Ellen R. Richie

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 γ-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.

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.

IL-7-producing stromal cells are critical for lymph node remodeling

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.

IL7-hCD25 and IL7-Cre BAC transgenic mouse lines: new tools for analysis of IL-7 expressing cells.

IL‐7 is a cytokine that is required for T‐cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL‐7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL‐7 synthesis, specific cell types involved and molecular mechanisms regulating IL‐7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL‐7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL‐7.hCD25 BAC transgene, detected by reactivity with anti‐hCD25 antibody, mimicked endogenous IL‐7 expression. Fetal and adult tissues from crosses between IL‐7.Cre transgenic mice and Rosa26R or R26‐EYFP reporters demonstrated X‐gal or YFP staining in tissues known to express endogenous IL‐7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL‐7 producing cells in various tissues and to manipulate gene expression selectively in IL‐7 expressing cells. genesis 47:281–287, 2009.

Structure and function of the thymic microenvironment

Organs are more than the sum of their component parts--functional competence requires that these parts not only be present in the appropriate proportions, but also be arranged and function together in specific ways. The thymus is an excellent example of the connection between cellular organization and organ function. Unlike more familiar organs, such as lung or kidney, the thymus is not organized into easily identifiable structures such as tubes and ordered cell layers, but instead is a complex meshwork of microenvironments through which T cell progenitors migrate, receiving signals that instruct them to differentiate, proliferate, or die. Proper thymic organization is essential to the optimal production of a functional T cell repertoire. During aging, the thymus undergoes involution, largely due to degradation of the TEC microenvironmental compartment, which then fails to support optimal thymocyte development resulting in reduced output of naive T cells. This review will summarize the current state of understanding of the composition and organization of thymic microenvironments and the mechanisms that promote their proper development and function.

Thymus Medulla Formation and Central Tolerance Are Restored in IKKα−/− Mice That Express an IKKα Transgene in Keratin 5+ Thymic Epithelial Cells

Medullary thymic epithelial cells (mTECs) play an essential role in establishing central tolerance due to their unique capacity to present a diverse array of tissue restricted Ags that induce clonal deletion of self-reactive thymocytes. One mTEC subset expresses keratin 5 (K5) and K14, but fails to bind Ulex europaeus agglutinin-1 (UEA-1) lectin. A distinct mTEC subset binds UEA-1 and expresses K8, but not K5 or K14. Development of both mTEC subsets requires activation of the noncanonical NF-κB pathway. In this study, we show that mTEC development is severely impaired and autoimmune manifestations occur in mice that are deficient in IκB kinase (IKK)α, a required intermediate in the noncanonical NF-κB signaling pathway. Introduction of an IKKα transgene driven by a K5 promoter restores the K5+K14+ mTEC subset in IKKα−/− mice. Unexpectedly, the K5-IKKα transgene also rescues the UEA-1 binding mTEC subset even though K5 expression is not detectable in these cells. In addition, expression of the K5-IKKα transgene ameliorates autoimmune symptoms in IKKα−/− mice. These data suggest that 1) medulla formation and central tolerance depend on activating the alternative NF-κB signaling pathway selectively in K5-expressing mTECs and 2) the K5-expressing subset either contains immediate precursors of UEA-1 binding cells or indirectly induces their development.

Cyclin D2 Overexpression in Transgenic Mice Induces Thymic and Epidermal Hyperplasia whereas Cyclin D3 Expression Results Only in Epidermal Hyperplasia

In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells.

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 hCD3ε transgenic and hCD3ε/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 Polycomb-group gene eed regulates thymocyte differentiation and suppresses the development of carcinogen-induced T-cell lymphomas

The mouse Polycomb-group gene, embryonic ectoderm development (eed), appears to regulate cellular growth and differentiation in a developmental and tissue specific manner. During embryogenesis, eed regulates axial patterning, whereas in the adult eed represses proliferation of myeloid and B cell precursors. The present report demonstrates two novel functional activities of eed: alteration of thymocyte maturation and suppression of thymic lymphoma development. Mice that inherit the viable hypomorphic 17Rn51989SB eed allele sustain a partial developmental block at or before the CD4−CD8−CD44−CD25+ stage of thymocyte differentiation. Furthermore, mice that are homozygous or heterozygous for the hypomorphic eed allele have an increased incidence and decreased latency of N-methyl-N-nitrosourea-induced thymic lymphoma compared to wild-type littermates. These findings support the notion that Polycomb-group genes exert pleiotrophic effects dictated by developmental stage and cellular context.

Enzymatic Activity Is Required for the in Vivo Functions of CARM1

CARM1 is one of nine protein arginine methyltransferases that methylate arginine residues in proteins. CARM1 is recruited by many different transcription factors as a positive regulator. Gene targeting of CARM1 in mice has been performed, and knock-out mice, which are smaller than their wild-type littermates, die just after birth. It has been proposed that CARM1 has functions that are independent of its enzymatic activity. Indeed, CARM1 is found to interact with a number of proteins and may have a scaffolding function in this context. However, CARM1 methylates histone H3, PABP1, AIB1, and a number of splicing factors, which strongly suggests that its impact on transcription and splicing is primarily through its ability to modify these substrates. To unequivocally establish the importance of CARM1 enzymatic activity in vivo, we generated an enzyme-dead knock-in of this protein arginine methyltransferase. We determined that knock-in cells and mice have defects similar to those seen in their knock-out counterparts with respect to the time of embryo lethality, T cell development, adipocyte differentiation, and transcriptional coactivator activity. CARM1 requires its enzymatic activity for all of its known cellular functions. Thus, small molecule inhibitors of CARM1 will incapacitate all of the enzymes cellular functions.