Small interference ITGA6 gene targeting in the human thymic epithelium differentially regulates the expression of immunological synapse-related genes

The thymus supports differentiation of T cell precursors. This process requires relocation of developing thymocytes throughout multiple microenvironments of the organ, mainly with thymic epithelial cells (TEC), which control intrathymic T cell differentiation influencing the formation and maintenance of the immunological synapse. In addition to the proteins of the major histocompatibility complex (MHC), this structure is supported by several adhesion molecules. During the process of thymopoiesis, we previously showed that laminin-mediated interactions are involved in the entrance of T-cell precursors into the thymus, as well as migration of differentiating thymocytes within the organ. Using small interference RNA strategy, we knocked-down the ITGA6 gene (which encodes the CD49f integrin α-chain) in cultured human TEC, generating a decrease in the expression of the corresponding CD49f subunit, in addition to modulation in several other genes related to cell adhesion and migration. Thymocyte adhesion to TEC was significantly impaired, comprising both immature and mature thymocyte subsets. Moreover, we found a modulation of the MHC, with a decrease in membrane expression of HLA-ABC, in contrast with increase in the expression of HLA-DR. Interestingly, the knockdown of the B2M gene (encoding the β-2 microglobulin of the HLA-ABC complex) increased CD49f expression levels, thus unraveling the existence of a cross-talk event in the reciprocal control of CD49f and HLA-ABC. Our data suggest that the expression levels of CD49f may be relevant in the general control of MHC expression by TEC and consequently the corresponding synapse with developing thymocytes mediated by the T-cell receptor.     

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.