Induction Of Immune Tolerance By UL144 Gene Modified Dendritic Cells

Lymphocyte activation involves signals delivered through primary antigen receptors -either the T-cell receptor(TCR) or B-cell receptor-as well as secondary signals delivered through an array of co-stimulatory and inhibitory receptors that regulate the extent,quality and duration of lymphocyte activation.For T cells,the co-stimulatory and inhibitory cell-surface receptors include CD28,cytotoxic T-lymphocyte antigen 4 (CTLA4) and their homologues,and several members of the tumour-necrosis factor(TNF) receptor(TNFR) family.The co-stimulatory receptors of the immunoglobulin superfamily include CD28 and inducible T-cell co-stimulator(ICOS),and the inhibitory receptors include CTLA4,programmed cell death 1(PD1) and a recently identified molecule known as B- and T-lymphocyte attenuator(BTLA).Until recently,it was thought that all these immunoglobulin superfamily members interacted with members of the B7 family of cell-surface receptors.Indeed,expression of B7-family members by antigen-presenting cells(APCs) and peripheral tissues regulates T-cell activation in the context of inflammatory stimuli.Likewise,until recently,most TNFR-family members were thought to interact only with TNF-family ligands,with exceptions including nerve growth-factor receptor(NGFR) binding neurotropins,and herpesvirusentry mediator (HVEM) binding herpes simplex virus type 1 glycoprotein D(HSV 1 gD),which contains an immunoglobulin domain.A subset of TNFRs,including 4-1BB,CD27,CD30,HVEM and OX40 function to positively co-stimulate T cells during the late phase of activation3. With the exception of HVEM and CD27,these receptors are not expressed by naive T cells,but are induced during T-cell activation.Expression of their TNF ligands is also induced on APCs by inflammatory stimuli.Recently,it was found that crosstalk occurs between the immunoglobulin superfamily and the TNFR family of co-stimulatory molecules.This finding resulted from the unexpected discovery that the immunoglobulin-domain-containing receptor BTLA binds the TNFRfamily member HVEM in both mice and humans.This interaction is all the more unusual because BTLA has been described to inhibit T-cell responses,whereas HVEM has been described to activate them.Several studies have recently explored this interaction,both from a structural and a biological perspective,in vitro and in vivo.The binding site on HVEM for BTLA is conserved in the orphan TNFR,UL144,present in human CMV.UL144 binds BTLA,but not LIGHT,and inhibits T cell proliferation, selectively mimicking the inhibitory cosignaling function of HVEM.BTLA is constitutively expressed on most CD4⁺ and CD8⁺ T cells and its expression progressively decreases upon T cell activation.Polarized Th1 and Th2 cells contained both BTLA-positive and BTLA-negative populations.Therefore,in this study,we think that expression of UL144 on DCs mediated by gene transfer may attribute to the production of tolerogenic DCs,and these DCs may inhibit effective T cells directly or indirectly through regulatory T cells.Under the inflammation condition,we want to know whether the UL 144 expressive DCs could influence the polarization,activation,proliferation and differentiation of T cells in vitro and prevent or therapy an autoimmune disease using experimental autoimmune myocarditis as model.This study may provide beneficial enlightenment for clinical therapy of myocarditis.In this study,through routine molecular biological techniques,we constructed recombinant adenoviral vector containing the UL144 gene fragment.Recombinant adenoviruses were generated by packaging of HEK293 and purified by velocity density gradient centrifugation in caesium chloride solutions.Then we conducted in vitro gene transfer,infecting HeLa with recombinant adenoviruses to identify UL144 gene by RT-PCR.And we analyzed surface major histocompatibility complex(MHC) and costimulatory molecule expression of dendritic cells infectted with AD-CMV-UL144. Simultaneously,theses DCs were cultured with OVA- stimulused CD4⁺T cells to test its immunostimulatory activity.After production of EAM model by immunized with myosin, UL144 expressive DCs was injected to mice's veins at day 7～8 and day 14～16 to evaluate the value of the virus in prevention of the induction or the progression of EAM.The main results and consulsions were as follows:Partâ… .Construction of UL144 gene recombinantr eplication-deficient adenovirus.UL144 gene was amplified from hCMV DNA which was extracted from CMV-Ag positive serum.After the fragment was inserted into the shuttle plasmid pAdTrack-CMV by multiple clonsites in pMD18-T vector,the linearized shuttle plasmid pAdTrack-CMV by Pmeâ… and skeleton vector pAdEasyl co-transformed into E.coli DH5α.Through homologous recombination we constructed recombinant adenoviral vector containing the UL144 gene fragment.Recombinant adenoviruses were generated by packaging of HEK293 after the linearization of adenoviral vector.Infecting HeLa with recombinant adenoviruses,UL144 gene was identified by RT-PCR.The use of recombinant virus for gene therapy requires rational quantity evaluation for the viral vector titer.In the part,we purified the adenovirus by velocity density gradient centrifugation in caesium chloride solutions.The activity of AD-CMV-UL144 was evaluated by Reed- Muench assay with resulting 50%tissue culture infective doses(TCID₅₀/mL) of 3×10¹⁰/mL,and AD-GFP with 1×10¹⁰/mL.Partâ…¡.The impact of recombinant adenovirus-mediated UL144 expression on mice myeloid dendritic cells.The aims of this part of experiments were to analysis the phenotype,cytokine secrete and the ability of antigen presenting of UL144 expression DC.Mice myeloid DC was isolated from bone marrow and cultured in RPMI1640 containing GM-CSF and IL-4.After six days,cells were purified using monoclonal antibody labeled magnetic bead,and then transfected with recombinant adenovirus.The supernatant were collected for cytokines detection and cell membrane proteins were analyzed by flow cytometry.The data showed that the expressive level of CD40,CD80(B7.1),CD86(B7.2),and Ia^d(MHC-â…¡) was lower on UL144 expression DC than on those control adenovirus transfected DCs.The TNF-αand IL-6 in supernatant were much lower in group of UL144 expression DCs,although there is no significant difference in other cytokines among those groups.We also found the UL144 expression DCs can resist LPS-mediated mature.[³H]-TdR incorporation not only showed the capacity of antigen-presenting of UL144 expression DCs is significant lower than controls,but also suggested that it could inhibit proliferation of active T cells. In summary,all of our data showed the UL144 expression DCs had the characters of tolerogenic DCs in some degree,and it might be beneficial for ameliorating some autoimmune diseases.Partâ…¢.The impact of recombinant adenovirus-mediated UL144 expressive DCs on experimental autoimmuno myocarditis.Balb/c mice were immunized on day 0 and 7 with purified porcine cardiac myosin to establish experimental autoimmune myocarditis.Recombinant adenovirus transfected homotype DCs was exposed to myosin and administered intravenously at day 7 and day 14, and mice were killed on day 21 to study effects of inhibition of T cell activation.The heart histological examination and concentration of serum cTnI evaluation were performed to evaluate the severity of the disease.Cytokine expression in the spleen mononuclear cells culture and T cell proliferation against cardiac myosin were analyzed.Flow cytometry revealed that the subgroup of Th cells and the expression of active markers on immune cells in spleen,lymphoid node and peripheral blood.The serum of total IgG and specific anti-myosin antibody were assayed to evaluate humoral immunity of EAM mice.Among the groups with different treatment,the inflammatory responses and resulting myocardial injury were significantly ameliorated by recombinant adenovirus Ad-CMV-UL144 transfected DCs.The major phenomena included that lessen of heart lesion,decreasing of serum cTnI,specific anti-myosin antibody,spleen cells proliferation degree against myosin,and down-regulation of active T cells,expression of CD69 or CD25 on T or B cells in spleen and lymphoid nodes.These data indicated that EAM development can be attenuated by specific antigen reactive UL144 expressive DCs during the immune response phase.