The Study On The Induction Of Immunotolerance To Naive T Cell Derived From CD34～+ Cell By MSCs In Vitro

Objection: Graft-versus-host disease (GVHD) is the major complication after allogeneic Bone marrow transplantation (BMT) that affect prognosis. The conditioning of the recipient results in damage of host tissues and secretion of inflammatory cytokines (such as IFN-r IL-2) ,which initiated GVHD .This indicates a central role of cytokines in the immunopathophysiology of acute GVHD. BM -derived mesenchymal stem cells (MSCs) have not only the multipotential differentiation ability ,but also appear to facilitate posttransplantation hematopoietic recovery and play an important role in the immunoregulation , However ,the characteristics and the mechanisms of this inhibition are hardly known to us. The newly generated T cells that haven't contact with any antigen after selection in the thymus is the real Naive T cells. Antigen presenting cell (APC ) will present the antigen to Naive T cells particularly ,thus donor derived Naive T cells is the initiate cells of GVHD. Our study was designed to explore the mechanism of MSCs' immunoloregulation more directly by co-culture of allogeneic MSCs with Naive T cells differentiated from cord blood CD34+ cells in vitro, and observe the effect of MSCs on cytokine production by Naive T cells.Method : Mononuclear cells (MNC)were extracted from heparinized bone marrow sample by density gradient centrifugation over a 1.073 g/ml percoll , remove the nonadherent cells after 48h by medium change. After 3 passages, the irradiated MSCs were used as feeder layer. CD34+ cells isolated from cord blood by sequential immunomagnetic bead selection were cultivated with TSC supplemented with IL-12, FL and IL-2. The suspended cells were harvested by gently aspiration andassayed for phenotype by flow cytometry after 5 weeks. After Co-culture of MSCs and Naive T cells for 7 days , variance of cytokine produced by Naive T cells in supernatant were analyzed by emzyme-linked immunoassays.At the same time ,variety of T cell phenotype were determined by flow cytometry.Result: Human bone marrow-derived MSCs were culture-expanded successfully . Some congeneous phenotypes expressed on most third passages cells were found by flow cytometric: CD166+ ,CD105+ CD14- and CD34- CD45- . The immature hematopoietic progenitor cells(CD34+ lineage-)seeded on TSC undergo T lymphopoiesis in a manner that recapitulates T cells ontogeny in vivo. The phenotypes of suspended cells after 5 weeks were assayed as :CD4+ 65.2%, CD8+ 52.7%, CD3+78.8%, CD45RA 88.9% . On the 7th day of co-culture, we found a mild proliferation of T cells after co-culture with MSCs under microscop. A reduced IFN- r production (p<0.05) and increased IL-2 (p<0.05) were found in supernatant by ELISA, but neither IL-4 nor IL-10 were detected. However ,CD8+ cell augment were found after 7 days cultureConclusions : This study report the immunogenicity of MSCs .We show here that MSCs can elicit allogeneic response by comparing the cells count before and after co-culture with Naive T cells , We also found a reduced IFN- Y production (p<0.05) and increased IL-2 (p<0.05) in the supernatant. So we deduce that MSCs may exert Immunomodulatory affected by proliferation of CD8+ T reg cells ,which inhibit the production of cytokine by alloreactive T cells that lead to GVHD. The result may provide new clues to explain immunoregulatory mechanism of MSCs.