Teresa Cejalvo

Sonic Hedgehog Is Produced by Follicular Dendritic Cells and Protects Germinal Center B Cells from Apoptosis

The Hedgehog (Hh) signaling pathway is involved in the development of many tissues during embryogenesis, but has also been described to function in adult self-renewing tissues. In the immune system, Sonic Hedgehog (Shh) regulates intrathymic T cell development and modulates the effector functions of peripheral CD4+ T cells. In this study we investigate whether Shh signaling is involved in peripheral B cell differentiation in mice. Shh is produced by follicular dendritic cells, mainly in germinal centers (GCs), and GC B cells express both components of the Hh receptor, Patched and Smoothened. Blockade of the Hh signaling pathway reduces the survival, and consequently the proliferation and Ab secretion, of GC B cells. Furthermore, Shh rescues GC B cells from apoptosis induced by Fas ligation. Taken together, our data suggest that Shh is one of the survival signals provided by follicular dendritic cells to prevent apoptosis in GC B cells.

Sonic Hedgehog Regulates Early Human Thymocyte Differentiation by Counteracting the IL-7-Induced Development of CD34+ Precursor Cells

The Hedgehog (Hh) family of signaling molecules normally functions in the development of numerous tissues by regulating cellular differentiation and proliferation. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate the potential role of Sonic hedgehog (Shh) in human intrathymic T cell maturation. Results show that the expression of the two components of the Hh receptor, Patched and Smoothened, is mostly restricted to CD34+ precursor cells that are committing to the T cell lineage. Shh significantly increased the viability of CD34+ T cell precursors modulating bcl-2 and bax protein expression, and also inhibited their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with Shh resulted in an arrested thymocyte differentiation and an accumulation of CD34+ progenitor cells. This effect was mainly attributed to the ability of Shh to counteract the IL-7-induced proliferation and differentiation of CD34+ cells. Shh down-regulated in the precursor cell population the expression of IL-7R as well as stromal-derived factor-1 chemokine receptor, CXCR4, and inhibited IL-7-dependent STAT5 phosphorylation. Therefore, Shh may function as a maintenance factor for intrathymic CD34+ precursor cells.

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2‐Fc or ephrinB1‐Fc fusion proteins to fetal thymus organ cultures established from 17‐day‐old fetal mice decreases the numbers of both double‐positive (CD4+CD8+) and single‐positive (both CD4+CD8– and CD4–CD8+) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4+CD8+ thymocytes, we have also demonstrated that ephrinB1‐Fc proteins are able to disorganize the three‐dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB‐Fc treatment also decreases the formation of cell conjugates by CD4+CD8+ thymocytes and TEC as well as the TCR‐dependent signaling between both cell types. Finally, immobilized EphB2‐Fc and ephrinB1‐Fc modulate the anti‐CD3 antibody‐induced apoptosis of CD4+CD8+ thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three‐dimensional organization of TEC.

EphB2-mediated interactions are essential for proper migration of T cell progenitors during fetal thymus colonization

The ephrin‐Eph ligand receptor pair is known to control the repulsion/adhesion process in different tissues, including the immune system. Herein, we evaluated the role of EphB2 receptors in T cell progenitor migration during in vitro thymus colonization and to ECM or chemokine stimuli. EphB2 and their ligands, ephrin‐B1 and ephrin‐B2, are expressed in BM‐derived progenitors, and EphB2−/− cells had diminished thymus colonization capacity. Conversely, EphB2LacZ cells, which maintain a preserved ephrin‐binding domain, were capable of colonizing WT thymuses similarly to WT progenitors, highlighting the importance of reverse signals transmitted to normal fetal thymus. However, the EphB2 receptor expressed by microenvironmental cells also drives progenitor immigration, as recolonization of EphB2‐deficient fetal thymuses was compromised profoundly. Additionally, we observed lower depositions of ECM and chemokines on EphB2‐deficient thymuses but no changes in their receptor expression on BM‐derived progenitors and developing thymocytes. Migration of EphB2‐deficient progenitors and thymocytes was also reduced through ECM or chemokine stimuli. Furthermore, ephrin‐B1 costimulation also inhibited haptotaxis and chemotaxis of WT but not EphB2LacZ cells, demonstrating the specific involvement of EphB2 signaling on T cell progenitor migration. Our data suggest the relevance of a nonactivated EphB2 for regulating T cell progenitor migration and its modulation upon ephrin‐B engagement.

Bone morphogenetic protein-2/4 signalling pathway components are expressed in the human thymus and inhibit early T-cell development.

T‐cell differentiation is driven by a complex network of signals mainly derived from the thymic epithelium. In this study we demonstrate in the human thymus that cortical epithelial cells produce bone morphogenetic protein 2 (BMP2) and BMP4 and that both thymocytes and thymic epithelium express all the molecular machinery required for a response to these proteins. BMP receptors, BMPRIA and BMPRII, are mainly expressed by cortical thymocytes while BMPRIB is expressed in the majority of the human thymocytes. Some thymic epithelial cells from cortical and medullary areas express BMP receptors, being also cell targets for in vivo BMP2/4 signalling. The treatment with BMP4 of chimeric human–mouse fetal thymic organ cultures seeded with CD34+ human thymic progenitors results in reduced cell recovery and inhibition of the differentiation of human thymocytes from CD4− CD8− to CD4+ CD8+ cell stages. These results support a role for BMP2/4 signalling in human T‐cell differentiation.

Alterations in the thymocyte phenotype of EphB‐deficient mice largely affect the double negative cell compartment

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T‐cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4− CD8−) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44− CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25−) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15‐day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T‐cell subsets defined by CD4/CD8 marker expression, in all cases.

On the role of Eph signalling in thymus histogenesis; EphB2/B3 and the organizing of the thymic epithelial network

In the current study, we extend our own previous results on the thymocyte phenotype of EphB2 and/or EphB3 deficient mice by analyzing the phenotype and the histological organization of their thymic epithelial stroma. All studied adult EphB-deficient thymi showed profound alterations with respect to the wild-type (WT) ones. Each mutant exhibited a specific phenotype, but also showed common features including occurrence of K5+K8+MTS10+ immature medullary epithelial cells, numerous K5-K8-MTS20+ cells and K5+K8+ cells in the thymic cortex and cortical and medullary K5-K8- areas devoid of epithelial cell markers. In addition, comparative analysis of WT and EphB-deficient embryonic and newborn thymi demonstrated that the observed adult phenotype was a consequence of the gradual accumulation of early phenotypic and morphological defects, becoming more severe at the end of embryonic life and in newborn animals. Together, these results confirm a role for EphB2 and EphB3 in thymus morphogenesis. The obtained data are discussed from the point of view of the recognized role played by these two Ephs in the homeostasis of other epithelia and their possible relationships with molecules known to be involved in thymic epithelial cell development.

Eph/Ephrin-Mediated Interactions in the Thymus

In the present study, we review available information on the relevance of Eph and ephrins in numerous processes occurring in the thymus that regulate not only T cell differentiation but also thymic epithelial cell (TEC) development and organization. Eph/ephrins are a large family of receptors and ligands involved in organogenesis and homeostasis of adult tissues. They are extensively expressed in the thymus and seem to be involved in the colonization of lymphoid progenitor cells and their migration throughout the thymic parenchyma necessary to provide an adequate topological location of developing thymocytes in the epithelial network that ensures their correct differentiation. In addition, EphB2 and EphB3 play a cell-autonomous role in regulating the transitions of double-negative to double-positive cells and of double-positive to single-positive thymocytes and the lack of these molecules or their ligands ephrin B1 and ephrin B2 induces profound alterations of the TEC maturation and in the arrangement of epithelial network. We emphasize that these results are largely reflecting the role played by this family of molecules in controlling thymocyte-TEC interactions within the thymus.

The Eph/ephrinB signal balance determines the pattern of T-cell maturation in the thymus

In order to carry out an in‐depth study of the roles of EphB receptors in T‐cell development and to determine the specific relevance of forward and reverse signals in the process, we established severe combined immunodeficient (SCID) mice chimeras with wild‐type (WT) or EphB‐deficient bone marrow cells. The obtained results demonstrate that EphB2 contributes more significantly than EphB3 in the control of CD4−CD8− (DN)–CD4+CD8+ (DP) progression, and that reverse signals generated in SCID mice receiving EphB2LacZ precursors, which express the EphB2 extracellular domain, partially rescue the blockade of DN cell maturation observed in EphB2‐null chimeras. In addition, increased apoptotic DP thymocytes occurring in EphB2 and/or EphB3 SCID chimeras also contribute to the reduced proportions of DP cells. However, EphB2LacZ chimeras do not show any changes in the proportions of apoptotic DP cells, thus suggesting that there is a role for ephrinB reverse signaling in thymocyte survival. The maturation of DP to CD4+CD8− or CD4−CD8+ seems to need EphB2 forward signaling and EphB3; a fact that was confirmed in reaggregates formed with either EphB2‐ or EphB3‐deficient DP thymocytes and WT thymic epithelial cells (TECs). The DP thymocyte–TEC conjugate formation was also affected by the absence of EphB receptors. Finally, EphB‐deficient SCID chimeras show profoundly altered thymic epithelial organization that confirms a significant role for EphB2 and EphB3 receptors in the thymocyte–TEC crosstalk.

Interplay between BMP4 and IL-7 in human intrathymic precursor cells

Bone morphogenetic proteins (BMPs) play a pivotal role during vertebrate embryogenesis and organogenesis, and have also been described to function in regulating cell fate and determination in self-renewing tissues in adults. Recent results have demonstrated that the different components of the BMP2/4 signaling pathway are expressed in the human thymus. In this study, we provide evidence that BMP4 and IL-7 interplay is important in the maintenance of the human thymic progenitor population. Intrathymic CD34+ cells express BMP receptors (BMPRIA, BMPRIB, ActRIA, BMPRII), signal transduction molecules (Smad1, 5, 8 and 4), and produce BMP4. Neutralization of endogenous BMP4 by treatment with the antagonist Noggin reduces thymic precursor cell survival, and the addition of exogenous BMP4 decreases their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with BMP4 inhibits cell expansion, arrests thymocyte differentiation, and leads to the accumulation of human CD34+ precursor cells. This effect is mainly attributed to the ability of BMP4 to counteract the IL-7-induced proliferation and differentiation of CD34+ cells. BMP4 down-regulates in the precursor cell population the expression of CD127 and inhibits the IL-7-dependent STAT5 phosphorylation. In addition, BMP signaling is promoted by IL-7. Our results also demonstrate that in thymic progenitors BMPs act downstream of Sonic Hedgehog, previously described to function as a maintenance factor for human intrathymic CD34+ precursor cells.