| Summary: | 碩士 === 輔仁大學 === 生命科學系碩士班 === 95 === In utero hematopoietic stem cell transplantation (IUHSCT) was used as strategy to replace bone marrow transplantation without the need of toxic conditioning therapy. The major limitation of IUT to date is limited chimerism and lack tolerance to alloantigen. Dendritic cells (DCs) are considered very potent antigen-presenting cells for T cell activation, but strongly tolerogenic as precursor DC. The major problem on failure of transplantation is that implant is rejected by recipient. Rejection of implant is mediated by type I T helper cells of host. TGF-β1 and IL-10 are kind of suppression cytokines. Related researches have reported that both cytokines inhibited the Th1 cell activation and DC maturation. Herein, we firstly investigated the expression level of TGF-β1 and IL-10 in 13-14-day fetus. Then, we measured the effects of TGF-1 or TGF-1+IL-10 cultured donor derived DCs on allogenic T cell responses by in vitro mix lymphocyte reaction assay (MLR). Furthermore, by using a MHC-mismatched mouse model, we examine the effects of TGF-1 and TGF-1+IL-10 DCs on in utero mice bone marrow transplantation. Results indicated that 13~15 days of pregnancy fetus posses the normal expression of TGF- receptor (40%) and IL-10 receptor (3.2%) on the surface of immune cells and production of TGF-1 cytokine (120pg/ml). Furthermore, 13-15 days of fetus exhibited the proliferate capability in response to mitogen stimulation. In this study, GM-CSF-derived bone marrow CD11c positive cells with expression of CD40 low, CD80 low, CD86 low, B7-H1 hi, and B7-DC med were acquired and used as a source of dendritic cells . Those cells had shown a weak phagocytic activity and can be activated by LPS stimulation. After culturing with immune suppressive cytokine, TGF-1 cultured DCs showed less expression on co-stimulatory molecules CD40, CD80, CD86, B7-DC and MHC class II, while TGF-1+IL-10 cultured DC showed less CD40, CD86 and B7-DC expression. By using mix lymphocyte assay, TGF-1 and TGF-1+IL-10 treated DCs inhibited the allogenic T cell proliferation through decreased CD28 expression and increased PD-1 on T cells. In addition, small numbers of CD4+CD25+ Foxp3 T cells were induced in co-cultured TGF-1 and TGF-1+IL-10 treated DCs with allogenic T cells. Further analysis of T cell cytokine production in cultures of MLR had showed that both TGF-1 and TGF-1+IL-10 treated DCs decreased the production of IL-2, IL-4, IL-10, and IFN-however, the production of TNF- was increased in former and production of TGF-1 was increased in later, respectively. In animal experiment, co-injected immune suppressive cytokine treated-DCs with donor bone marrow cells had increased engraftment of donor cells, but did not increase the number of donor cells in fetus recipients. In conclusion, TGF-1 or TGF-1+IL-10 conditioned DCs have induced the negative regulation in response to allogenic T cells. Although this negative regulation increased the engraftment of donor cells in fetal recipients, but the percentage of donor cells in recipients was not improved in this application.
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