| Objective:To develop a new method to induce immune tolerance. In this study we transfected recipient rat (Lewis) bone marrow-derived immature dendritic cells (imDC) by recombinant adenovirus-mediated IKK2dn (Adv-IKK2dn), by which we induced CD4~+CD25-T cells, and then we studied the influence of CD4~+CD25-T cells to the immune reaction and analysised the changes of cytokines. Thus try to explain the characteristics and mechanisms of the immune tolerance mediated by these CD4~+CD25-T cells.Methods:This study was divided into two parts. In the first part, we generated recipient-derived imDC in vitro, IKK2dn was transfected into imDC to maintain their immature state, by which we induced CD4~+CD25-T cells. In the second part, the CD4~+CD25-T cells were separated by MACS, then we used the primal mixed lymphocyte reaction (MLR) to detect the influence of CD4~+CD25-T cells to immune reaction in MLR. Detailed experimental methods as follows:(1). Bone marrow-derived mononuclear cell, which were obtained from the tibias and femurs of Lewis rats, were induced into imDC in 6-well plates with recombinant rat granulocyte macrophage-colony stimulating factor (GM-CSF) (5ng/ml) and recombinant rat IL-4(5ng/ml). Collected cells on the fifth day. (2). Transfected recombinant adenovirus-0 (Adv-0) and recombinant adenovirus-mediated IKK2dn (Adv-IKK2dn) into rat bone marrow-derived imDC, then we obtained the Adv0-DC and IKK2dn-DC, after that the expressions of IKK2dn gene were detected by using western blotting analysis. After that, loaded with donor antigens. (3). The imDC transfected by AdV-IKK2dn and loaded with BN antigen were cultured with recipient-derived T cells. The productions of CD4~+CD25-T cells were analyzed with flowcytometry. (4). The CD4~+CD25-T cells were separated by using magnetic cell sorting system(negative selection and positive selection). (5). Five groups were involved in this study: negative control, positive control, Adv0-CD4~+T cells group (CD4~+T cells induced by Adv0-DC), IKK2dn-T cells group (CD4~+CD25-T cells induced by IKK2dn-DC). The donor spleen cells and third-party spleen cells were served as stimulate cells, recipient-derived T cells were served as reaction cells. The capacity of IKK2dn-T cells in adjusting the T cell proliferation response stimulated by allogeneic antigens was observed by MTT. And the levels of IL-2, IL-10, interferon-γ(IFN-γ) and TGF-βin the supernatants was detected by ELISA.Results:(1). In our experiments, we obtained enough number of lewis rat imDC with high purity. 3.0×107 cells could be extracted form each rat, the cell vitality detection was more than 95%. The imDC presented a growth state of semi-suspension with the characteristic of a few irregular branches on the surface and larger volume. Under the Transmission Electron Microscopy, we found the cells were round, rich in cytoplasm and organelles, less protuberance on the surface, which was in accordance with the characteristics of imDC. The imDC expressed a lower levels of CD80, CD86 and MHC-II molecule (19.1±2.15%, 17.0±1.60% and 27.0±2.21%, respectively), which were consistent with the results of our early studies. On the 14th day, the expressions of CD86 and MHC-II were 56.0±3.40% and 66.5±1.40%, compared with those on the 5th day,the difference was statistically significant (P all <0.05).(2). The green fluorescedce could be detected on the surface of imDC under the inverted fluorescence microscope after transfected by AdV-IKK2dn. We detected the molecular weight of 87kDa strips by using Western blot analysis, suggesting that imDC were transfected successfully. In contrast to untransfected imDC and Adv0-DC, the primal mixed lymphocyte responses results showed that allogeneic T cells proliferation induced by IKK2dn-DC was obviously low(P<0.01). (3). In contrast to untransfected imDC (control group) and Adv0-DC, recipient -derived imDC transfected by IKK2dn and loaded with donor splenocyte lysate could induce lewis rat T lymphocyte into CD4~+CD25-T cells. The expressions of CD25 in CD4~+T cells induced by IKK2dn-DC were low (18.5±1.40%), compared with those of the control group (72.2±3.30%) and the Adv0-DC group (63.7±1.80%), the difference were statistically significant (P all <0.05).(4). CD4~+CD25-T cells induced by recipient imDC transfected by IKK2dn gene and Loaded with donor antigens could inhibit recipient T cells proliferation caused by the stimulation of allogeneic antigens. At the higher ratio(CD4~+CD25-T/MLR), CD4~+CD25-T cells could not only suppress the proliferative response of recipient T cells toward BN antigens, but also reduce the proliferation of recipient T cells against third party stimulators. But at the lower ratio(CD4~+CD25-T/MLR), the inhibition had antigen specificity, the CD4~+CD25-T cells only suppressed the proliferative response toward BN antigens. In contrast to Adv0-CD4~+T cells group and the control group, there was a stronger secretion of IL-10 and TGF-β(P<0.05) and a weaker secretion of IL-2 and IFN-γ(P<0.05) in the supernatants in MLR.Conclusions:(1). Recipient-derived imDC transfected by IKK2dn and loaded with donor splenocyte lysate could induce lewis rat T lymphocyte into CD4~+CD25-T cells. Our in vitro studies found that this kind of T cells could inhibit T-cell response and induce immune tolerance.(2). The immune tolerance induced by CD4~+CD25-T cells has antigen specificity, this specificity is associated with the ratio of CD4~+CD25-T cells. The mechanism of this kind of immune tolerance may due to the secretion of high levels of cytokines IL-10, TGF-beta, and inhibiting T cells differentiating into activated T cells.(3). GM-CSF and IL-4, when with the concentration of 5ng/ml, can generate a large number of lewis rat immature dendritic cells with high purity, and this makes possible to induce immunological tolerance DC. (4). ImDC transfected by IKK2dn could restrain their maturation. ImDC transfected by IKK2dn and loaded with BN antigens can lower the allogeneic MLR and thus induce immune tolerance. |
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