The Effect of Mesenchymal Stem Cells on Preventing Graft-Versus-Host Disease After Allogeneic Hematopoietic Stem Cell Transplantation—the Effect of Mesenchymal Stem Cells on Active T Lymphocytes(in vitro)
Authors: Qiao Zhenhua , Ye Fang , Yang Tao , Zhang Li , Jia Ning 
Published: October 2019

Abstract
Abstract 5182

Objective
To study the immune regulatory effects of human bone marrow Mesenchymal stem cells(MSCs) on active T lymphocytes in vitro and to explore the new strategy to prevent Graft-Versus-Host Disease(GVHD) in allogeneic hematopoietic stem cell transplantation(allo-HSCT).

Methods
1. Mononuclear cells from human peripheral blood cells were isolated and cultured in the presence of phytohemagglutinin (PHA) (final concentration was 10μ g/ml) for different time (24hours, 36hours, 48hours, 72hours). 2. The ability of T lymphocyte proliferation and activation was measured by 3H-Thydimine incorporation. 3. MSCs from human bone marrow cells were isolated and cultured.The purity of MSCs were identified with morphological characterization by microphotograph and the phenotypes were tested by flow cytometry (FCM). 4.MSCs were planted in 6-well plates (2×104/well for group B,4×104/well for group C and 8×104/well for group D) and cocultured for 72 hours with T cells activated as the control group (group A). CD3+CD4+, CD3+CD8+, CD4+CD25+ and CD4+CD152+ expressed on T cells were analyzed by FCM after coculture with MSCs for 72 hours respectively.T lymphocyte proliferation after cocultured with MSCs was evaluated by 3H-Thydimine incorporation. The expressions of IL-2, sIL-2R and TGF-β1 proteins in these four groups was detected by ELISA.

Results
1. The ability of T lymphocyte proliferation in the same PHA concentration increased with the time changing. Its ability was strongest when culturing for 48 hours (P<0.01)and had no difference between 48 hours and 72 hours (P>0.05). 2. The phenotypes of MSCs were measured by FCM:they were positive on the expression of CD44, CD105, CD29 and FIK1 and negtive on the expression of CD33, CD34, CD45 and HLA-DR. 3. MSCs inhibited T lymphocyte proliferation and the inhibitory effect depended on the amount of MSCs:CD3+CD8+, CD4+CD25+ and CD4+CD152+ T cells cocultured with bone marrow MSCs (group B, group C, group D) increased obviously and a significant decrease of CD3+CD4+ T cell proliferation as compared with control group (T lymphocytes uncocultured with MSCs) (P<0.01), and there were no difference between group C and group D (P>0.05). In group B, C and D, IL-2 and sIL-2R levels were lower than control group (P<0.01) and TGF-β1 level was higher obviously than group A (P<0.01). Every of IL-2, sIL-2R and TGF-β1 expressions has difference during group B, C and D (P<0.05).

Conclusions
1. Mitogen PHA could increase proliferation function of T lymphocytes and this function was strongest when cultured for 48 hours in same concentration of PHA. 2. MSCs inhibit T lymphocyte proliferation and perform their immunosuppressive function by up-regulation of CD3+CD8+ T cells, CD4+CD25+ Tcells and CD4+CD152+ Tcells. MSCs decreased IL-2 and sIL-2R levels and increased TGF-β1 level in PHA-stimulated peripheral blood lymphocytes. 3. MSCs play a critical immune negative regulatory effect on preventing GVHD. MSCs pretreatment may be useful in the prevention of GVHD in allo-HSCT and provides a new strategy to induce transplantation tolerance.

Disclosures:
No relevant conflicts of interest to declare.