Study Of Applying Transfecting Immature Dendritic Cells With FOXP3 Gene Hyper-expression Gland Virus To Immunotolerance Of Experimental Autoimmune Encephalomyelitis

Objective: Multiple sclerosis (MS) is a T-cell-caused autoimmune disease chracterised by leuko-demyelinative process in the central nerve system. Experimental autoimmune encephalomyelitis (EAE) is a typical animal model for MS. Currently, its cause and pathogenesis are not clear yet. Considerable research has shown that the occurrence of MS and EAE is linked to the abnormal activation of T-cells, especially to CD4+T cells which play a key role in the pathogenesis of MS. An established pathogenesis is that the imbalance of Th1/Th2 can lead to MS. Th1 can worse MS by secreting such factors as IL-2, IL-12 and IFN-γ, while Th2 can relieve MS by secreting factors, such as IL-4, IL-5 and IL-10. Recently a new theory has been advanced that the imbalance of Th17/Treg can lead to MS. Th17 can worse inflammation by secreting IL-17, while Treg, regulatory T-cells, can suppress immune reaction and relieve inflammation. Therefore, research on the immunotolerance and its induction has become one of the current keys in the research into MS immunity.Dendritic cells (DC) have been the only antigen presenting cells that can stimulate the activation and proliferation of initial T-cells up to now. They are the initiator for specific immunoresponse and play a key role in the pathological immunoresponse of MS. Recent researches have discovered that DC serve as both immunoresponse and immunotolerance in the immunoregulation, and thus advancing the idea of tolerogenic dendritic cells (tDC). That is, when DC present antigen to T-cells, there may be two results: immunostimulation (immunogenicity) or immunosuppression (immunotolero- genicity). By their maturity level, DC are divided into immature DC (imDC) and mature DC (mDC), with different forms, cell phenotypes and functions. By modifying the cell surface molecules and the secretory cell factors, DC can regulate the differentiation of T-cells with antigen specificity and excite different immunoresponses. Different types of DC have different features: imDC can induce immunotolerance; mDC are highly able to present antigen and stimulate the activation of initial T-cells; genetically-modified DC and tDC can be used to treat autoimmune diseases, e.g. MS.Foxp3, also known as forkhead/winged helix transcription factor, is a FOXP3+Treg specific transcription factor,playing an important part in regulating Treg development and functioning. To change FOXP3 gene transfection cells into regulatory cells will make it possible to treat MS with immunotherapy. Indoleamine 2,3-xygenase,IDO, is a restriction enzyme in tryptophan catabolism. It can form low tryptophan environment in certain areas. The expression of IDO in DC is involved in the immunotolerance, when it induces the imcompetence of T-cells, inhibits the proliferation of them and promotes the apoptosis of them with tryptophan exhaustion and toxic metabolites. Recently it has been found that IDO can induce the production of Treg,and thus immunotolerance. They promote each other: IDO can induce the production of new Treg,and in turn, Treg can induce IDO expression. Tregs, by combining cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) on their surface with B7-1/B7-2 on DC surface,excite the high expression of IDO in DC.By studying how to apply FOXP3 gene hyper-expression gland virus to transfect imDC,and observing the biological features, phenotypes and the expression and secretory change of the transfected imDC(i.e. DC.FOXP3), and by observing the effect of DC.FOXP3 on the spleen CD4+T of EAE mouse models in vitro, and counter-acting the IDO effect with 1-methyl tryptophan (1-MT), an IDO supppressant,I intend to explore the way in which DC.FOXP3 induce immunotolerance with a view to providing a new insight into the prevention and treatment of MS.Methods:1 prepare and determe mouse FOXP3 gene hyper-expression recombinant gland virus vectors.Use FOXP3 genes as templates, amplify target genes with PCR, and copy them into gland virus vectors, pAV-MCMV-GFP-3FLAG. Determine the sequence, package, purify and assay gland virus, and test its titer. Test the expressions of the prepared plasmid and of transfected 293 cells with gland virus, respectively, under fluoroscope, and observe the GFP expression. Examine the FOXP3 protein expression in the 293 cells, using Western blotting.2 transfect mouse imDC with FOXP3 gene hyper-expression gland virus and assay Extrinsically separate DC from mouse marrow, culture and purify DC with microbeads, observe cell forms under light microscope, scanning and transmission electron micrographs. Determine the expressions of CD11c, MHCII, CD80 and CD86 with Flow cytometry(FCM). Determine mixed lymphocyte reaction (MLR) with CCK-8. Transfect mouse bone marrow source imDC with FOXP3 gene recombinabt gland virus, observe the green fluorescence under an inverted fluoroscope. Determine the transfection efficiency. Using RT-PCR and Western blotting respectively,determine the mRNA and protein expressions of imDC transfected with FOXP3 genes.3 Determine the effect of FOXP3 gene hyper-expression gland virus transfection on the biological features and phenotypes of mouse imDC. Having transfected imDC with FOXP3 gene recombinant gland virus,observe the forms of transfected imDC under scanning and transmission electron micrographs. Determine the expressions of CD11c, MHCII, CD80 and CD86 with FCM. Using realtimePCR, determine the expressions of various cell factors in mRNA of the transfected imDC. Using ELISA, determine the cytokine secretion of DC.FOXP3.4 Transfect imDC with FOXP3 gene hyper-expression gland virus to induce CD4+T immunotolerance in EAE mice.Give subcutaneous MOG35-55 to mice to build EAE models. Prepare pathological sections to observe the changes in the brain and myel of the mice. Separate CD4+T cells from the spleens of EAE mouse models, culture them along with DC.FOXP3. Using CD4+T cell counting and CCK8, determine the competence of DC.FOXP3 to stimulate the proliferation of CD4+T cells. Using AnnexinⅤ-7AAD, determine the effect of DC.FOXP3 on the apoptosis of CD4+T cells. Culture the DC.FOXP3 group along with CD4+CD25-T cells. Obtain the percentage of CD4+CD25+FOXP3+Treg cells in CD4+T cells with FCM. Separate the CD4+T cells with microbeads. Using realtimePCR, determine the expressions of various cell factors in mRNA of the CD4+T cells. Using ELISA, determine the cell factors in supernate. Apply the 1-MT, then count the number of CD4+T cells and the number of the apoptotic cells. Determine the changes in the number of CD4+CD25+FOXP3+Treg cells, the mRNA expressions of various cell factors in CD4+T cells, and the changes of the cell factors in supernate.Results:1 preparing and determining the mouse FOXP3 gene hyper-expression recombinant gland virus vectors were done successfully. Using PCR, amplify FOXP3 gene fragments, and then clone them to pAV-MCMV-GFP-3FLAG, a gland virus vector. Examine their sequence, which corresponds to FOXP3 gene sequence of the Pubmed gene pool. Packaging and assaying recombinant gland virus was performed successfully. The titer of gland virus was 1.25X1011PFU/ml. The intensity of fluorescence was strong under the fluoroscope, and GFP expression was observed. In the transfected 293 cells that were determined by Western blotting, positive straps of Foxp3-GFP-Flag symphysic protein were visualised, from which FOXP3 protein expression was basically judged.2 The results of transfecting mouse imDC with FOXP3 gene hyper-expression gland virus and assaying①changes in DC forms:under light microscope, on Day 5, a small number of spines were seen in the cells that had been cultured and the number of colonies increased, which is normal for imDC. Under scanning electronic microscope, imDC cells took the shapes of circle and circloid, and cell surface was covered with many folds and few short, thin spines. mDC cells took diverse forms, with large numbers of folds and branch-like spines that were uneven in thickness and relatively long. Under transmission electron micrographs, imDC cells took regular forms with short and few spines on the surface and most nuclei going sideways. There were relatively many pinobubbles and lysosome,and modest numbers of mitochondria, endoplasmic reticulum and ribosome in the cytoplasm. Some C-shaped lysosomes lost the regular form. MDC cell form was irregular,with relatively long spines on the surface, relatively large cell body, irregular nucleus. There were reduced cyst-like structures and lysosomes in cytoplasm, and large numbers of mitochondria, endoplasmic reticulum, ribosome and Golgi's body.②FCM detected:Microbeads seperated imDC and mDC both reached up to CD11c,with a purity of being over 90%. In imDC, cell surface molecules were presented with low expressions: MHCII(27.2%), CD80(27.5%), CD86(29.5%) respectively ,while in mDC, cell surface molecules were presented with remarkably high expressions: MHCI(I99%), CD80(98.8%), CD86(98.4%).③in MLR, the competence of DC to stimulate the proliferation of allogene T-cells:both imDC and mDC groups were better able with the increasing of DC percentage. the competence of same-reaction ratio imDC group to stimulate the proliferation of T-cells was remarkably lower than that of mDC group, the difference being significant statistically.④under inverted fluoroscope, green fluorescence was seen in imDC of the transfected target gene AD-FOXP3 group (i.e. DC.FOXP3)and transfected empty adenovirus AD-GFP group(i.e. DC.GFP), but this did not happen in the group that had not been transfected(i.e. DCN), which shows that FOXP3 hyper-expression gland virus transfection was successful,with a transfection efficiency over 90%.⑤RT-PCR detected FOXP3 mRNA expression in DC.FOXP3; Western blotting detected FOXP3 protein expression in DC.FOXP3; but neither FOXP3 mRNA expression nor FOXP3 protein expression could be detected in DC.GFP and DCN.3 The results of determining the effect of FOXP3 gene hyper-expression gland virus transfection on the biological features and phenotypes of mouse imDC.①under scanning and transmission electron micrographs, the immature manifestations of imDC remained unchanged either in DC.FOXP3 and DC.GFP or in DCN.②FCM showed that AD-FOXP3 transfection had no effect on the CD11c, MHC-II, CD80, and CD86 molecule expressions on the imDC surface, which largely remained immature.③QPCR showed that, on the mRNA level,compared with DCN and DC.GFP, FOXP3 expression was high in DC.FOXP3. In DC.FOXP3 the expressions of such Th1 cell factors as IFN-γand such Th17 cell factors as IL23, IL17 were considerably reduced, but the expressions of such Th2 cell factors as IL-10 and IDO expression were remarkably heightened.④ELISA showed that, compared with the control group, in DC.FOXP3 the secretions of such imDC supernate Th1 cell factors as IFN-γand such Th17 cell factors as IL17 were considerably reduced, but such Th2 cell factors as IL-10 increased.4 The results of transfecting imDC with FOXP3 gene hyper-expression gland virus to induce CD4+T immunotolerance in EAE mice.①EAE mouse models were successfully made. Under the light microscope, pathological section observations showed that, with HE staining, there was a lot of inflammatory cytic infiltration in the cerebrum and in the spinal marrow of the neck and the waist in the EAE mice; that there were oversleeve-like changes around the blood vessels, mainly distributing in the leukopart around the brain cavity and the intralumbar intumescent areas, The control group saw no abnormality.②having cultured imDC along with EAE mouse CD4+T, compared with DCN and DC.GFP,DC.FOXP3 could inhibit the proliferation of CD4+T cells in the mouse spleens, which could be seen from the reduction of CD4+T cells and the lowered ability of DC.FOXP3 to stimulate CD4+T proliferation by CCK-8. AnnexinⅤ-7AAD showed that DC.FOXP3 could speed the apoptosis of CD4+T cells.③having cultured imDC along with CD4+CD25-T cells, FCM detected that DC.FOXP3 could induce the conversion of CD4+CD25-T to CD4+CD25+FOXP3+Treg.④QPCR detected the mRNA expressions of various cell factors in the CD4+T cells in the co-culture system, which showed that: DC.FOXP3 inhibited the mRNA expressions of the Th1 transcripton, T-bet, and IFN-r, and the Th17 transcripton, RORγt, and IL-23, IL-17, and promoted the mRNA expressions of the Th2 transcripton, GATA-3, and IL-4 and Treg transcripton, FOXP3 and CTLA-4.⑤ELISA showed that, compared with DCN and DC.GFP,in DC.FOXP3 that had been co-cultured, the contents of IFN-r and IL-17 lowered in the supernate while those of IL-4 and IL10 increased.⑥in the DC.FOXP3 group that had been co-cultured and added with 1-MT, the number of CD4+T cells increased but fewer cells had withered and died; the polarisation on CD4+CD25-T cells became weaker; the transcripton and cell factor mRNA levels in the Th1 and Th17 cells inceased; the transcripton and cell factor mRNA levels in the Th2 and Treg cells deceased; ELISA detection of the secreted cell factor levels in the co-culture solution found that the contents of IFN-r and IL-17 increased while those of IL-4 and IL10 lowered.Conclusions:1 FOXP3 gene hyper-expression recombinant gland virus vectors can be successfully prepared.2 DC can be cultured,induced and amplified; the biological features and phenotypes of imDC and mDC can be assayed; FOXP3 gene hyper-expression imDC cellular models can be made.3 the phenotypes of AD-FOXP3 transfected imDC can not be altered; the expressions and secretions of Th1 and Th17 inflammatory factors can be weakened and those of Th2 inflammation-inhibitive factors strengthened. And IDO can be highly expressed with cells, thus intensifying the imDC tolerogenicity.4 AD-FOXP3 transfected imDC may inhibit the CD4+T-cell proliferation of the EAE mouse spleens, speed the CD4+T-cell apoptosis, induce the production of Treg, suppress the Th1 snd Th17 functioning, intensify the Th2 and Treg functioning, and reverse Th1/Th2 and Th17/Treg imbalances in vitro. 1-MT being applied, EAE mouse spleen CD4+T-cell functioning can be restored, which demonstrates that DC. FOXP3 intensify the immunotolerance by activating the IDO functioning.