Prevention And Treatment Of Experimental Autoimmune Thyroiditis By Adenovirus-mediated Gene Transfer Of CIITA Mutant

Major histocompatibility complex (MHC) class II molecules are the predominant presenters of exogenous antigens to CD4~+ T helper cells. Constitutive expression of class II MHC is restricted to "professional" antigen-presenting cells but can be induced on various tissues by gamma interferon. The class II transactivator (CIITA) expression appears to be an absolute requisite for expression of class II MHC, whether constitutive or inducible. Studies in recent years suggest a view of CIITA as a master regulator controlling expression of class II MHC genes. The regulation of CIITA largely determines the presence or absence of class II MHC and its degree of expression and thus the nature of the immune response. As CIITA is a single gene or protein that affects a large family of genes, it represents an ideal target for intervention in the MHC class II antigen presentation pathway and then for immune suppression in transplantation and autoimmunity. Expression of CIITA is mainly controlled by three different promoters named pI, pIIIand pIV. These promoters display transcriptional activity in cell type-specific manner.pIII is used primarily in B cells and to a lesser extent in some dendritic cell (DC)subsets. PI is highly specific for DCs. pIV is induced by IFN-γ in most cell types. Each promoter precedes a distinct first exon that is spliced alternatively to a shared second exon. This leads to the production of three types of transcript (type I, type III and type IV), each possessing a different 5' end and translated to three different CIITA proteins (type I CIITA, type III CIITA and type IV CIITA).A kind of protein molecule which has its structure changed and lost its normal biological function can often compete with its wild type protein and then block the biological function of wild type protein. This kind of protein mutant which could inhibit the biological function of its wild type protein was called dominant negative mutant. The dominant negative mutant of target gene whose expression is mediatedby vector can be used to inhibit the biological function of its wild type protein , and thus can be applied in prevention and treat of diseases in gene therapy. The N-terminal is the major active site of CIITA, and a CIITA mutant whose N-terminal is partly absent has the ability of competition with wild type CIITA and will eventually cause the decline or loss of the normal function of CIITA to activate the expression of MHC-II genes. Dominant negative mutant with recombinant adenovirus as vector of gene transfer, will strengthen its stability and penetration, and promote the effectiveness and feasibility of gene therapy.Experimental autoimmune thyroiditis (EAT) has proved to be a highly useful animal model of human Hashimoto's thyroiditis because its pathological characters and progression are similar to the latter. EAT is a kind of organ-specific autoimmune disease mediated by T cell immune, and characterized by thyroid epithelial cells' degeneration, necrosis, fibrosis, as well as infiltration of mononuclear cells. Activation of autoreactive T cells which induced by abnormal expression of MHCII, infiltration of CD/ T cells and macrophages, and increasing of pre-inflammatory factors is the major cause of thyroid lesion. Thus, it is imaginable that down-regulation of MHC II expression and presenting auto-antigen peptide by target suppression of CIITA expression can prevent the both induction and the progressive of EAT.The primary aim of the present study is to investigate: (1) the effects of inhibition and blockade of CIITA mutant on the constitutive and inducible expression of MHC class II molecules;(2) the effects of CIITA mutant gene transfer on the prevention and treat of EAT, as well as the related mechanisms.In this study, we designed and constructed a CIITA mutant with its absence of N-terminal from 325 to 678 base pairs using type IV CIITA cDNA as template. Using routine molecular biological techniques and PCR-SOE method, we constructed two kind of recombinant adenoviral vector containing the CIITA mutant, that is Ad-CMV-CIITAm and Ad-pIV-CIITAm. Recombinant adenoviruses were generated by packaging in HEK293 cells and purified by velocity density gradient centrifugation in caesium chloride solutions. Then we infected SVEC cell line and J774 cell line with recombinant adenoviruses, in order to observe the inhibitory effect on constitutive or inducible expression of MHC class II molecules: After induction ofEAT model by immunized with thyroglobulin, recombinant adenovirus was injected intravenously at day 0-2 or day 14-16 to evaluate the effect of CIITA mutant gene transfer on the induction and progression of EAT. This study includes three part:Part I: Construction, Purifying and Titre Measuring of Recombined Adenovirus Containing CIITA MutantFirstly, Using mouse type IV CIITA cDNA as template, we constructed a CIITA gene mutant with its absence of N-terminal from 325 to 678 base pairs by means of PCR-SOE and then the CIITA mutant was cloned into eukaryotic expression vector pIRES. Secondly we constructed a recombinant adenoviral backbone containing CIITA mutant named pAd-CMV-CIITAm and another recombinant adenoviral backbone containing CIITA mutant named pAd-pIV-CHTAm in which the transcriptional activity is controlled by CIITA pIV promoter. Then the two recombinant adenoviral backbones were successfully packaged into recombinant adenoviruses named Ad-CMV-CIITAm and Ad-pIV-CHTAm which have biological activity. The construction of Ad-CMV-CIITAm and Ad-pIV-CHTAm has made a foundation for in vitro and in vivo study on inhibition of expression of MHC-II molecules in the future .In the part, we also purified the recombinant adenoviruses by velocity density gradient centrifugation in caesium chloride solutions. The infectious activity of recombinant adenoviruses was evaluated by flow cytometry and TCID50 method, while their practical virus particles was determined by real-time PCR. The deviation between the two classes of methods is no more than 102, and the similar results were obtained between methods in the same class, which supports the conclusion that these high-titred and purified recombinant adenoviruses are stable. Because the two classes methods have some defection respectively, different method should be adopted to evaluate the quality of virus overly. In this study, we take virus practical particles as the dosage standard for the sake of repetition of consequent experiments.Part II: Study on Expression Regulation of CIITA Mutant Recombinant Adenoviruses and Their in vitro FunctionsFirstly, cell line SVEC were stimulated by recombinant mouse IFN-y and infected by recombinant adenoviruses. The result showed MHC II (IAk) expression on normal SVEC cells was negative ,but increased dramatically when stimulated by IFN-y. And as expected, the wild-type CIITA mRNA was also induced in a dosage-dependent manner. After SVEC cells were infected by recombinant adenoviruses containing CIITA mutant, Ad-CMV-CIITAm or Ad-pIV-CIITAm, the positive rate of SVEC cells expressing IAk molecule induced by IFN-y was decreased significantly when compared with the control Ad-GFP. These results suggested that Ad-CMV-CIITAm and Ad-pIV-CIITAm can inhibit inducible expression of class II MHC molecules in vitro.Secondly, cell line J774 were transfected by recombinant adenoviruses , with the stimulation by recombinant mouse IFN-y or not . The result showed MHC II (IAd) expression on normal J744 cells was positive, but increased significantly when stimulated by IFN-y. After J774 cells were transfected by recombinant adenoviruses Ad-CMV-CIITAm, the positive rate of J774 cells expressing IAd molecule was decreased significantly, with IFN-y or not, when compared with the control Ad-GFP. But transfection by recombinant adenoviruses Ad-pIV-CIITAm could not downregulate the IAd expression on J774 cells when without IFN-y , because CIITA mutant mRNA were induced in a IFN-y-dependent manner when measured by real-time PCR . These results proved that CIITA mutant could inhibit constitutive and inducible expression of class II MHC molecules in vitro, and that pIV promoter's transcriptional activity was activated by IFN-y.Part III: Study on Prevention and Treat of Experimental Autoimmune Thyroiditis by Adenovirus-mediated Gene Transfer of CIITA mutantFemale CBA/J mice were immunized on day 0 and 7 with purified porcine thyroglobulin to establish disease model of experimental autoimmune thyroiditis. Recombinant adenovirus was administered intravenously on day 0-2 or 14-16, and mice were killed on day 28 to study effects of inhibition of CIITA mutant . The thyroid histological examination were performed to evaluate the severity of the disease. Cytokine secretion in the spleen mononuclear cells culture and T cellproliferation against thyroglobulin were analyzed. The subgroups of T cells and the expression of I-Ak and inducible costimulator (ICOS) on immune cells in spleen and peripheral blood were analyzed by flow cytometry. The expression of MHC II in thyroid tissue was determined by immunohistochemical staining. The total IgG and specific anti-thyroglobulin autoantibody in serum were assayed by ELISA to evaluate humoral immunity of EAT mice. Cytokines secretion level in the spleen mononuclear cells culture stimulated by thyroglobulin were analyzed by ELISA.In prophylactic experiment, the inflammatory responses and thyroid injury were dramatically inhibited by recombinant adenovirus Ad-CMV-CIITAm treatment. The major phenomena included less thyroid lesion, lower titre of specific anti-thyroglobulin autoantibody, lower level of IFN-y and IL-2 but higher IL-4, decreased spleen cells proliferation degree against thyroglobulin, negative immunohistochemical staining of MHC II in thyroid tissue, and down-regulation of activated T cells, expression of I-Ak and ICOS on T cells. The results from therapeutic experiment are similar to those from prophylactic experiment, which indicated that EAT development can be attenuated by CIITA mutant treatment during the immune response phase.When using Ad-pIV-CIITAm instead of Ad-CMV-CIITAm to prevent or treat EAT, the effects is comparable to those of Ad-CMV-CIITAm, which supports the conclusion that the IFN-y-reactive CIITA mutant expression could effectively prevent and treat EAT.How to treat autoimmune thyroiditis in humans remains a major clinical problem. Our results, which demonstrated the efficacy of Ad-CMV-CIITAm and Ad-pIV-CIITAm on blockade of T cell activation through MHC II-antigen peptide-TCR complex during both the antigen priming phase and immune response phase of EAT, would have important implications for clinical treatment of autoimmune thyroiditis.