The Inhibition Mechanism Of T Lymphocyte Immune Reaction By Human Bone Marrow-Derived Mesenchymal Stem Cells

Objectives: By dynamic analyzing the effect of human bone marrow-derived mesenchymal stem cells (MSCs) on allogeneic T lymphocytes for intracellular cytokine mRNA expression of interleukin-2(IL-2), interferon-γ(IFN-γ), IL-4 and IL-10, we further studied the inhibition mechanism of T lymphocyte immune reaction mediated by MSCs, so as to provide experimental data for clinical applications such as autoimmune diseases and graft-versus-host disease(GVHD) management. Methods: Human bone marrow-derived MSCs were isolated and cultured for proliferating to passage cells, and the purity of MSCs was identified with the spindle-fibrobla- stic morphology characterized by microphotograph and the phenotypes tested by flow cytometry. The stromal feeder layers of 1×10~5 per well of MSCs dealed with mitomycin were cocultured with allogeneic T lymphocytes isolated from peripheral blood by Ficoll Hypaque and nylon cotton column. At 12 hours, 24 housr, 48 hours, 72 hours, 96 hours and 5 days, respectively, T lymphocytes were collected and RNA was extracted, then cDNA was gained by reverse transcription method, so IL-2, IFN-γ, IL-4 and IL-10 mRNA levels in T cells were detected by Fluorescent Quantitative PCR(FQ-PCR), thus we could observe the cytokines changing tendency and determine the detection time for further experiment. Subsequently 1×10~3, 1×10~4 or 1×10~5 per well of MSCs were respectively cocultured with 1×10~6 per well of allogeneic T cells for group A, and were cocultured with mixed lymphocyte reaction system composing of 1×10~6 of peripheral blood mononuclear cells and 1×10~6 of T cells per well for group B, and then the mRNA levels of cytokines IL-2, IFN-γ, IL-4 and IL-10 were measured by FQ-PCR at 72 hours. Finally, 25%, 50% or 75% concentrations of the supernatant of MSCs were added to PHA-activated allogeneic T cells for group C, and the mRNA levels of the same cytokines were analyzed by FQ-PCR after cocultured for 72 hours.Results: Mononuclear cells isolated from bone marrow were inoculated to the flasks. Fibroblastic-like cells grew slowly within 2 to 4 days, then proliferated into fibroblast colony quickly and uniformed morphology characterization arranging in whorl or parallel. The cells reached 95% of confluence in 2 weeks. The passage cells had the same morphology feature with the primary and grew quickly. The growth curve of passage cells showed the cells reached to logarithm growth period at 3rd day, reached to peak at 6th day and then to platform period. According to the flow cytometry assay, MSCs at third passage were uniformly positive for CD166, CD105, CD29 and CD13, while negative for CD34, CD45 and HLA-DR. The purities of MSCs were above 95%. The inductions of mRNA levels of IL-2, IFN-γ, IL-4 and IL-10 were stable on the whole at 72 hour in our MLC systems that the levels of IL-2, L-4 and IL-10 increased while IFN-γ reduced (P<0.05, respectively) by quantitave of MSCs. In group A, MSCs could restrain the expression of IL-2 and IFN-γ levels but promote the levels of IL-4 and IL-10 compared with control group (P<0.05, respectively), all of which expressed in a dose dependent way(P<0.05). In group B, the levels of IL-2, IL-4 and IL-10 increased(P<0.05), while IFN-γ decreased(P<0.05) in each cocultured group with MSCs compared with control group (F<0.05, respectively), moreover IFN-γ and IL-10 production changed in a dose dependent way(P<0.05), however it did not occur in IL-2 and IL-4 (P>0.05) . In group C, different concentrations of MSCs supernatant could supress the expression of IL-2 and IFN-γ but promote the expression of IL-4 compared with control group (P<0.05, respectively) , and all of the effects depended on concentrations (P<0.05); on the other hand, the effection on IL-10 expression was not significant(P>0.05).Conclusions: ①MSCs can regulate the balance between T helper 1 (Th1) and T helper 2 (Th2) subsets, which may require cell-to-cell contact and soluble factors produced by Th1 and Th2. ②MSC-induced suppression is a complex mechanism that affecting cytokines expression of Th1 and Th2 and may function differently depending on distinct stimuli that MSCs could inhibit the intracellular cytokines expression of Th1 so as to enhance Th2 expression in PHA-activating group, while the increase of IL-2 expression suggests the proliferating state of T cells stimulated by allogeneic antigen. ③The characteristics of immune regulation of MSCs would provide some experimental data for potential clinical treatment in allograft rejection, GVHD and autoimmune disease.