratin induces cell cycle arrest in proliferating cells in vitro and in vivo. However, apart from its well known function of providing epithelial cells with resilience to mechanical trauma, little is known about its possible roles in nondividing cells. To investigate what these might be, transgenic mice were generated in which the expression of K10 was driven by bovine K6beta gene control elements (bK6(beta)hK10). The transgenic mice displayed severe abnormalities in the tongue and palate but not in other K6-expressing cells such as those of the esophagus, nails, and hair follicles. The lesions in the tongue and palate included the cytolysis of epithelial suprabasal cells associated with an acute inflammatory response and lymphocyte infiltration. The alterations in the oral mucosa caused the death of transgenic pups soon after birth, probably because suckling was impaired. These anomalies, together with others found in the teeth, are reminiscent of the lesions observed in some patients with pachyonychia congenita, an inherited epithelial fragility associated with mutations in keratins K6 and K16. Although no epithelial fragility was observed in the bK6(beta)hK10 oral epithelia of the experimental mice, necrotic processes were seen. Collectively, these data show that the carefully regulated tissue- and differentiation-specific patterns displayed by the keratin genes have dramatic consequences on the biological behavior of epithelial cells and that changes in the specific composition of the keratin intermediate filament cytoskeleton can affect their physiology, in particular those of the oral mucosa.     

Thymic epithelial requirement for γδ T cell development revealed in the cell ablation transgenic system with TSCOT promoter

In order to investigate the role of thymic epithelial cell (TEC) subsets during T-cell development, we established a new transgenic system, enabling inducible cell-specific ablation as well as marking the TEC subsets using bicistronic bacterial nitroreductase and EGFP genes. Two different lengths of the TSCOT promoter in transgenic mice, named 3.1T-NE and 9.1T-NE, drive EGFP expression into TECs. In adult life, EGFP expression was located in the medulla with a smaller 3.1 kb TSCOT promoter, while it was maintained in the cortex with a 9.1 kb promoter, suggesting putative TEC specific as well as compartment specific cis elements within two promoters. Nitroreductase induced cell death was specific without bystander killing upon the treatment of prodrugs such as nitrofurantoin and metronidazol. The degree of cell death was dependent on the dose of the prodrug in the cell and the fetal thymic organ cultures (FTOCs). Fetal thymic stromal populations were analyzed based on the expression levels of EpCAM, MHCII, CDR1 and/or UEA-1. EGFP expression patterns varied among subsets indicating the differential TSCOT promoter activity in each TEC subset. Prodrug treatment in FTOCs reduced the numbers of total and subsets of thymocytes. A CD4⁺CD8⁺ double positive cell population was highly susceptible in both transgenic lines. Surprisingly, there was a distinct reduction in γδ T cell population only in the 9.1T-NE thymus, indicating that they require a NTREGFP expressing TEC population. Therefore, these results support a division of labor within TEC subsets for the αβ and γδ lineage specification.     

Impaired NF-kappa B activation and increased production of tumor necrosis factor alpha in transgenic mice expressing keratin K10 in the basal layer of the epidermis

Both the diversity and the precisely regulated tissue- and differentiation-specific expression patterns of keratins suggest that these proteins have specific functions in epithelia besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through the inhibition of Akt kinase activity. Given the roles of Akt in NF-kappa B signaling and the importance of these processes in the epidermis, a study was made into the possible alterations of the NF-kappa B pathway in transgenic mice expressing K10 in the proliferative basal layer. It was found that the inhibition of Akt, mediated by K10 expression, leads to impaired NF-kappa B activity. This appears to occur through the decreased expression of IKK beta and IKK gamma. Remarkably, increased production of tumor necrosis factor alpha and concomitant JNK activation was observed in the epidermis of these transgenic mice. These results confirm that keratin K10 functions in vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner but also influence tissue homeostasis.     

A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter

Using a previously described human keratin 14 (K14) promoter, we created mice expressing a peptide Ag (OVAp) in epithelial cells of the skin, tongue, esophagus, and thymus. Double transgenic mice that also express a TCR specific for this Ag (OT-I) showed evidence for Ag-driven receptor editing in the thymus. Surprisingly, such mice exhibited a severe autoimmune disease. In this work we describe the features of this disease and demonstrate that it is dependent on CD8 T cells. Consistent with the Ag expression pattern dictated by the human K14 promoter, an inflammatory infiltrate was observed in skin and esophagus and around bile ducts of the liver. We also observed a high level of TNF-alpha in the serum. Given that Ag expression in the thymus induced development of T cells with dual TCR reactivity, and that dual-reactive cells have been suggested to have autoimmune potential, we tested whether they were a causal factor in the disease observed here. We found that OT-I/K14-OVAp animals on a recombinase-activating gene-deficient background still suffered from disease. In addition, OT-I animals expressing OVA broadly in all tissues under a different promoter did not experience disease, despite having a similar number of dual-specific T cells. Thus, in this model it would appear that dual-reactive T cells do not underlie autoimmune pathology. Finally, we extended these observations to a second transgenic system involving 2C TCR-transgenic animals expressing the SIY peptide Ag with the hK14 promoter. We discuss the potential relationship between autoimmunity and self-Ags that are expressed in stratified epithelium.     

Ontogeny and regulation of IL-7-expressing thymic epithelial cells

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1(-/-) mice are developmentally and functionally distinct.     

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.     

Identification of MiR-205 As a MicroRNA That Is Highly Expressed in Medullary Thymic Epithelial Cells

Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.     

Increased expression of keratin 16 causes anomalies in cytoarchitecture and keratinization in transgenic mouse skin

Injury to epidermis and other stratified epithelia triggers profound but transient changes in the pattern of keratin expression. In postmitotic cells located at the wound edge, a strong induction of K6, K16, and K17 synthesis occurs at the expense of the keratins produced under the normal situation. The functional significance of these alterations in keratin expression is not known. Here, we report that overexpression of a wild-type human K16 gene in a tissue-specific fashion in transgenic mice causes aberrant keratinization of the hair follicle outer root sheath and proximal epidermis, and it leads to hyperproliferation and increased thickness of the living layers (acanthosis), as well as cornified layers (hyperkeratosis). The pathogenesis of lesions in transgenic mouse skin begins with a reorganization of keratin filaments in postmitotic keratinocytes, and it progresses in a transgene level-dependent fashion to include disruption of keratinocyte cytoarchitecture and structural alterations in desmosomes at the cell surface. No evidence of cell lysis could be found at the ultrastructural level. These results demonstrate that the disruption of the normal keratin profile caused by increased K16 expression interferes with the program of terminal differentiation in outer root sheath and epidermis. They further suggest that when present at sufficiently high intracellular levels, K16, along with K6 and K17, appear capable of inducing a reorganization of keratin filaments in the cytoplasm of skin epithelial cells.     

Laminin-211 controls thymocyte--thymic epithelial cell interactions

Thymocyte differentiation occurs within the thymic microenvironment, consisting of distinct cell types and extracellular matrix (ECM) elements. One of these ECM proteins is laminin. Previous experiments showed that laminin mediates interactions between thymocytes and thymic epithelial cells (TEC) in mice. Since, laminin comprises a family of related isoforms, we searched for laminin isoform expression in the human thymus. We found constitutive gene expression of various laminin chains in TEC preparations, comprising laminin-111 and laminin-211 isoforms. Immunocytochemistry revealed a selective laminin-211 distribution in the thymic lobules. In vitro functional assays revealed that laminin-211 enhances TEC/thymocyte adhesion and thymocyte release from thymic nurse cells, as well as the reconstitution of these complexes. Conversely, these interactions are blocked by monoclonal antibodies specific for laminin-211 and the laminin receptor VLA-6. Our results reinforce the notion that distinct laminin isoforms in the human thymus are relevant for lymphoepithelial interactions.     

Differentiation patterns of CD4/CD8 thymocyte subsets in cocultures of fetal thymus and lymphohemopoietic cells from c-fos transgenic and normal mice.

This study examined the involvement of c-fos protooncogene in thymocyte development from lymphohemopoietic T cell progenitors, within the thymic microenvironment. We first analyzed the thymocytes developing in vitro in the fetal thymus from the c-fos transgenic mice and found a high proportion of CD4+ single positive (SP) cells. We then seeded either fetal liver or bone marrow (BM) cells from normal donors onto lymphocyte-depleted fetal thymus explants of c-fos transgenic mice. The results showed an increased proportion of mature CD4+ SP and decreased CD4+CD8+ double positive (DP) cells. A similar pattern of CD4/CD8 thymocyte subsets was observed when either thymus or BM cells from c-fos transgenic mice developed within a normal thymic stroma. The kinetics of thymocyte development in organ culture (from Days 3 to 11) suggested that the SP cells obtained under these conditions may have bypassed the CD4+CD8+ DP phase. It appears that the altered pattern of thymocyte development manifested in adult c-fos transgenic mice can be induced by the early embryonic thymic stroma, and may also involve cells in the lymphohemopoietic tissues.     

MHC non-restricted, CD95-independent apoptosis of immature thymocytes induced by thymic epithelial cells

The interaction of thymocytes with thymic epithelial cells in the absence of an exogenous antigen was studied in vitro. Thymic, but not splenic epithelial cells induced apoptosis of thymocytes. A thymic epithelial cell line (TEC) induced apoptosis of thymocytes but not of splenic T-cells. The target population for TEC-induced death were immature CD4(+)8(+) (double positive), but not mature single positive thymocytes. TEC also induced DNA fragmentation in day 18 foetal thymocytes, most of which are CD4(+)8(+) cells. Radiation leukemia virus (RadLV)-transformed thymic lymphoma clones expressing various phenotypes reflected this sensitivity, in that a CD4(+)8(+)3(+) clone apoptosed by thymic epithelial cells or TEC. Other, single positive or double negative clones were resistant. Thymocytes from C3H (H-2(k)), C57BL/6 (H-2(b)) and Balb/C (H-2(d)) mice apoptosed equally in response to either C57BL/6 thymic epithelial cells or TEC (H-2(b) x H-2(d)). Likewise, thymocytes from MRLIpr((-/-)) and B6Ipr((-/-)) mice, which do not express CD95 were also apoptosed by TEC.The data suggest that thymic epithelial cells induce MHC non-restricted, Fas-independent apoptosis of immature thymocytes. This response may reflect a mechanism through which thymocytes expressing TcR with no affinity to self MHC/peptide complexes are eliminated.     

B7-1 overexpression by thymic epithelial cells results in transient and long-lasting effects on thymocytes and peripheral T helper cells but does not result in immunodeficiency.

The B7-1 (CD80) molecule provides costimulatory function for the activation of T helper lymphocytes upon encounter with antigen. To investigate the role of this molecule in thymocyte maturation, we have generated transgenic (Tg) mice in which CD80 expression is driven by the keratin 14 promoter (K14). This overexpression of CD80 resulted in the loss of detectable cell surface CD28 expression on thymocytes and a significant reduction in both the surface T cell receptor expression and the ratio of CD4(+) to CD8(+) single-positive thymocytes in Tg animals compared to nontransgenic (non-Tg) controls. While many of these defects were transient, the significant decrease in CD4(+) versus CD8(+) T cell ratio persisted peripherally. Peripheral T cells from these Tg mice were found to be significantly hyporesponsive to T cell mitogens and in mixed leukocyte reaction, effects that our data indicate are due to reduced IL-2 production by Tg T cells upon activation. Despite these functional defects, immunization with both complex and simple protein antigens produced no differences in the proliferative or humoral responses to these antigens between Tg and non-Tg groups. These data indicate that thymic CD80 signaling results in the deletion of significant numbers of CD4(+) T cells but does not culminate in antigen-specific immunodeficiency.     

Cutting edge: thymocyte-independent and thymocyte-dependent phases of epithelial patterning in the fetal thymus

Thymic epithelial cells (TECs) in adult mice have been classified into distinct subsets based on keratin expression profiles. To explore the emergence of TEC subsets during ontogeny, we analyzed keratin 8 and keratin 5 expression at several stages of fetal development in normal C57BL/6J mice. In addition, thymic epithelial development and compartmentalization were explored in recombination-activating gene 2/common cytokine receptor gamma-chain-deficient and Ikaros-null mice that sustain early and profound blocks in thymocyte differentiation. The results demonstrate that initial patterning of the thymic epithelial compartment as defined by differential keratin expression does not depend on inductive signals from hematopoietic cells. However, thymocyte-derived signals are required during late fetal stages for continued development and maintenance of TEC subsets in the neonate and adult.     

Overexpression of CRABPI in suprabasal keratinocytes enhances the proliferation of epidermal basal keratinocytes in mouse skin topically treated with all-trans retinoic acid

We investigated whether ectopic expression of CRABPI, a cellular retinoic acid binding protein, influenced the actions of all-trans retinoic acid (ATRA) in transgenic (TG) mice. We targeted CRABPI to the basal vs. suprabasal layers of mouse epidermis by using the keratin 14 (K14) and keratin 10 (K10) promoters, respectively. Greater CRABPI protein levels were detected in the epidermis of adult transgenic(+) mice than in transgenic(-) mice for both transgenes. In adult mouse skin CRABPI overexpression in the basal or suprabasal keratinocytes did not cause morphological abnormalities, but did result in decreased CRABPII mRNA levels. Ectopically overexpressed CRABPI in suprabasal keratinocytes, but not in basal keratinocytes, enhanced the thickening of the epidermis induced by topical ATRA treatments (10 microM, 400 microl for 4 days) by 1.59+/-0.2-fold (p<0.05). ATRA treatment (10 microM) resulted in a 59.9+/-9.8% increase (p<0.05) in the BrdU labeling index in K10/FLAG-CRABPI TG(+) mice vs. TG(-) mice. Retinoid topical treatments reduced p27 and CYP26A1 mRNA levels in TG(+) and TG(-) mouse skin in K14 and K10/FLAG-CRABPI transgenic mice. As epidermal basal keratinocyte proliferation is stimulated by paracrine growth factors secreted by ATRA activated suprabasal keratinocytes, our results indicate that CRABPI overexpression in suprabasal keratinocytes enhances the physiological functions of ATRA.     

A reassessment of the effect of activated Notch1 on CD4 and CD8 T cell development

The Notch signaling pathway plays an important role in the early steps of T cell development and in the generation of T cell tumors, but its role in the CD4 vs CD8 lineage decision is controversial. Notch1 is not essential for CD4 or CD8 T cell development; however, there are suggestions that multiple Notch family members may act in a redundant fashion during thymic development. In theory, expressing a constitutively activated form of Notch in CD4(+)CD8(+) thymocytes could provide clues about the normal role of Notch in developing CD4 and CD8 T cells. Unfortunately, two different studies of transgenic mice expressing activated forms of Notch1 (Notch1IC) led to conflicting conclusions. In this study, we re-examine the effect of the two Notch1IC transgenes on thymocyte development. We find that both Notch1IC transgenic lines display a decrease in CD4 single positive (SP) thymocytes and a corresponding increase in CD8 SP thymocytes. The enhanced development of CD8 SP thymocytes is dependent on either class I or II MHC. Thus, data from two different Notch1IC transgenic lines indicate that Notch activity promotes CD8 and inhibits CD4 SP development. We suggest that the discrepancies in previous reports of Notch1IC transgenic mice are due to differences in the propensity of the two different transgenic lines to develop tumors.     

Competition for specific intrathymic ligands limits positive selection in a TCR transgenic model of CD4+ T cell development

Efficient positive selection of a broad repertoire of T cells is dependent on the presentation of a diverse array of endogenous peptides on MHC molecules in the thymus. It is unclear, however, whether the development of individual TCR specificities is influenced by the abundance of their selecting ligands. To examine this, we analyzed positive selection in a transgenic mouse carrying a TCR specific for the human CLIP:I-Ab class II complex. We found that these mice exhibit significantly reduced CD4+ T cell development compared with two other transgenic mice carrying TCRs selected on I-Ab. Moreover, many of the selected cells in these mice express endogenous and transgenic receptors as a consequence of dual TCRalpha expression. Dramatic enhancement of the selection efficiency is observed, however, when fewer transgenic cells populate the thymus in mixed bone marrow chimeras. These results suggest that positive selection is limited by the availability of selecting peptides in the thymus. This becomes apparent when large numbers of thymocytes compete for such peptides in TCR transgenic animals. Under such conditions, thymocytes appear to undergo further TCRalpha gene rearrangement to produce a receptor that may be selected more efficiently by other thymic self-peptides.