Effect Of Solube CD83 And BTLA On The Co-stimulatory Capacity Of Dendritic Cells And Soluble CD83 Prolongs Survival Time Of Skin Allo-grafts In Mouse

I IntroductionRejection of grafts is main cause of allograft loss. The principal mechanism underlying the rejection is the vigorous adaptive immune response mounted by recipient T lymphocytes against allogeneic antigen. Antigen presenting cells (APCs), including dendritic cells (DCs), stimulating T cell activation is critical in allo-direct recognision and allo-indirect recognition. DCs are the most powerful APCs known today. They play a major role in the regulation of cell-mediated immune responses by interaction with T cells and can induce tolerance to self- or allo-antigens in both thymus and in lymphoid organs. Mature DC with high expression of major histocompatibility complex (MHC) class II molecules, co-stimulatory molecules (CD40, CD80, and CD86) and adhesion molecules (CD54, CD58), has powerful co-stimulatory action to self or allo- T cells. Whereas immature DC, with only moderate expression of MHC molecules and no or low levels of co-stimulatory molecules and adhesion molecules, has been regarded as useful for the induction of transplantation tolerance A popular model to explain the requirement of co-signals in T-cell activation isthe two-signal hypothesis. In this model, signal one is derived from the TCR aftertriggering by antigenic opeptide presented by MHC molecules and signal two isdelivered through co-stimulation. Interaction of the two signals induces optimalactivation of T cells. Lack of a co-stimulatory signal leads to decreased responses and,in some cases, even induces tolerance or anergy. However, this two-signal model hasbeen recently challenged by the discovery of new co-inhibitory pathways and theircrucial roles in the control of T- cell activation. Current experimental results supportthe idea that the TCR signal is requires for the induction of activation and tolerance/anergy, but co-stimulators and co-iahibitors have an essential role in these responses.Thus, Chen Liepin propose a co-signalling network modle to explain T cell activationor tolerance/anery. The central theme of this model is that the TCR signal does notdecide or govern the direction of T- cell response and that the outcome of an antigenspecific T cell response is guided by co-stimulators and/ or co-inhibitors, which areoften simultaneously provided to T cells by APCs and other host cells. According toco-signalling network modle, blockage of the co-stimulatory signal or enhancement ofthe co-inhibitory signal will lead to T cell tolerance/ anergy.CD83 is the best known marker for mature human DC. Although the precise function of CD83 is not known, its selective expression and upregulation with the costimulators CD80 and CD86 suggests CD83 had an important role in the induction of immune responses. Isolation of cDNA encoding CD83 revealed that it is a 45-kDa type 1 membrane glycoprotein member of the Ig superfamily. The structure of CD83 is significant homologies with the B7 ancestral gene family that includes B7-1 and B7-2. Recently, Fujimoto et al reported that CD83-deficient (CD83^-/-) mice had a specific block in CD4⁺ single-positive thymocyte development. The lack of CD83 resulted in a selective 75%-90% reduction in peripheral CD4⁺ T cells. With soluble CD83, Scholler et al found that CD83-Ig fusion protein inhibited T cell proliferation, and Lechmann et al reported that soluble CD83 protein significantly down-regulated the expression of CD80 and CD83 on DCs and completely inhibited DC-mediating T cell stimulation in a concentration-dependent manner. These findings suggest strongly that CD83 may be a co-stimulatory molecule for T- cell activation, and soluble CD83 can block T cell response both in T cell level, which inhibits T cell activation, and in DC levels, which down-regulates the expression of co-stimulator on DC, and therefor inhibits DC mediating T cell activation.BTLA (B and T lymphocyte attenuator) is CD28 family member and is similar functionally and structurally to PD-1 and CTLA-4, is found recently, which expresses on T and B cell mainly. BTLA exerts inhibitory effects on B and T lymphocytes. It remains expressed on Th1 but not Th2 cells, suggesting that BTLA may specifically down-regulate Th1-mediated inflammatory responses. BTLA^-/- T cells show an increased response to anti-CD3 in vitro. Polarized BTLA^-/- Thl cells have about a two-fold increase in proliferation to antigen. Whether BTLA specifically regulates Thl effector function is not yet clear. BTLA^-/- B cells show modestly increased responses to anti-IgM, and BTLA^-/- mice show a three-fold increase in hapten-specific antibody responses and enhanced susceptibility to EAE. The relative roles of BTLA in negatively regulating T cell and B cell responses and peripheral B and T cell tolerance remain to be determined. Similar to CTLA-4 and PD-1, BTLA was first thought to bind a B7-like Ig family molecule B7-H4. However, Sedy and Gonzalez show that BTLA interacts with HVEM recently. HVEM is now thought to be a co-stimulator, based on its ability to induce the activation of NF-kB, Jun N-terminal kinase (JNK) and adapter protein 1 (AP1) engaged with LIGHT. Thus, HVEM not only bind BTLA as a ligand, but also bind LIGHT as a receptor. However, BTLA binds HVEM at CRD1, in a region distinct from LIGHT, but in the same region as herpesvirus gD, a site on the opposite face to where LIGHT binds. It has been a decision that HVEM-BTLA pathway exert a inhibitory signal norientationally to B and T cell, but the reverse signal of HVEM-BTLA pathway exerting to HVEM expression cell is unknown. According to the bifunction of soluble CD83, not only inhibits T cell activation but also down-regulate co-stimulatory capacity of DC, the first objective of this study is to explore the possibility of soluble mouse CD83-Ig in the prolonging of survival time of allo-skin grafts in mouse, which will provide a solid experimental evidence to clinical therapy for graft rejetion with soluble CD83.The second objective of this study is to clone and to expresse mouse BTLA, and study the effect of the reverse signal of HVEM-BTLA pathway on the expression of co-stimulatory molecules, so as to establish a solid fundament for treatment of graft rejection targeting HVEM-BTLA pathway.II Clone of mouse CD83 and BTLA and human IgG1α Fc gene ,and construction of expression vectorscDNA of mouse CD83 (mCD83) and mouse BTLA (mBTLA) were reverse transcribed from the total RNA of mouse spleen by RT -PCR, and human IgG1α Fc (hIg) gene cDNA was reverse transcribed from the total RNA of human peripheral blood mononuclear cell (PBMC) by RT-PCR too. Then the fragment of hIg gene was cloned into the eukaryotic expression system pCDNA3.1(+) to construct pcDNA-hIg vector, and the extracellular domain gene of mCD83 or mBTLA was inserted into pcDNA-hlg vector upstream hIgFe, respectively, to constructe eukaryotic expression vector pmCD83-hIg or pmBTLA-hIg, and the DNA inserts was further confirmed by restriction enzyme digestions and DNA direct sequencing. The results showed that we cloned mCD83, mBTLA and hIg gene succesfully, and the expression vectors pmCD83-hIg and pmBTLA-hIg were constructed correctly confirmed by restriction enzyme digestions and DNA sequencing.III Preparation and biologic effects study of Soluble mCD83-hIg fusion protein By using Lipofectamine^TM000, pmCD83-hIg plasmid was transfected into COS-7 cells, and soluble mCD83-hIg fusion protein were expressed in COS-7 cells confirmed by ELISA and Western blot analysis using the cultural supernatants. The relative amount of soluble CD83-Ig was determined by ELISA with the human IgG standard preparations. mRNA for recombinant CD83-Ig expression was analyzed by RT-PCR using primers from the sequence of mCD83 and hIg fusion gene mCD83-hIg. The soluble mCD83-hIg fusion protein from the cultural supernatants was purified by protein A-Sepharose affinity chromatography. The results showed that COS-7 cells transfected with pmCD83-hIg secreted mCD83-hIg into cultural medium. Western blot analysis using a polyclonal antibody specific for mCD83 detected a 39.8 kD protein, which is in agreement with the molecular weight of addition mCD83 extracellular domain and hIg Fc. A 120 bp fragment was amplified from the total RNA of pmCD83-hIg transfected COS-7 cells by RT-PCR corresponding to anticipated size. The amount of soluble mCD83-hIg resulted from 7-day cultures ranged from 25μg/L to 380 μg/L determined by ELISA, and the fusion protein display a single band at 39.5 kD in SDS-PAGE analysis after purified by protein A-Sepharose 4B affinity chromatography. The results showed that the quality of soluble mCD83-hIg fusion is suitable for further experiment.Mouse dendritic cell line (DC2.4) served as the DC cell model. The morphous of DC2.4 display a typical dendritic profile under light microscopy and it express high levels of CD83, CD80 and CD86 determined by flow cytometery, indicating a matured DC phenotype for DC2.4 cells.The effect of mCD83-hIg on the expression of co-stimulatory molecules and the cell cycle and apoptosis of DC2.4 were analysis by flow cytometry, and the secretion of IL-12 from DC2.4 under stimulation of LPS was tested by ELISA. Then the allo-stimulatory capacity of DC2.4 treated with mCD83-hIg were analyzed by mixed leukocyte reaction (MLR).The results showed that mCD83-hIg down-regulated the expression of co-stimulatory molecules, CD80, CD86 and CD83 on DC2.4, and inhibited IL-12 secretion from DC2.4, and so as to down-regulated its co-stimulatory capacity. But we failed to detect the any effect of soluble mCD83-hIg on cell cycling and apoptosis on DC2.4.One-way MLC was employed to determain the inhibitory effect of mCD83-hIg on the proliferation of allo-T cells and the secretion of cytokines from allo-T cells with spleen T cells from BALB/c mice as responders and splenocyte from C57BL/6 mice as stimulator. It was found that mCD83-hIg have had an inhibitory effect on allogenic T cell proliferation and the production of IL-2 and IFN-γ, but we failed to detect the differences of IL-4 and IL-10 between the mCD83-hIg treated MLC and the controls.The results of this section indicated that soluble mCD83-hIg down-regulated T cell allo- responses via its inhibitory effec both on DC and T cell.IV Soluble mCD83-hIg prolong the survival time of allo-skin graftsin mouseThe allo-skin graft model was established with C57BL/6 mouse as donor and BALB/c mouse as recipient. Soluble mCD83-hIg was injected via vena caudalis on the day before transplantation, and the first day and the third day after transplantation. Two groups of mice received human IgG and equal volume of PBS respectively, served as controls. The grafts survival time was determined by naked eye observation and by histological analysis. The mean survival time of grafts in mCD83-hIg treated group was (12.57 ± 2.07) day, which was significantly longer than that of allografts in the human IgG [(8.57± 1.51) day] or PBS [(8.71 ± 1.11) day] control group.At the seventh day of skin transplantation, some mice in every group were sacrificed. The sera were prepared from vena angularis blood for cytokine determination, the spleen T cells were purified for restimulatory assay, and the skin grafts were taken off for histological analysis and cytokines RT-PCR. The results showed that IFN-γ and IL-2 production were strongly reduced in the sera and the T cells proliferation capacity were decreased in mCD83-hIg treated mice, significantly. But we failed to detected any changes of IL-4 and IL-10. We also found the mRNA transcription of IFN-γ and IL-2 decrease strikingly in allografts, but the mRNA of IL-4 and IL-10 had not changes too, in the mice treated with mCD83-hIg. These results corresponded to the data from in vitro experiment. Histological analysis showed that the allo-skin grafts from mCD83-hIg treated mice had less inflammatory infiltration.The results indicated that soluble mCD83-hIg prolong the survival time of all-skin grafts in mouse via its inhibitory effect on the polarization of Thl cells and production of Thl type cytokines.V Expression of recombinant GST-mBTLA fusion protein andpreparation of anti- GST-mBTLA serum in rabbitThe recombinant prokaryotic expression vector pGEX-4T-2/mBTLA to express glutathion S- transferring enzyme-extracellular domain of mBTLA (GST-mBTLA) was constructed by cloning the extracellular domain gene of mBTLA into pGEX-4T-2 vextor and then trasformed into E coli BL21 (DE3). The fusion protein GST-mBTLA was expressed under the induction of 1 mmol/L IPTG, and then was extracted from inclusion body and purified through Glutathione Sepharose 4B chromatography column. The purified recombinant GST-mBTLA fusion protein was immunized with complete Freund adjuvant into rabbit to prepare anti-GST-mBTLA serum. The fusion protein GST-mBTLA was expressed in E coli BL21 (DE3) successfully. The molecular weight of the fusion protein was 43.0 kDa determined by SDS-PAGE, which was corresponding to expectation. The titer of the anti-serum were 1:16 and 1:3200 determined by double agarose diffusion and by ELISA, respctively.VI Effcet of soluble mouse BTLA on the expression of B7-1 and B7-2on dendritic cellsDC2.4 stable transfectants for pmBTLA-hIg secreted a soluble fusion protein (mBTLA-hIg) into cultural medium. Western blot analysis of cultural medium using a polyclonal antibody specific for mBTLA detected a 43.3kD protein, which is in agreement with the molecular weight of addition mBTLA and hIg Fc. A 175 bp fragment was amplified from the total RNA of pmBTLA-hIg transfected DC2.4 by RT-PCR with the primers from the sequence of mBTLA-hIg fusion gene corresponding to anticipated size. The amount of soluble mBTLA-hlg resulted from 6-day of cultures of 1×10⁴ cells was determined by ELISA and ranged from 45μg/L to 230μg/L.DC2.4 stable transfectants for pmBTLA-hIg were cultured for 72 hour, then the cells were digested by trypsin, washed thrice with PBS, and then analysis by FCM with the stain of FITC labelled anti-human IgG to determine whether mBTLA-hlg bind to DC 2.4. The results showed that DC2.4 stable transfected with pmBTLA-hlg were positive with the stain of anti-human IgG, indicating mBTLA-hlg fusion protein could bind to DC2.4. The expression of co-stimulatory molecules on pmBTLA-hIg transfected DC2.4 was analyzed by FCM too. Surprisingly, we found for the first time that the expression of B7-1 on DC2.4 stable transfectants for mBTLA-hIg was up-regulated, but the expression of B7-2 was decreased. Furthermore, we found that recombinant GST-mBTLA fusion protein up-regulated B7-1 expression on DC2.4 too, but we failed to detect effect of GST-mBTLA on the expression of B7-2 on DC2.4. These results indicated that soluble mBTLA, including mBTLA-hIg secreted from pmBTLA-hlg transfected DC2.4 and GST-mBTLA had regulatory effect on the expression of B7-1 and B7-2 on DC. Finaly, we found that anti-GST-mBTLA serum could block the effect of mBTLA on expression of B7-1 and B7-2 on DC2.4.The results from this section indicated that soluble mBTLA had regulatory effect on the co-stimulatory capacity of DC possibly, and therefor regulated DC-mediated T cell response further.VII Conclusion1. Soluble mCD83-hIg coule inhibit DC expression of co-stimulator and production of IL-12, as a result inhibit co-stimulatory capacity of DC. Soluble mCD83-hIg could inhibit allo- T cell proliferation and production of IL-2 and IFN-γ also. Thus. Soluble mCD83-hIg down-regulated T cell responses at both T cell and DC levels.2. Soluble mCD83-hIg could prolong the survival time of allo-skin grafts in mouse, which suggest that mCD83-hIg is a good immunosuppressor for treatment of graft rejetion and autoimmune disease clinically.3. Soluble mBTLA had a regulatory action on the expression of B7 molecules on DC, which maybe resulted from the reverse signal of HVEM-BTLA pathway.