Small interference ITGA6 gene targeting in the human thymic epithelium differentially regulates the expression of immunological synapse-related genes (original) (raw)
Related papers
BMC Genomics, 2013
Background: The thymic epithelium is the major microenvironmental component of the thymus, the primary lymphoid organ responsible for the generation of T lymphocytes. Thymic epithelial cells (TEC) control intrathymic T cell differentiation by means of distinct types of interactions. TEC constitutively produce chemokines and extracellular matrix ligands (such as laminin and fibronectin) and express corresponding receptors, which allow thymocytes to migrate in a very ordered fashion. We previously showed that laminin mediates TEC/thymocyte interactions in both mice and humans. More recently, we used RNAi technology to knock-down the ITGA5 gene (which encodes CD49e, the integrin α-chain subunit of the fibronectin receptor VLA-5) in cultured human TEC. Using a similar strategy, herein we knocked-down the ITGA6 gene, which encodes CD49f, the α-chain of two integrin-type laminin receptors, namely VLA-6 (α6β1) and α6β4. Results: We first confirmed that RNAi-induced knock-down of the ITGA6 gene was successful, at both transcription and translational levels, with a significant decrease in the membrane expression of CD49f, apart from CD49b, CD49c and CD49d, ascertained by cytofluorometry on living TEC. We also demonstrated that such knock-down promotes a decrease in cell adhesion to laminin. Using quantitative PCR, we demonstrated that gene expression of other integrin α-chains were concomitantly down-regulated, particularly those which form other laminin receptors, including ITGA1, ITGA2 and ITGA7. Interestingly enough, LAMA1 gene expression (whose corresponding protein chain is part of laminin-111) was largely increased in ITGA6 knocked-down TEC cultures. Lastly, the network complexity of gene expression under ITGA6 influence is much broader, since we found that other cell migrationrelated genes, namely those coding for various chemokines, are also modulated when IGTA6 is knocked-down. Conclusion: The data presented herein clearly show that down regulation of ITGA6 gene in the human thymic epithelium triggers a complex cascade of effects upon the expression levels of several other cell migration-related genes, including extracellular matrix and chemokine ligands and receptors. Taken together, these data unravel the concept that the expression of genes involved in controlling of thymocyte migration by the thymic microenvironment should be regarded as complex networks, so that a defect in the expression of one single gene may reflect in an amplified cascade with functional consequences for TEC adhesion onto the natural ligand and potential consequences upon the normal patterns of TEC/thymocyte interactions.
BMC Genomics, 2010
Background: The 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. Results: Herein, 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/CD8defined thymocyte subsets.
Developmental Immunology, 2000
T cell precursors homed to thymus develop in close contact with stromal cells. Among them, thymic epithelial cells (TEC) are known to exert dominant roles in their survival and functional shaping. Key molecules mediating TEC/thymocytes interactions include cytokines and growth factors secreted by the two cell types and adhesion receptors mediating cell contact. Signaling events triggered in thymocytes by adhesion to epithelial cells have been extensively investigated, whereas little is known on the opposite phenomenon. We have previously investigated this issue in a co-culture system composed of TEC cultures derived from human normal thymus and heterologous thymocytes. We demonstrated that thymocytes adhere to TEC involving [ and [34 integrins and induce the clustering of (z3[ and 6134 heterodimers at the TEC surface. In addition thymocyte adhesion was followed by activation of NF-zB and NF-IL6 gene transciption factors and enhanced IL-6 production. The two latter phenomena were reproduced by the cross-linking of the 3, 6, [31 and [34 integrins, thus implying that the 3131 and z6134 heterodimers can signal during thymocyte adhesion. We have extended our previous work investigating in the same experimental setting the inducing activity of non stimulated or activated policlonal or clonal mature T cells as representative of the more mature thymocyte subset. We found that adhesion of unstimulated T cell i) involved 1, but not 4 integrin functions at the surface ii) induced the clustering of 3[31, but not c21 heterodimers at the TEC surface and iii) up-regulated the nuclear binding activity of NF-r,B transcription factor and the IL-6 secretion. We propose that 31 and 64 heterodimers are induced to cluster at the TEC surface recognizing yet unknown cellular ligands differentially expressed during T cell development.
Developmentally regulated interactions of human thymocytes with different laminin isoforms
Immunology, 2002
The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different a, three b and three c subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin a1 chain (e.g. LN-1) were not present in the human thymus, laminin isoforms containing the a2 chain (LN-2/4) or the a5 chain (LN-10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin a4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN-5 isoform containing the a3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4 x CD8 x thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN-10/11, but not to LN-1, LN-2/4 or LN-5. Adhesion of these thymocytes to LN-10/11 was mediated by the integrin a6b1. During further development, the cortically localized CD4 + CD8 + thymocytes have lost the capacity to adhere to laminin-10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8 + thymocytes which were mainly found in the medulla were able to bind to LN-5 which is expressed by medullary epithelial cells. Interactions of CD8 + thymocytes with LN-5 were integrin a6b4-dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.
Blood, 1998
T-cell precursors develop within the thymus in contact with multiple supportive elements, among which thymic epithelial cells (TEC) are known to exert a dominant role in their homing, survival, and functional differentiation. All these functions are supported by cell-cell contacts and cytokine release. Signaling events triggered in lymphoid cells by adhesion to TEC are well characterized, but little is known about the opposite phenomenon. To address this issue, we derived cultures of TEC from human normal thymus. TEC monolayers were cocultured with thymocytes and immunostained with monoclonal antibodies (MoAbs) to integrin (2, 3, 4, and 6) and beta (beta1 and beta4) chains. Optical and confocal analysis showed that integrins were polarized on TEC at discrete surface locations: 6beta4 lined the basal surface of TEC monolayers, whereas 3beta1 was found mostly at TEC-TEC contacts; it is noteworthy that both 3beta1 and 6beta4 became highly enriched also at the boundaries with adherent t...
1996
T cell development in the thymus requires the establishment of stable interactions with cellselecting elements such as the cortical epithehum followed by a regulated movement of selected progenitors to the medulla. Cell adhesion and migration are mediated by integrins in a number of biological systems though little is known regarding their function in the thymus. We demonstrated previously that immature CD31~ l~ double positive human thymocytes adhere avidly to FN via the integrin, VLA4. We now demonstrate that the interaction of mature CD3hiCD69 hi thymic subsets with FN triggers migration rather than firm adhesion. Migration requires the engagement of VLA4 in cooperation with VLA5 and both receptors regulate the persistence and directionality of movement. While migration capability is linked to maturation state, ligand concentration determines the efficiency of migration. In fact, FN and the alternatively sphced CS1 site are predominant in the thymic medulla, suggesting an instructive role of this ECM protein in vivo. Our studies identify a novel VLA4 and VLA5/FNmediated pathway likely to be involved in regulating cell traffic between the cortex and medulla of the thymus. Moreover, the data provides evidence that VLA4 exists in at least two functional states at distinct stages ofT cell development. While different states of VLA4 activation have been described on cell lines, this represents the first evidence supporting a biological significance for this integrin property. p ositioning of developing thymocytes within the thymic cortex and medulla as well as the regulation of their ability to move from one region to another are crucial to T cell differentiation. Thus a stable interaction with cortical epithelial cells facihtating the recognition of MHC/peptide complexes is a prerequisite for the positive selection of immature double positive T cells (1). In fact, there is evidence that positive selection within the cortex is limited by contact with these selecting elements (2, 3) underscoring the importance of migratory events leading T cell progenitors to the epithehum. Subsequent events of maturation, including negative selection, appear to occur in the medulla involving bone marrow-derived antigen-presenting cells as well as a phenotypically distinct medullary epithelium (4). Implicit in these compartment-specific events is the programmed detachment of developing thymocytes from the cortical epithelium and the regulated movement of those committed to the mature single positive phenotype to the medulla. While much has been learned about the function of the T cell receptor (TCPQ and the costimulatory mole-cules, CD4 and CD8, these molecules cannot directly mediate stable cell-cell adhesion or migration. In contrast, the role of interactions mediated by integrin adhesion molecules expressed by thymocytes with extracellular matrix (ECM) 1 proteins such as fibronectin (FN) in thymocyte motihty, has not been elucidated.
Blood, 1998
T-cell precursors develop within the thymus in contact with multiple supportive elements, among which thymic epithelial cells (TEC) are known to exert a dominant role in their homing, survival, and functional differentiation. All these functions are supported by cell-cell contacts and cytokine release. Signaling events triggered in lymphoid cells by adhesion to TEC are well characterized, but little is known about the opposite phenomenon. To address this issue, we derived cultures of TEC from human normal thymus. TEC monolayers were cocultured with thymocytes and immunostained with monoclonal antibodies (MoAbs) to integrin (2, 3, 4, and 6) and β (β1 and β4) chains. Optical and confocal analysis showed that integrins were polarized on TEC at discrete surface locations: 6β4 lined the basal surface of TEC monolayers, whereas 3β1 was found mostly at TEC-TEC contacts; it is noteworthy that both 3β1 and 6β4 became highly enriched also at the boundaries with adherent thymocytes. ...
Differing roles for B7 and intercellular adhesion molecule-1 in negative selection of thymocytes
Journal of Experimental Medicine, 1996
To ensure self tolerance, immature thymocytes with high binding affinity for self peptides linked to major histocompatibility complex (MHC) molecules are eliminated in situ via apoptosis (negative selection). The roles of two costimulatory molecules, B7-1 and intercellular adhesion molecule-1 (ICAM-1), in negative selection was examined by studying apoptosis of T cell receptor transgenic CD4+8+ thymocytes cultured with specific peptides presented by MHC class I-transfected Drosophila cells. When coexpressed on these cells, B7-1 and ICAM-1 act synergistically and cause strong class 1-restricted negative selection of thymocytes. When expressed separately, however, B7-1 and ICAM-1 display opposite functions: negative selection is augmented by B7-1, but is inhibited by ICAM-1. It is notable that B7-1 is expressed selectively in the thymic medulla, whereas ICAM-1 is expressed throughout the thymus. Because of this distribution, the differing functions of B7-1 and ICAM-1 may dictate the s...
A Novel Integrin Involved in Thymocyte-Thymic
Summary Thymocytes differentiate in the thymic microenvironment into immunocompetent T cell through the interaction with a variety of accessory cells, including thymic epithelial cells (TEC) . TEC play an important role in the selection process presenting self antigens in association with major histocompatibilitycomplex(MHC) molecules to the maturingT cells . TheT cell receptor recognizes the selfantigen-MHC complex, but other surface molecules help stabilize this interaction. Thus, the CD2/LFA3 and LFA-1/intercellular adhesion molecule 1 pairs have been shown to participate in the binding between lymphoid cells and TEC. Here we describe an integrin of the very late activation antigen subfamily composed by the known Nt chain and by a novel ci chain. This adhesion molecule is expressed on the surface of medullary TEC and is involved in the adhesion between TEC and thymocytes, but not peripheral blood T lymphocytes.