| Objectives: To analyze and confirm the contribution of TGF-β1 on the function of CD4+CD25+ regulatory T cells (Tregs) in vitro. Furthermore,the important purpose of this study is to identify and compare in vivo suppressive effect of CD4+CD25+Tregs generated in vitro through TGF-β1 on reversing and preventing murine lupus-like syndrome of chronic graft-versus-host disease (cGVHD). Therefore, a better understanding of signals for the generation and development of CD4+CD25+Tregs will probably result in a new immunotherapy for systemic lupus erythematosus.Methods: CD4+CD25-T and CD4+CD25+T cells of BALB/c murine were separated with MACS and the purity was analyzed by FCM. First of all, we set up different cell culture systems. Total RNA was prepared from cultured cells with Trizol reagent and reverse transcribed into cDNA. And Foxp3 expression was detected by PCR. Also freshly isolated CD4+CD25+Tregs, in vitro-expanded CD4+CD25+Tregs (eTregs) and in vitro -induced CD4+CD25+Tregs (iTregs) conditioned with TGF-β1 were assessed by their abilities to inhibit the proliferation of CD4+CD25-T cell, CPM (cycles per minute) value was detected. Furthermore, we generated murine lupus-like syndrome of cGVHD by parental lymphocyte engraftment, and CB6F1 recipients were monitored for clinical signs by the following parameters: weight of body, red blood cell, white blood cell and platelet, serum anti-dsDNA and ANA autoantibodies measured by ELISA, proteinuria detected by albustix, pathologic changes of kidney tested through light microscope and electron microscope. To assess the reverse effect of different Tregs on murine lupus-like syndrome of cGVHD, freshly isolated CD4+CD25+Tregs, eTregs and iTregs through TGF-β1 were injected into CB6F1 mice after 2 wk of disease induction. And to analyze the preventive effect in vivo, different doses of iTregs were injected on 1 day before disease induction. Statistical analysis was performed by Mann-Whitney rank sum test or Student t test or ANOVA test depending on the dataset using SPSS 11.5 software. GraphPad Prism 4.0 software was used to analyze survival data, survival fractions were calculated by Kaplan-Meier method and compared by log rank test.Results: The purity of BALB/c CD4+CD25-T and CD4+CD25+T cells were >98%. Freshly isolated CD4+CD25+T cells were unresponsive to anti-CD3 mAb and syngeneic antigen-presenting cells (irradiated C57BL/6 splenic cells), but it could expand up to 4- to 5-fold if IL-2 was added to the culture system. And adding IL-2 and TGF-β1 would lead to only 1- to 2-fold expansion on cells. Compared with the culture system including freshly isolated CD4+CD25-T cells, anti-CD3 mAb and syngeneic APCs, the expansion of cells was 1- to 2-fold in the culture system added with IL-2, but it was only 30% to 40%-fold in the culture system conditioned with IL-2 and TGF-β1. As to the level of Foxp3, freshly isolated CD4+CD25-T cells expressed little Foxp3, as well as CD4+CD25-T cells cultured with anti-CD3 mAb, syngeneic APCs and IL-2 in the absence of TGF-β1. When TGF-β1 was added to the culture protocol above, it led to a significant elevated Foxp3 expression. When these iTregs were re-stimulated with anti-CD3 mAb and APCs, this population did not proliferate in response to the stimulators, which was similar to freshly isolated CD4+CD25+Tregs. The dose-response analysis showed that CD25 and Foxp3 expression were rising with the increase of TGF-β1. And 1 ng/ml TGF-β1 was the most appropriated dose. Freshly isolated CD4+CD25+Tregs, eTregs and iTregs had visible effect on inhibiting the proliferation of CD4+CD25-T cells in vitro. iTregs and responder cells mixed with a different ratio (1:1, 1:2, 1:5, 1:10 or 1:20), could suppress the alloresponse markedly. Furthermore, the addition of graded numbers of iTregs resulted in a positive dose-dependent manner. When anti-TGF-β1 was added to the system of mixed lymphocyte reaction, it would partially block the Foxp3 expression and might impair the suppressive function of iTregs. After parental lymphocyte engraftment, proteinuria, weight loss, reduced RBC (red blood cell), WBC (white blood cell) and PLT (platelet), anti-dsDNA and ANA autoantibodies, glomerulonephritis occured on CB6F1 mice. Our results showed that in vitro-expanded and -induced Tregs by TGF-β1 could retroconverse the morbidity of cGVHD with lupus-like syndrome mice which have already developed autoantibody. And iTregs had more efficient inhibition than that of freshly isolated or eTregs in reversing the clinical symptom of the diseased mice. When comparing the protective effect among different groups which were injected with 25×106 or 5×106 or 1×106 iTregs respectively, the difference was distinguished. And the best inhibition on autoimmunity was mediated by 25 million iTregs, and there was no pathologic effect in this group.Conclusions: We established an essential correlation between TGF-β1 and CD4+CD25+Tregs'activation, expansion, as well as induction from precursor cells. It indicates that TGF-β1 signaling is required to maintain the suppressive effect of CD4+CD25+Tregs in vitro and in vivo. Importantly, iTregs have more inhibitive function on reversing an established disease and preventing the onset of lupus-like syndrome of cGVHD. In a word, the present study raises a possibility that CD4+CD25+Tregs induced by TGF-β1 in vitro have the potential that is used as an immunotherapy agent to control SLE.
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