Daniella Arêas Mendes-da-Cruz

Intrathymic T-cell migration: a combinatorial interplay of extracellular matrix and chemokines?

Cell migration is crucial for intrathymic T-cell differentiation. Chemokines and extracellular matrix proteins per se induce thymocyte migration, and recent data suggest a combinatorial role for these molecules in this event. For example, thymocyte migration induced by fibronectin plus CXCL12/SDF1-alpha (stromal cell-derived factor1-alpha) is higher than that elicited by the chemokine alone. If such interactions are relevant in the thymus, abnormal expression of any of these ligands and/or their corresponding receptors will lead to defects in thymocyte migration. At least in the murine model of Chagas disease, this seems to be the case. Therefore a better knowledge of this complex biological circuitry will provide new clues for understanding thymus physiology and designing therapeutic strategies targeting developing T cells.

Molecular mechanisms governing thymocyte migration: combined role of chemokines and extracellular matrix

Cell migration is crucial for thymocyte differentiation, and the cellular interactions involved now begin to be unraveled, with chemokines, extracellular matrix (ECM) proteins, and their corresponding receptors being relevant in such oriented movement of thymocytes. This notion derives from in vitro, ex vivo, and in vivo experimental data, including those obtained in genetically engineered and spontaneous mutant mice. Thymic microenvironmental cells produce both groups of molecules, whereas developing thymocytes express chemokine and ECM receptors. It is important that although chemokines and ECM proteins can drive thymocyte migration per se, a combined role of these molecules likely concurs for the resulting migration patterns of thymocytes in their various differentiation stages. In this respect, among ECM moieties, there are proteins with opposing functions, such as laminin or fibronectin versus galectin‐3, which promote, respectively, adhesion and de‐adhesion of thymocytes to the thymic microenvironment. How chemokines and ECM are produced and degraded remains to be more clearly defined. Nevertheless, matrix metalloproteinases (MMPs) likely play a role in the intrathymic ECM breakdown. It is interesting that these molecules also degrade chemokines. Thus, the physiological migration of thymocytes should be conceived as a resulting vector of multiple, simultaneous, or sequential stimuli, involving chemokines, adhesive, and de‐adhesive ECM proteins. Moreover, these interactions may be physiologically regulated in situ by matrix MMPs and are influenced by hormones. Accordingly, one can predict that pathological changes in any of these loops may result in abnormal thymocyte migration. This actually occurs in the murine infection by the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. In this model, the abnormal release of immature thymocytes to peripheral lymphoid organs is correlated with the higher migratory response to ECM and chemokines. Lastly, the fine dissection of the mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells. The most important function of the thymus is to generate T lymphocytes, which once leaving the organ, are able to colonize specific regions of peripheral lymphoid organs, the T cell zones, where they can mount and regulate cell‐mediated, immune responses. This intrathymic T cell differentiation is a complex sequence of biological events, comprising cell proliferation, differential membrane protein expression, gene rearrangements, massive programmed cell death, and cell migration. In this review, we will focus on the mechanisms involved in controlling the migration of thymocytes, from the entrance of cell precursors into the organ to the exit of mature T cells toward peripheral lymphoid organs. Nevertheless, to better comprehend this issue, it appeared worthwhile to briefly comment on some key aspects of thymocyte differentiation and the tissue context in which it takes place, the thymic microenvironment.

Altered thymocyte migration during experimental acute Trypanosoma cruzi infection: combined role of fibronectin and the chemokines CXCL12 and CCL4

We previously showed migration disturbances in the thymus during experimental infection with Trypanosoma cruzi, the causative agent of Chagas disease. These changes were related to the enhanced expression of extracellular matrix ligands and receptors, leading to the escape of immature cells to the periphery. Here, we analyzed the expression and role of selected chemokines (CXCL12 and CCL4) and their receptors (CXCR4 and CCR5) in regulating thymocyte migration in conjunction with extracellular matrix during acute T. cruzi infection. We found increased chemokine deposition in the thymus of infected mice when compared to controls, accompanied by enhanced co‐localization with fibronectin as well as up‐regulated surface expression of CXCR4 and CCR5 in thymocytes. We also noticed altered thymocyte migration towards the chemokines analyzed. Such an enhancement was even more prominent when fibronectin was added as a haptotatic stimulus in combination with a given chemokine. Our findings suggest that thymocyte migration results from a combined action of chemokines and extracellular matrix (ECM), which can be altered during pathological conditions such as T. cruzi infection, and may be at the origin of the changes in the T cell repertoire seen in this pathological process.

The Thymic Orchestration Involving Aire, miRNAs, and Cell–Cell Interactions during the Induction of Central Tolerance

Developing thymocytes interact sequentially with two distinct structures within the thymus: the cortex and medulla. Surviving single-positive and double-positive thymocytes from the cortex migrate into the medulla, where they interact with medullary thymic epithelial cells (mTECs). These cells ectopically express a vast set of peripheral tissue antigens (PTAs), a property termed promiscuous gene expression that is associated with the presentation of PTAs by mTECs to thymocytes. Thymocyte clones that have a high affinity for PTAs are eliminated by apoptosis in a process termed negative selection, which is essential for tolerance induction. The Aire gene is an important factor that controls the expression of a large set of PTAs. In addition to PTAs, Aire also controls the expression of miRNAs in mTECs. These miRNAs are important in the organization of the thymic architecture and act as posttranscriptional controllers of PTAs. Herein, we discuss recent discoveries and highlight open questions regarding the migration and interaction of developing thymocytes with thymic stroma, the ectopic expression of PTAs by mTECs, the association between Aire and miRNAs and its effects on central tolerance.

RNA interference-mediated knockdown of CD49e (α5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

BackgroundThe thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin α5β1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody.ResultsHerein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets.ConclusionConceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Developing T-cell migration: role of semaphorins and ephrins

Cell migration is a crucial event for normal T‐cell development, and various ligand/receptor pairs have been implicated. Most of them, including chemokines and extracellular matrix proteins, have attractant properties on thymocytes. We discuss herein two further groups of ligand/receptor pairs, semaphorins/neuropilins and ephs/ephrins, which are constitutively expressed by thymocytes and thymic microenvironmental cells. Evidence shows that the corresponding interactions are relevant for developing T‐cell migration, including the entry of bone marrow progenitor cells, migration of CD4/CD8‐defined thymocyte subpopulations triggered by chemokines and/or extracellular matrix proteins, and thymocyte export. Conceptually, the data summarized here show that thymocyte migration results from a complex network of molecular interactions, which generate not only attraction, but also repulsion of migrating T‐cell precursors.—Mendes‐da‐Cruz, D. A., Stimamiglio, M. A., Muñoz, J. J., Alfaro, D., Terra‐Granado, E., Garcia‐Ceca, J., Alonso‐Colmenar, L. M., Savino, W., Zapata, A. G. Developing T‐cell migration: role of semaphorins and ephrins. FASEB J. 26, 4390–4399 (2012). www.fasebj.org

Semaphorins and neuropilins: new players in the neuroendocrine control of the intrathymic T‐cell migration in humans

Cell migration is a key event for proper intrathymic T‐cell differentiation, and several ligand–receptor interactions contribute to the well‐co ordinated movement of developing thymocytes within the thymic lobules. Herein we summarize recent data that place semaphorin 3A (Sema3A) and its receptor neuropilin 1 (NRP1) as further players in the physiological process of cell migration in the human thymus. These molecules, as well as class A plexins (necessary for the intracellular signalling transduction triggered by Sema3A–NRP1 ligation), are constitutively expressed by both developing thymocytes and components of the thymic microenvironment, including epithelial and dendritic cells. Functionally, Sema3A decreases the adhesion of human thymocytes on thymic epithelial cell monolayers and exerts per se a dose‐dependent chemorepulsive effect on human thymocytes. Moreover, Sema3A inhibits chemoattractant migratory responses induced by other ligands, including fibronectin, laminin and CXCL12 (chemokine CXC motif ligand 12). These data should be placed in the context of the concept that migration of developing T cells is a multivectorial system, in which the resulting migration vector derives from a balance of several simultaneous and/or sequential ligand–receptor pair interactions. Accordingly, semaphorins and neuropilins can be considered as further players in the system.

Growth hormone modulates migration of thymocytes and peripheral T cells

In the context of immunoneuroendocrine cross talk, growth hormone (GH) exerts pleiotropic effects in the immune system. For example, GH‐transgenic mice, as well as animals and humans treated with GH, exhibit enhanced cellularity in the thymus. GH also stimulates the thymic microenvironment, augmenting chemokine and extracellular matrix (ECM) production, with consequent increase in ECM‐ and chemokine‐driven thymocyte migratory responses. Peripheral T cell migration triggered by laminin or fibronectin was enhanced in cells from GH‐transgenic versus wild‐type control adult mice, as seen for CD4+ and CD8+ T cells from mesenteric lymph nodes. Migration of these T lymphocytes, triggered by the chemokine CXCL12, in conjunction with laminin or fibronectin, was also enhanced compared with control counterparts. Considering that GH can be used as an adjuvant therapy in immunodeficiencies, including AIDS, the concepts defined herein, that GH enhances developing and peripheral T cell migration, provide new clues for future GH‐related immune interventions.

Fibronectin receptor defects in NOD mouse leucocytes: possible consequences for type 1 diabetes.

Integrins of the very late antigen (VLA) family mediate leucocyte traffic to lymphoid organs under physiological conditions and in chronic inflammatory situations such as autoimmunity. Accordingly, the current thinking is of a positive correlation between VLA expression and capability of the generation of autoimmunity. Herein we discuss recent findings on the defective expression of integrin‐type fibronectin receptors α4β1 (VLA‐4) and α5β1 (VLA‐5) in the non‐obese diabetic (NOD) mouse, a murine model of autoimmune insulin‐dependent diabetes mellitus. As compared with normal animals, NOD thymocytes (including the CD4+CD25+ regulatory T cells) exhibit a decrease in the membrane expression of α5β1, resulting in a functional impairment of fibronectin‐mediated interactions, including cell migration. Interestingly, thymocytes that are trapped within the giant perivascular spaces seen in NOD thymus are consistently α5β1 negative, suggesting that the progressive arrest of mature cells can be related to the α5β1 defect. Peripheral T cells also exhibit decreased α5β1 membrane expression and impaired fibronectin‐driven migration. Additionally, we observed a defect in α4β1 fibronectin receptor expression in NOD macrophages. Peritoneal, bone marrow‐derived‐precursor, as well as thymic macrophages of NOD mice showed an impaired upregulation of α4‐integrin chain expression, dependent on the level of macrophage maturation. Overall these data lead to the notion that NOD leucocytes bear distinct fibronectin receptor‐mediated cell migration defects, which may be involved in the pathogenesis and/or pathophysiology of the autoimmune events seen in NOD mice. Further studies will be helpful to define whether or not this concept can be applied for other autoimmune diseases.

Laminin-Mediated Interactions in Thymocyte Migration and Development.

Intrathymic T-cell differentiation is a key process for the development and maintenance of cell-mediated immunity, and occurs concomitantly to highly regulated migratory events. We have proposed a multivectorial model for describing intrathymic thymocyte migration. One of the individual vectors comprises interactions mediated by laminins (LMs), a heterotrimeric protein family of the extracellular matrix. Several LMs are expressed in the thymus, being produced by microenvironmental cells, particularly thymic epithelial cells (TECs). Also, thymocytes and epithelial cells express integrin-type LM receptors. Functionally, it has been reported that the dy/dy mutant mouse (lacking the LM isoform 211) exhibits defective thymocyte differentiation. Several data show haptotactic effects of LMs upon thymocytes, as well as their adhesion on TECs; both effects being prevented by anti-LM or anti-LM receptor antibodies. Interestingly, LM synergizes with chemokines to enhance thymocyte migration, whereas classe-3 semaphorins and B ephrins, which exhibit chemorepulsive effects in the thymus, downregulate LM-mediated migratory responses of thymocytes. More recently, we showed that knocking down the ITGA6 gene (which encodes the α6 integrin chain of LM receptors) in human TECs modulates a large number of cell migration-related genes and results in changes of adhesion pattern of thymocytes onto the thymic epithelium. Overall, LM-mediated interactions can be placed at the cross-road of the multivectorial process of thymocyte migration, with a direct influence per se, as well as by modulating other molecular interactions associated with the intrathymic-trafficking events.