Abstract
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.
Highlights
The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation
We transfected into the human thymic epithelial cells (TEC) line a CD49e-small interference RNA (siRNA) that is complementary to the CD49e gene or a control scrambled siRNA, both conjugated to alexa-488
To determine whether expression of the CD49e integrin subunit was suppressed on the cell surface of TEC line following transfection with CD49e-siRNA cells were stained with a monoclonal CD49e-specific antibody conjugated to phycoeritrin
Summary
The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation, eventually leading to the migration of positively selected thymocytes to the T-dependent areas of peripheral lymphoid organs. This differentiation process involves sequential expression of a variety of membrane proteins and rearrangements in T-cell receptor genes. Most potentially self-reactive thymocytes are negatively selected by clonal deletion, whereas some are rescued from death through positive selection, eventually yielding the vast majority of the Tcell repertoire [1] In both positive and negative selection events, cell-cell adhesion between developing thymocytes and non-lymphoid microenvironmental cells of the organ is mandatory [1]. It is possible that supramolecular ECM arrangements function as a conveyor belt, allowing an ordered migration of thymocytes within the organ [5]
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