Panel B reveals that there are no changes in the expression of CD29 (1 integrin subunit) mRNA, as determined by real-time quantitative PCR 48 hours after transfection. cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is Gallic Acid 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 51 (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 Gallic Acid 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. Conclusion Conceptually, 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. Background 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 MDS1 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 T-cell 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]. The thymic microenvironment which is tridimensional network composed of non-lymphoid cells including thymic epithelial cells (TEC) C the most conspicuous cellular elements C dendritic cells, macrophages and, to a lesser extent fibroblasts, as well as extracellular matrix (ECM) [2,3]. Developing thymocytes interact with thymic microenvironment in a defined spatial order. This is evident from the different localization of the individual stages of thymocyte development. The most immature, CD4-CD8- double negative (DN) thymocytes are found beneath the subcapsular epithelium of the thymic lobules, while the more mature, CD4+CD8+ double positive (DP) stages can be detected throughout the cortical region. The more differentiated CD4+ or CD8+ single positive (SP) thymocytes are mainly found in the medulla [2]. There is evidence that extracellular matrix (ECM) molecules play a fundamental role in localizing the different thymocyte stages in the thymus [4]. Interestingly, it is possible that supramolecular ECM arrangements function as a conveyor belt, allowing an ordered migration of thymocytes within the organ [5]. Fibronectin (FN) is one ECM ligand constitutively expressed in all mammalian thymuses so far analyzed, including mice and humans [6-8]. Particularly in the human thymus both FN isoforms were detected within the thymic lobules [9]. Two integrin-type fibronectin Gallic Acid specific receptors are consistently expressed by developing thymocytes: VLA-5 (51, CD49e/CD29), which recognizes the RGD-containing fibronectin binding site and VLA-4 (41, CD49d/CD29) that identify the CS1 segment of fibronectin, derived by.