In Vitro And In Vivo Studies On Inhibition Of T Lymphocyte Functions By The Soluble HVEM-Ig Fusion Protein Combined With CIITA Mutant Recombinant Adenoviruses

T lymphocytes play critial roles in transplant rejective responses.T cells require 2 signals for an effective immune response.The first signal is generated by the interaction of the T-cell receptor(TCR) with a cognate peptide-major histocompatibility complex(MHC) on the surface of an antigen-presenting cell(APC).T cells that are stimulated via the TCR alone fail to produce appropriate cytokines,are unable to sustain proliferation,and will often undergo apoptosis.The second signal is a costimulatory signal required to prevent apoptosis and induce full activation and differentiation into effector and memory T cells. Because full T-cell activation requires 2 signals,blocking T-cell costimulatory molecules has been supposed to be a strategy utilized to induce tolerance in solid organ transplantation.BTLA,whose only ligand is HVEM,is a new costimulatory molecule belonging to immunoglobulin(Ig) family.Many studies have indicated that HVEM-BTLA pathway delivers inhibitory signals reducing proliferation and cytokine production of T cells. Recent studies have also suggested that expression of MHC-Ⅱ,the predominant presenters for exogenous antigens,are mainly controlled by the classⅡtransactivator(CⅡTA).Since regulation of CⅡTA largely determines the expression intensity of MHC-Ⅱ,CⅡTA might represents an ideal target for intervention therapy of graft rejection through the classⅡantigen presentation pathway.In this study,a HVEM eukaryotic expression system in Chinese hamster ovary(CHO) cells was first constructed,and after further amplification and purification,the soluble fusion protein containing extracellular region of mouse HVEM and mouse IgG2a Fc region(HVEM-Ig) were then obtained.After being identified for the physical properties, HVEM-Ig was then used for in vitro and in vivo biologic function studies on HVEM-BTLA costimulatory pathway.We also administered CⅡTA mutant recombinant adenoviruses to inhibit the expression of MHC-Ⅱmolecules in the presence or absence of HVEM-Ig,observing their synergia biological effect on T cells.Based on these results,we will further,but not in this paper,discuss the feasibility of using HVEM-Ig combined with CⅡTA mutant recombinant adenoviruses for the immunotherapy of graft rejection.PartⅠConstruction of HVEM-Ig expression system and identification of the acquired fusion proteinThe extracellular part of DNA sequence encoding mouse HVEM was amplified by RT-PCR from splenic mononuclear cells,and the mouse IgG2a Fc domain was cloned from WT-1 cells.The two domains were then inserted into pSecTag2 to get the eukaryotic expression plasmid pSecTag2-HVEM-Ig.The recombined plasmids were stably transfected into CHO cells and all the cells were then cultured in the presence of 600μg/ml Hycromycin B to select the HVEM-Ig-expressing clones.Established cell lines were then screened by ELISA with anti-mouse IgG2a Fc antibody.HVEM-Ig high-expressing clones (＞1 pg/cell·24h) were cultured continuously to detect their expression stability,and 5 clones that stably expressed more than 1 pg/cell·24h fusion protein,as a result,were selected and used for further large scale amplification in roller bottle procession system.The CHO/HVEM-Ig cells were first cultured in 1640/10%FCS until adherence,and then in serum-free medium for another 5～8 d.The supematants were harvested,and HVEM-Ig fusion protein was purified with rProtein A-Sepharose FF affinity chromatograph according to description provided by the manufacturer.The obtained fusion protein was then used for further identification of physico-chemical property and biological function.Series of experiments were performed to confirm the expression product to be HVEM-Ig fusion protein.In SDS-PAGE,about 45 kD specific protein band was identified in reducing condition,while about 90 kD and 120 kD two specific protein bands were detected in non-reducing condition.These data indicated that the expression product existed as a mixture of bisulfide-binding dimer and trimer which was similar to the natural expression patterns of HVEM in vivo.Western Blot assay discovered that the soluble protein could also be specifically detected by anti-mouse HVEM antibody,confirming that it was an HVEM-Ig fusion protein.PartⅡConstruction of Ad-CⅡTA and its biological activities in vitro Firstly,The CⅡTA mutant gene lacking 325 to 678 base pairs on N-terminal was obtained according to cDNA of typeⅣCⅡTA by means of PCR-SOE.Secondly,The recombinant adenoviral backbone containing CⅡTA mutant gene was obtained through homologous recombination.Finally CⅡTA mutant recombinant adenoviruses(Ad-CⅡTA) were generated by packaging of HEK293 and purified by density gradient ultracentrifugation with caesium chloride solutions.The activity of virus was evaluated by TCID50.For evaluation of the possible inhibitive function on expression of MHC-Ⅱmolecules,Ad-CⅡTA were transfected into Hela cell lines,and the positive rate of HLA-DR molecules expressed on HeLa induced by IFN-γ,decreased by 40～45%(mean 42.5%),and mean fluorescent intensity(MFI) by 62～67%(mean 64.5%) compared with the control. These data indicated that CⅡTA mutant inhibited inducible expression of MHC-Ⅱmolecules.PartⅢBiological effects of HVEM-Ig and/or Ad-CⅡTA on T cellsThe single-direction mixed lymphocyte reaction(MLR) was performed to evaluate the reactivity of T lymphocyte.Splenic T cells isolated from BALB/c mouse were used as responder cells,while splenic mononuclearcells from C57BL/6 pretreated with mitomycin as stimulator cells.Different concentration of HVEM-Ig or control IgG were added and the cells were incubated for 3 days before responsive rates were detected using[~3H]-TdR method.And the results demonstrated a dose-dependent inhibition of HVEM-Ig on T-cell proliferation.Cytokines in the culture supernatants determined by corresponding ELISA kit discoved that the levels of TNF-α,IFN-γ,IL-2 and IL-10 in HVEM-Ig group were lower than those in the control groups(P＜0.05),meanwhile levels of IL-4 was similar in all these groups.In vivo study of allo-reactive T cell proliferation were performed using vital dye CFSE, the typical property of which was that the agent would be distributed equally to daughter cells upon division,which has also enabled it to become a very useful tool to study lymphocyte division kinetics and differentiation in a variety of in vivo systems.Mice were allocated into three groups:no treatment control,IgG+Ad-GFP,HVEM-Ig+Ad-CⅡTA,and CFSE-labeled BALB/c spleen cells were injected i.v.together with soluble protein and Ad-CⅡTA into C57BL/6 mice that were preirradiated by 9Gyγray.72h after transfer,spleen cells were harvested for detecting CD4~+CFSE~+ and CD8~+CFSE~+ by FACS,and the proliferation of CD4~+ and CD8~+ T cells were analyzed using cellquest software.The results showed that HVEM-Ig plus Ad-CⅡTA significantly inhibit the proliferation of both CD4~+T cell and CD8~+ T cells as compared to the IgG+Ad-GFP controls.Conclusion:In vitro study demonstrated that HVEM-Ig fusion protein inhibited T cells proliferation while Ad-CⅡTA down-regulated expression of MHC-Ⅱmolecules.The combined application of HVEM-Ig and Ad-CⅡTA obviously inhibited allogenetic T cells proliferation responses in vivo.Our data provided the primary evidence that immunotherapy and gene therapy,refering to HVEM-Ig or Ad-CⅡTA therapy respectively in this paper,have synergistic effect on the inhibition of allogenetic T cell responses,which might suggest a new strategy for controlling graft rejection.