Anna L. Furmanski

Non-obese diabetic mice select a low-diversity repertoire of natural regulatory T cells

Thymus-derived Foxp3+ natural regulatory CD4 T cells (nTregs) prevent autoimmunity through control of pathogenic, autoreactive T cells and other immune effector cells. Using T cell receptor (TCR) transgenic models, diversity within this lineage has been found to be similar to that of conventional CD4 T cells. To determine whether balanced TCR diversity may be perturbed in autoimmunity, we have analyzed receptor composition in C57BL/6 and autoimmune non-obese diabetic (NOD) mice. The natural regulatory and conventional CD4 repertoires of C57BL/6 had similar diversities. Despite the apparently normal thymic development of the NOD nTreg lineage, TCR diversity within the selected repertoire was markedly restricted. Detailed analysis of TCRα and -β chain composition is consistent with positive selection into the natural regulatory lineage being under stringent audition for interaction with MHC class II/self-peptide. The NOD MHC region, including the unique H2-Ag7 class II molecule, partly accounts for the reduction in diversity, but additional NOD genetic contribution(s) are required for complete repertoire compaction. Mechanistic links between MHC, autoimmunity, and nTreg diversity identified in this study are discussed.

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.

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.

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.

Regulation of murine normal and stress-induced erythropoiesis by Desert Hedgehog.

The function of Hedgehog signaling in hematopoiesis is controversial, with different experimental systems giving opposing results. Here we examined the role of Desert Hedgehog (Dhh) in the regulation of murine erythropoiesis. Dhh is one of 3 mammalian Hedgehog family proteins. Dhh is essential for testis development and Schwann cell function. We show, by analysis of Dhh-deficient mice, that Dhh negatively regulates multiple stages of erythrocyte differentiation. In Dhh-deficient bone marrow, the common myeloid progenitor (CMP) population was increased, but differentiation from CMP to granulocyte/macrophage progenitor was decreased, and the mature granulocyte population was decreased, compared with wild-type (WT). In contrast, differentiation from CMP to megakaryocyte/erythrocyte progenitor was increased, and the megakaryocyte/erythrocyte progenitor population was increased. In addition, we found that erythroblast populations were Dhh-responsive in vitro and ex vivo and that Dhh negatively regulated erythroblast differentiation. In Dhh-deficient spleen and bone marrow, BFU-Es and erythroblast populations were increased compared with WT. During recovery of hematopoiesis after irradiation, and under conditions of stress-induced erythropoiesis, erythrocyte differentiation was accelerated in both spleen and bone marrow of Dhh-deficient mice compared with WT.

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.

Direct BMP2/4 signaling through BMP receptor IA regulates fetal thymocyte progenitor homeostasis and differentiation to CD4+CD8+ double-positive cell

BMP2/4 signaling is required for embryogenesis and involved in thymus morphogenesis and T-lineage differentiation. In vitro experiments have shown that treatment of thymus explants with exogenous BMP4 negatively regulated differentiation of early thymocyte progenitors and the transition from CD4−CD8− (DN) to CD4+CD8+ (DP). Here we show that in vivo BMP2/4 signaling is required for fetal thymocyte progenitor homeostasis and expansion, but negatively regulates differentiation from DN to DP cell. Unexpectedly, conditional deletion of BMPRIA from fetal thymocytes (using the Cre-loxP system and directing excision to hematopoietic lineage cells with the Vav promoter) demonstrated that physiological levels of BMP2/4 signaling directly to thymocytes through BMPRIA are required for normal differentiation and expansion of early fetal DN thymocytes. In contrast, the arrest in early thymocyte progenitor differentiation caused by exogenous BMP4 treatment of thymus explants is induced in part by direct signaling to thymocytes through BMPRIA, and in part by indirect signaling through non-hematopoietic cells. Analysis of the transition from fetal DN to DP cell, both by ex vivo analysis of conditional BMPRIA-deficient thymocytes and by treatment of thymus explants with the BMP4-inhibitor Noggin demonstrated that BMP2/4 signaling is a negative regulator at this stage. We showed that at this stage of fetal T-cell development BMP2/4 signals directly to thymocytes through BMPRIA.

Role of Hedgehog signalling at the transition from double-positive to single-positive thymocyte.

In the thymus, developing T cells receive signals that determine lineage choice, specificity, MHC restriction and tolerance to self‐antigen. One way in which thymocytes receive instruction is by secretion of Sonic hedgehog (Shh) from thymic epithelial cells. We have previously shown that Hedgehog (Hh) signalling in the thymus decreases the CD4:CD8 single‐positive (SP) thymocyte ratio. Here, we present data indicating that double‐positive (DP) thymocytes are Hh‐responsive and that thymocyte‐intrinsic Hh signalling plays a role in modulating the production of CD4+ (SP4), CD8+ (SP8) and unconventional T‐cell subsets. Repression of physiological Hh signalling in thymocytes altered the proportions of DP and SP4 cells. Thymocyte‐intrinsic Hh‐dependent transcription also attenuated both the production of mature SP4 and SP8 cells, and the establishment of peripheral T‐cell compartments in TCR‐transgenic mice. Additionally, stimulation or withdrawal of Hh signals in the WT foetal thymus impaired or enhanced upregulation of the CD4 lineage‐specific transcription factor Gata3 respectively. These data together suggest that Hh signalling may play a role in influencing the later stages of thymocyte development.

Role of endogenous annexin-A1 in the regulation of thymocyte positive and negative selection.

We have recently shown that endogenous Annexin-A1 (AnxA1) plays a homeostatic regulatory role in mature T cells by modulating the strength of TCR signaling. In this study we investigated the role of endogenous AnxA1 in thymocyte maturation. Analysis of AnxA1-/- thymocyte populations at the immature CD4-CD8- double negative (DN) stage showed a proportional decrease in the DN1 and an increase in the DN3 subsets compared to control littermates. There were no significant differences in thymocyte numbers or proportions of CD4+ and CD8+ single positive (SP) populations between Anx1-/- and AnxA1+/+ mice. However, when we crossed AnxA1-/- mice onto HY-TCR transgenic mice, we observed an increase in CD4+CD8+ double positive (DP) and CD4 SP cells in male AnxA1-/-/HY-TCR compared to AnxA1+/+/HY-TCR. Conversely, female AnxA1-/-/HY-TCR mice showed an increase in DP and a decrease in CD8 (SP) cells compared to female AnxA1+/+/HY-TCR. Biochemical analysis of the signaling pathways responsible for these effects showed a decrease in anti-CD3-induced Erk phosphorylation and NF-κB activation in AnxA1-/- thymocytes compared to control littermates. Together these findings demonstrate a role for endogenous AnxA1 in regulating both positive and negative selection of the TCR repertoire. These results suggest that targeting AnxA1 expression or function in T cells could represent a useful approach for the development of novel therapies for the treatment of autoimmune diseases.