Ariadne L. Hager-Theodorides

Bone Morphogenetic Protein 2/4 Signaling Regulates Early Thymocyte Differentiation

Bone morphogenetic protein (BMP)2 and BMP4 are involved in the development of many tissues. In this study, we show that BMP2/4 signaling is involved in thymocyte development. Our data suggest that termination of BMP2/4 signaling is necessary for differentiation of CD44+CD25−CD4−CD8− double negative (DN) cells along the T cell lineage. BMP2 and BMP4 are produced by the thymic stroma and the requisite BMP receptor molecules (BMPR-1A, BMPR-1B, BMPR-II), and signal transduction molecules (Smad-1, -5, -8, and -4) are expressed by DN thymocytes. BMP4 inhibits thymocyte proliferation, enhances thymocyte survival, and arrests thymocyte differentiation at the CD44+CD25− DN stage, before T cell lineage commitment. Neutralization of endogenous BMP2 and BMP4 by treatment with the antagonist Noggin promotes and accelerates thymocyte differentiation, increasing the expression of CD2 and the proportion of CD44−CD25− DN cells and CD4+CD8+ double-positive cells. Our study suggests that the BMP2/4 pathway may function in thymic homeostasis by regulating T cell lineage commitment and differentiation.

Reduced Thymocyte Development in Sonic Hedgehog Knockout Embryos

The Hedgehog family of secreted intercellular signaling molecules are regulators of patterning and organogenesis during animal development. In this study we provide genetic evidence that Sonic Hedgehog (Shh) has a role in the control of murine T cell development. Analysis of Shh−/− mouse embryos revealed that Shh regulates fetal thymus cellularity and thymocyte differentiation. Shh is necessary for expansion of CD4−CD8− double-negative (DN) thymocytes and for efficient transition from the earliest CD44+CD25− DN population to the subsequent CD44+CD25+ DN population and from DN to CD4+CD8+ double-positive cells.

The role of morphogens in T-cell development

The Hedgehog (Hh) and Wnt family proteins, and the bone morphogenetic proteins (BMPs) 2 and 4, act as morphogens during vertebrate embryogenesis and organogenesis by regulating patterning and cell fate. They have recently been found to have a role in regulating cell fate and determination in self-renewing tissues in adults, such as the immune system and haematopoietic system. This Review presents studies on the role of Sonic Hh (Shh), Wnts and BMP2/4 in the regulation of thymocyte development. Shh and BMP2/4 act as negative regulators of thymocyte development. By contrast, Wnt signalling, through beta-catenin, has a positive role in the control of T-cell development, such that an absence or reduction in the Wnt signal leads to a reduction in cell number and cell proliferation rate and differentiation to the CD4+CD8+ double-positive stage.

The Gli3 Transcription Factor Expressed in the Thymus Stroma Controls Thymocyte Negative Selection Via Hedgehog-Dependent and -Independent Mechanisms

The Hedgehog (Hh) responsive transcription factor Gli3 is required for efficient thymocyte development in the fetus. In this study we show that Gli3, not detected in adult thymocytes, is expressed in the murine fetal and adult thymus stroma. PCR array analysis revealed Cxcl9, Rbp1, and Nos2 as novel target genes of Gli3. We show that Gli3 positively regulates the expression of these genes, most likely by suppressing an intermediate repressor. Deletion of autoreactive thymocytes depends on their interactions with the thymus stroma. Repression of the proapoptotic gene Nos2 in Gli3 mutants coincides with reduced apoptosis of double positive thymocytes undergoing negative selection in vitro and in vivo, and the production of autoreactive thymocytes. Taken together these data indicate that Gli3 controls thymocyte apoptosis and negative selection possibly via the regulation of Nos2. Defective Gli3 expression in the thymus stroma also resulted in decreased CD5 expression on mature thymocytes and inappropriate production of MHC class I-selected CD4+ cells, both consistent with reduced TCR signal strength. Overall our data indicate that Gli3 expressed in the thymus stroma regulates negative selection and TCR signal strength via Hh-dependent and -independent mechanisms, with implications for autoimmunity.

Indian hedgehog (Ihh) both promotes and restricts thymocyte differentiation

We show that Indian Hedgehog (Ihh) regulates T-cell development and homeostasis in both fetal and adult thymus, controlling thymocyte number. Fetal Ihh(-/-) thymi had reduced differentiation to double-positive (DP) cell and reduced cell numbers compared with wild-type littermates. Surprisingly, fetal Ihh(+/-) thymi had increased thymocyte numbers and proportion of DP cells relative to wild type, indicating that Ihh also negatively regulates thymocyte development. In vitro treatment of thymus explants with exogenous recombinant Hedgehog protein promoted thymocyte development in Ihh(-/-) thymi but inhibited thymocyte development in Ihh(+/-), confirming both positive and negative regulatory functions of Ihh. Analysis of Rag(-/-)Ihh(+/-) thymi showed that Ihh promotes T-cell development before pre-T-cell receptor (pre-TCR) signaling, but negatively regulates T-cell development only after pre-TCR signaling has taken place. We show that Ihh is most highly expressed by the DP population and that Ihh produced by DP cells feeds back to negatively regulate the differentiation and proliferation of their double-negative progenitors. Thus, differentiation from double-negative to DP cell, and hence the size of the DP population, is dependent on the concentration of Ihh in the thymus. Analysis of Ihh conditional knockout and heterozygote adult mice showed that Ihh also influences thymocyte number in the adult.

Sonic hedgehog negatively regulates pre-TCR–induced differentiation by a Gli2-dependent mechanism

Hedgehog signaling regulates differentiation, survival, and proliferation of the earliest double-negative (DN) thymocytes, but its importance at later stages of T-cell development is controversial. Here we use loss- and gain-of-function mouse models to show that Shh, by signaling directly to the developing thymocyte, is a negative regulator of pre-TCR-induced differentiation from DN to double-positive (DP) cell. When hedgehog signaling was reduced, in the Shh(-/-) and Gli2(-/-) thymus, or by T lineage-specific transgenic expression of a transcriptional-repressor form of Gli2 (Gli2DeltaC(2)), differentiation to DP cell after pre-TCR signal transduction was increased. In contrast, when Hh signaling was constitutively activated in thymocytes, by transgenic expression of a constitutive transcriptional-activator form of Gli2 (Gli2DeltaN(2)), the production of DP cells was decreased. Gene expression profiling showed that physiologic Hh signaling in thymocytes maintains expression of the transcription factor FoxA2 on pre-TCR signal transduction.

Splenomegaly and Modified Erythropoiesis in KLF13–/– Mice

To study the function of the Krüppel-like transcription factor KLF13 in vivo, we generated mice with a disrupted Klf13 allele. Although Klf13–/– mice are viable, fewer mice were present at 3 weeks than predicted by Mendelian inheritance. Viable Klf13–/– mice had reduced numbers of circulating erythrocytes and a larger spleen. The spleen contained an increased number of Ter119medCD71hi, Ter119hiCD71hi, and Ter119hiCD71med cells but not Ter119hiCD71– cells, indicating an increase in less mature erythroblasts. A higher proportion of the Ter119medCD71hi cells were proliferating, indicating that the mice were under a degree of erythropoietic stress. These data indicate that KLF13 is involved in the normal control of erythropoiesis.

Non-redundant role for the transcription factor Gli1 at multiple stages of thymocyte development.

The Hedgehog (Hh) signalling pathway influences multiple stages of murine T-cell development. Hh signalling mediates transcriptional changes by the activity of the Gli family of transcription factors, Gli1, Gli2 and Gli3. Both Gli2 and Gli3 are essential for mouse development and can be processed to function as transcriptional repressors or transcriptional activators, whereas Gli1, itself a transcriptional target of Hh pathway activation, can only function as a transcriptional activator and is not essential for mouse development. Gli1-deficient mice are healthy and appear normal and non-redundant functions for Gli1 have been difficult to identify. Here we show that Gli1 is non-redundant in the regulation of T-cell development in the thymus, at multiple developmental stages. Analysis of Gli1-deficient embryonic mouse thymus shows a role for Gli1 to promote the differentiation of CD4-CD8- double negative (DN) thymocytes before pre-TCR signal transduction, and a negative regulatory function after pre-TCR signalling. In addition, introduction of a Class I-restricted transgenic TCR into the adult Gli1-deficient and embryonic Gli2-deficient thymus showed that both Gli1 and Gli2 influence its selection to the CD8 lineage.

Repression of Hedgehog signal transduction in T-lineage cells increases TCR-induced activation and proliferation

Hedgehog proteins signal for differentiation, survival and proliferation of the earliest thymocyte progenitors, but their functions at later stages of thymocyte development and in peripheral T-cell function are controversial. Here we show that repression of Hedgehog (Hh) pathway activation in T-lineage cells, by expression of a transgenic repressor form of Gli2 (Gli2δC2), increased T-cell differentiation and activation in response to TCR signalling. Expression of the Gli2δC2 transgene increased differentiation from CD4+CD8+ to single positive thymocyte, and increased peripheral T cell populations. Gli2δC2 T-cells were hyper-responsive to activation by ligation of CD3 and CD28: they expressed cell surface activation markers CD69 and CD25 more quickly, and proliferated more than wild-type T-cells. These data show that Hedgehog pathway activation in thymocytes and T-cells negatively regulates TCR-dependent differentiation and proliferation. Thus, as negative regulators of TCR-dependent events, Hh proteins provide an environmental influence on T-cell fate.

A Novel Role for Hedgehog in T-Cell Receptor Signaling: Implications for Development and Immunity

The Hedgehog (Hh) signaling pathway is a key regulator of both embryonic development and homeostasis of adult tissues, including thymus and blood. In the thymus, Hh signals for differentiation, survival and proliferation in the early stages of T cell development, before TCR gene rearrangement. Our recent data has shown that Hh signaling also modulates T cell receptor (TCR) signal strength in more mature T lineage cells. We showed that constitutive activation of the Hh pathway in thymocytes (by transgenic expression of the transcriptional activator form of Gli2) decreased TCR signal strength with profound consequences for the thymus - allowing self-reactive T cells to escape deletion and altering T cell CD4/CD8 lineage decisions. In contrast, in the Sonic Hh deficient thymus, TCR signaling was increased, again influencing both TCR repertoire selection and CD4/8 lineage commitment. In peripheral T cells, the transcriptional changes induced by activation of the Hh signaling pathway lead to reduced T cell activation. Hh signaling also attenuated ERK phosphorylation and proliferation in mature T cells on TCR ligation. Modulation of TCR signal strength by Hh pathway activation has importance for immunity as the presence or absence of Hh in the environment in which a T cell is activated would shape the immune response.