Mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation in vitro and in vivo. One of the ways by which MSCs may modulate immune responses is by the induction of CD4+CD25+FOXP3+ regulatory T cells (Treg), which suppress alloreactivity in vitro and prevent or attenuate GVHD in animal models. TGF-b (TGFB1 gene), one of the factors secreted by MSC, is known to induce the expression of FOXP3 and to drive the generation of Tregs from CD4+CD25− T cells. T cell activation is characterized by the expression of many surface molecules, and CD69 is one of the earliest markers, transiently expressed following activation. CD69 is selectively expressed at sites of chronic inflammation and recent in vivo and in vitro results indicate that this receptor may modulate the inflammatory response, by inducing TGF-b production. Despite the importance of secreted factors, cell to cell contacts promote increased lymphocyte immune modulation. Interestingly, TGF-b is known to induce the expression of b-IG-H3 (TGFBI gene), a secreted extracellular matrix adaptor protein, whose expression is higher on hematopoietic stem cells adherent to MSC. Given the potential role of CD69 as a regulatory molecule, we explored the effects of MSC on the expression of CD69, TGFB1, FOXP3 and TGFBI on co-cultured T-cells. Peripheral blood mononuclear cells (PBMC) from 6 individuals were co-activated by anti-CD3/CD28 beads and cultured either in the presence or in the absence of MSC (5:1) adhered to the bottom of culture wells. IL2 (20U/ml) was added in the 3rd day for full activation. Cultured PBMC were collected 1, 3 and 5 days after activation. Percentage of CD69+ cells and proliferation of activated lymphocytes (cell division tracking by CFSE) were evaluated by flow cytometry on gated CD3+ lymphocytes. Transcripts levels of TGFB1, TGFBI, FOXP3 and IL10 were determined by real time PCR and normalized using ACTB as an endogenous control. Relative expression levels were calculated in comparison with activated PBMC cultivated alone at the 5th day. Proliferation of lymphocytes co-cultured with MSC was significantly inhibited. As expected, activation of lymphocytes cultured alone was evidenced by the expression of CD69 in 5% of cells in the first day (mean percentage), followed by a decrease in the subsequent days (4% and 3%, respectively). Interestingly, lymphocytes co-cultured with MSC displayed a completely different pattern, with a similar initial activation (7%) which was followed by significant increase in the 3rd day (16%), maintained in the 5th day (14%). Furthermore, TGFBI and IL10 were both expressed at significantly higher levels on PBMC co-cultured with MSC, compared to PBMC alone, in all days evaluated. In addition, their transcript levels decreased faster on PBMC cultured alone. In the other hand, TGFB1 and FOXP3 levels in PBMC cultured alone or in the presence of MSC were similarly higher in the 1st day, but decreased till the 5th day, when their levels were slightly, but significantly higher on PBMC co-cultured with MSC, compared to PBMC cultured alone. We demonstrate for the first time that co-culture with MSC causes a sustained increase of the CD69 marker on CD3+ lymphocytes, which is accompanied by increased levels of TGFB1, TGFBI, FOXP3 and IL10 on total PBMC. Our results are in line with the proposed immunoregulatory role of CD69. In addition, higher TGFBI levels on PBMC may increase lymphocyte adherence to MSC, thus favoring immune modulation. This work was supported by FINEP, CNPq and FAPESP.

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