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Objective To investigate the therapeutic effect of CTLA4Ig gene on experimental autoimmune thyroiditis (EAT) by the use of portable synthetic costimulatory molecules of cytotoxic T lymphocyte antigen 4 (CTLA-4) antagonist (CTLA4Ig) eukaryotic expression vector.Methods Thirty C57BL/6J female mice were divided into three groups,named EAT model group (EAT,n =10),CTLA4Ig-treatment group (CTLA4Ig-EAT,n =10) and control group(n =10).At 28 day after first immunization,plasmids mixture with pCI or pCI/CTLA4Ig were injected into thyroid tissues of EAT and CTLA4Ig-EAT by surgery,respectively.Serum,thyroid tissues and spleens were collected as samples.Thyroid autoantibody and expression of interleukin (Th)1,Th2 related cytokinesby were measured by real-time quantitative PCR and ELISA.Results Compared with EAT group,the expression of CTLA-4 in thyroid of CTLA4Ig-EAT group was elevated double folds (P =0.038),and the expression of Th1 cytokine interferon γ and intercellular adhesion molecule-1 decreased significantly (P =0.016,0.042).Meanwhile,Th2 cytokine IL-4 was increased after CTLA4Ig treatment (P =0.044).The same changes were seen in spleen tissues and serum.There was no significant difference in terms of TPOAb between EAT and treated group.Conclusion Local thyroid injection of CTLA4Ig gene shows the therapeutic effect to same degree on EAT through adjusting the underlying Th1/Th2 imbalance.
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CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation.
Subject(s)
Animals , Cricetinae , Female , Blotting, Western , Cell Proliferation , Cricetulus , Cysteine , DNA , Drug Delivery Systems , Electroporation , Graft Survival , Islets of Langerhans , Islets of Langerhans Transplantation , Liposomes , Lymphocyte Culture Test, Mixed , Lysine , Ovary , Polymerase Chain Reaction , Proteins , TransplantsABSTRACT
Objective To investigate the effect of CTLA4-Ig chimera protein on mice mortality, histopathological changes, viral fiters, expression of CTLA4 protein on infiltrated T lymphocyte and the balance of Thl/Th2 in mice myocarditis caused by coxsackie virus B3 (CVB3). Methods A total of 106 four to six week-old male BALB/c mice were used in the experiments, which were divided into CTLA4-Ig group (n = 16), CVB3 group (n=40), IgG group (n =40) and normal control group(n = 10) randomly. The mice in CVB3 group, IgG group and CTLA4-Ig group were inoculated intraperitoneally with 0. 15 ml CVB3 and the mice in norreal control group with 0. 15 ml Eagle. The mice in IgG group and CTLA4-1g group were inoculated with IgG (0. I mg/kg) and CTLA4-Ig(0. 1 mg/kg) at 6 h and 72 h post inoculation(p, i. ), respectively, The surplus mice in each group were sacrificed at day 7 p.i. Light microscope was used to quantify the inflammation. The expression of CVB3 mRNA in mycardium were semi-quantified by real-time quantitative polymerase chain reaction (RQ-PCR). The expression of CTLA4 protein were analyzed by immunohistochemistry. The levels of IL-2, IL-4 and 1FN-γ in serum were measured by ELISA. Results The mice mortality, histopathological score and CVB3 mRNA in CTLA4-Ig group were lower than that in CVB3 group ( P < 0.05, P < 0.01, P < 0. 05, respectively). The expression of CTLA4 was significantly increased in CTLA4-Ig therapy group (P < 0.05 ). The serum level of IFN-γ of mice in CVB3 group were significantly higher than that in normal control group( P < 0.01 ). The serum level of IL-4 of mice in CVB3 group were much lower than that in normal control group( P < 0.01 ). The serum level of IL-2 in CVB3 group had no statistical significance with that in normal control group ( P > 0.05 ). The serum level of IFN-γ in mice of CTLA4-Ig group were much lower than that in CVB3 group ( P <0.01 ) and lgG group (P < 0. 01 ). The serum level of IL-4 of mice in CTLA4-Ig group were significantly higher than that in CVB3 group (P<0.01) and IgG group (P<0.01). The serum level of IL-2 in CTLA4-Ig group had no statistical significance with that in CVB 3 control group and lgG group ( P > 0. 0 5 ) . Conclusion CTLA4-Ig may relieve inflammation and reduce mice mortality by blocking the costimulation signals for T lymphocyte activation and reinforcing Th2 response.
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[Objective] To investigate the inhibitory effect of CTLA-4Ig fusion protein on atherosclerosis in the mice with an apolipoprotein-E gene defect fed on cholesterol diet.[Methods] Twenty-five male 10-week-old ApoE-/- mice were selected and fed on cholesterol diet for 4 weeks,5 out of which were executed at random as control group and their pathological sections were kept to observe the early fatty streaks.The other 20,divided into CTLA-4Ig treatment group,PBS group,IgG1 group,and blank group at random,5 in each.Three groups were given intraperitoneal injection of CTLA-4Ig (10 μg per time),PBS (100 μL per time),Rat-IgG1 (10 μg per time) respectively,twice a week,for 8 weeks.The blank group has no treatment.Followed by 8-week treatment,the whole aorta from the root to crotch of iliac artery was separated after anesthesia with the intraperitoneal injection of 1 % pentobarbital.Subsequently,the area ratio of plaque and lumen,the thickness ratio of endangium and tunica media,the lipid-soaking extent intra-plaque and the content of collagen fibrils and smooth muscle cells intra-plaque were analyzed by image-processing soft.[Results] After fed on cholesterol diet for 4 weeks,there were obviously atherosclerosis in the aorta in the ApoE-/- mice.There were typical atherosclerotic plaque in ApoE-/- mice fed on cholesterol diet after another 8 weeks.The area ratios of plaque and lumen in CTLA-4Ig group,PBS group,IgG1 group,and blank group were 0.27 ± 0.08,0.40 ± 0.08,0.43 ± 0.08,and 0.46 ± 0.10,and obviously increased than those in control group (0.05 ± 0.01,P ＜ 0.05).The thickness ratios of endangium and tunica media in four groups were 2.6 ± 0.6,6.0 ± 0.9,5.7 ± 0.8,and 5.9 ± 0.6 and obviously increased than those in control group (0.5 ± 0.1,P ＜ 0.05).The lipid-soaking extent intra-plaque in experimental groups were 26.0 ± 3.0,40.8 ± 5.7,40.6 ± 3.0,and 43.2 ± 5.7,and were obviously increased than those in control group (7.2 ± 1.4,P ＜ 0.05 ).It was found that the area ratio of plaque and lumen,the thickness ratio of endangium and tunica media,and the lipid-soaking extent intra-plaque in CTLA-4Ig group were significantly lower than those in PBS group,IgG1 group,and blank group (P ＜ 0.05),but there was no significant difference in those between the PBS group,IgG1 group,and blank group (P ＞ 0.05).The content of collagen fibrils in CTLA-4Ig group were 16.0 ± 1.1 and higher than those in PBS group,IgG1 group,and blank group (8.6 ± 1.2,9.2 ± 1.5,and 9.0 ± 1.3,P ＜ 0.05).The content of smooth muscle cells in plaque in CTLA-4Ig group were 11.8 ± 1.0 and higher than those in PBS group,IgG1 group,and blank group (7.8 ± 0.8,7.5 ± 0.9,and 7.3 ± 0.7,P ＜ 0.05).There was no significant difference in content of collagen fibrils and smooth muscle cells between the PBS group,IgG1 group,and blank group (all P ＞ 0.05).[Conclusion] CTLA-4Ig fusion protein could evidently inhibit the atherosclerosis progression and enhance the stability of plaque through increasing the content of collagen fibrils produced by smooth muscle cells intra-plaque in ApoE-/- mice fed on cholesterol diet.
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Objective:To investigate the clinic efficacy of gene transfected miniature pig skin as dressings to cure superficial Ⅱ degree burns of face in infants.Methods:Forty inpatients suffering from superficial Ⅱ degree face burn were included in our study.The ages of patients were 0.5 to 11 years and wound areas were 0.5% to 1.5% TBSA.A prospective randomized study was undertaken to evaluate the efficacy of CTLA4Ig gene transfected miniature pig skin while exposure therapy in traditional was taken as control.The healing time,scar,pain,skin graft and the rate of infection of wounds were observed.Results:The CTLA4Ig gene transfected miniature pig skin groups had less healing time,less pain and less infection rate.Conclusion:It is feasible that the CTLA4Ig gene transfected miniature pig skin is used as an effective and efficient cover to treat superficial Ⅱ degree burn wounds on face of infants.
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Rheumatic diseases are characterized by immunological dysfunction.T-cell medicated immune response plays a very important role in the development and persistence of rheumatic disease.CTLA-4 is an important co-stimulatory molecules expressed on T cells.It has inhibitory effect on T cell-mediated immune response.Plenty of evidences have demonstrated abnormalities in CTLA-4 alleles and proteins in patients with rheumatic diseases.Therefore,CTLA-4 has become an target in treating rheumatic diseases.CTLA-4Ig has been developed as an fusion protein which is composed of extrA-cellular domain of human CTLA-4 and the Fc fragment of human IgG1.So far,only data about CTLA-4Ig in treating rheumatoid arthritis are available.These data showed that CTLA-4Ig could relieve the symptoms of rheumatoid arthritis and retard the development of the disease.It is safe and effective.In addition,effectiveness was demonstrated even in patients who failed to response to anti-TNF monoclonal antibodies.Therefore,CTLA-4Ig is an encouraging therapy for rheumatic diseases.However,because of very short history,long-term study is needed to fully understand its role in the treatment of rheumatoid arthritis.
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To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.
Subject(s)
Mice , Male , Animals , Skin Transplantation/immunology , Mice, Inbred BALB C , Interleukin-2/immunology , Immunoconjugates/administration & dosage , Graft Survival/immunology , Drug Combinations , CD40 Ligand/immunology , Bone Marrow Transplantation/immunology , Antibodies/administration & dosageABSTRACT
To facilitate the establishment of mixed chimerism with limited dose of bone marrow (BM) cells, and to achieve tolerance in skin graft model, combined blocking of costimulatory pathway and IL-2 pathway was used in minimally myeloablative model using busulfan. BM cells (2.5 x 10(7)) of BALB/c were injected into C57BL/6 mice at day 0 with full thickness skin graft after single dose injection of busulfan (25 mg/kg) on day-1. Recipients were grouped and injected the anti-CD154, CTLA4-Ig, anti-IL-2R at days 0, 2, 4, and 6 according to protocol. Mixed macrochimerism were induced in groups treated with anti-CD154+anti-CTLA4-Ig, anti-CD154+anti-IL-2R, and anti-CD154+anti-CTLA4 Ig+anti-IL-2R. Three groups having chimerism enjoyed prolonged graft survival more than 6 months. Superantigen deletion study revealed deletion of alloreactive T cells in combined blockade treated groups. In graft versus host disease model using CFSE staining, CD4+ T cell and CD8+ T cell proliferation were reduced in groups treated with CTLA4-Ig or anti-IL-2R or both in combination with anti-CD154. However, anti-IL-2R was not so strong as CTLA4-Ig in terms of inhibition of T cell proliferation. In conclusion, IL-2 pathway blocking combined with anti-CD154 can establish macrochimerism with limited dose of BM transplantation and induce specific tolerance to allograft.
Subject(s)
Mice , Male , Animals , Skin Transplantation/immunology , Mice, Inbred BALB C , Interleukin-2/immunology , Immunoconjugates/administration & dosage , Graft Survival/immunology , Drug Combinations , CD40 Ligand/immunology , Bone Marrow Transplantation/immunology , Antibodies/administration & dosageABSTRACT
In order to investigate the effects of mouse CTLA4Ig gene-modified dendritic cells (DCs) on the survival of the corneal allografts in rats, the plasmid PG\CTLA4Ig was transfected into DCs of F344 rats mediated by LipofectamineTM 2000. The expression of CTLA4Ig was detected by immunofluorescent microscopy. The effects of donor DCs on the proliferation of T cells in Lewis rats (recipients) were tested by by CCK8. Corneal transplantation was performed from F344 rats to Lewis rats. The DCs modified with CTLA4Ig gene were injected into the Lewis rats on the day 0 and 3 after transplantation. The movement of the DCs after modification in vivo was observed by immunofluorescent microscopy, and the survival of corneal allografts was evaluated by Holland criterion. The results showed that the CTLA4Ig-modified DCs could restrain the proliferation of allogenetic T cells. The CTLA4Ig-modified DCs prolonged survival of corneal allografts. (P＜0.01). It was suggested that the injection of CTLA4Ig gene-modified DCs could obviously inhabit the allograft rejection and prolong the survival of corneal allografts.
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Objective To explore the curative effect and safety of CTLA-4Ig transgene for experimental autoimmune encephalomyelitis (EAE). Methods The adenovirus loaded CTLA-4Ig was injected into Wistar rat EAE models through the lateral cerebral ventricle. The clinical symptomatic and electrophysiological changes were observed to judge the efficiency of CTLA-4Ig. Results After injection of AdCTLA-4Ig into the lateral cerebral ventricle of Wistar rat EAE models, the time of EAE onset was delayed and the incidence rate was significantly reduced. And the transductive function tested by brain-stem auditory evoked potential (BAEP) and somatosensory evoked potential (SEP) was obviously improved. Conclusion CTLA-4Ig is effective in managing EAE.
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Objective To examine the role of adenovirus cytotoxic T lymphocytic associated antigen 4(Ad-CTLA4Ig) in treatment of induced Sjogren syndrome(SS) in mice.Methods SS was induced in 30 BALB/c mice by challenging with the mixture of homologous antigen from submandibular gland tissues and complete freunds adjuvant(CFA).Two hours after challenge,Ad-CTLA4Ig was intraperitoneally injected in the experimental mice(n=10),while thymic peptide in control mice(n=10).Morphological changes of submandibular gland,water intake and static total saliva flow rate of each group were observed.Results There was no obvious pathological change in Ad-CTLA4Ig treated group,in which the static total saliva flow rate was significantly higher than that in control groups(P
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BACKGROUND: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. METHODS: We have used CHO-dhfr cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. RESULTS: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. CONCLUSION: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , B7 Antigens , Blotting, Western , Cell Line , Electrophoresis, Polyacrylamide Gel , Encephalomyelitis, Autoimmune, Experimental , Graft Rejection , Graft vs Host Disease , Homicide , Immunosuppression Therapy , Lymphocyte Culture Test, Mixed , Lymphocytes , Silver Staining , Staphylococcal Protein A , T-LymphocytesABSTRACT
BACKGROUND: Atopic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Several reports have suggested an important role for costimulation through the CD28/CTLA4 (cytotoxic T lymphocyte-associated antigen 4)-B7 (CD80/CD86) pathway in allergen activation of T cells in animal models of allergen-induced asthma, because B7-CD28/ CTLA4 interaction can promote the differentiation and development of the Th2 lymphocyte subset. OBJECTIVE: In the present study, we intended to investigate a potential role of humanized CTLA4-Ig on the inhibition of T and B cell activation by blocking B7/CD28 interactions. METHOD: For this purpose we produced humanized CTLA4-Ig fusion protein by transfection to CHO cell and examined its inhibitory effects for activated T and B cell responses. We evaluated the inhibitory effect of MLR (mixed lymphocyte reaction) and con A-stimulated T cell proliferation. And we assayed wheather B cell was inhibited by stimulation of costimulatory signal in LPS-induced B cell response and PFC assay. RESULT: In vitro assay, humanized CTLA4-Ig fusion protein inhibited T cell-specific immune response in dose-dependent manner: CTLA4-Ig inhibited allogeneic stimulation in murine MLR, and the proliferation of T cell by the stimulation of Con A. But CTLA4-Ig did not inhibit directly the proliferative response of B cell by the stimulation of LPS. In addition, in vivo assay, CTLA4-Ig inhibited the production of antibody from B cell, which was presented by plaque-forming cell (PFC) assay. CONCLUSION: These findings suggest that humanized CTLA4-Ig is effective to inhibit the proliferation of activated T cell directly by blocking B7/CD28 costimulation. And humanized CTLA4-Ig influences antibody-producing capacity of B cell indirectly by regulating T cell.
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Animals , Cricetinae , Humans , Abatacept , Antibody Formation , Asthma , Cell Proliferation , CHO Cells , Lymphocyte Subsets , Lymphocytes , Models, Animal , Mucous Membrane , T-Lymphocytes , TransfectionABSTRACT
AIM: To study the effect of rhCT-LAIg on human T lymphocytes. METHODS: Primary mixed lymphocyte reaction (MLR), secondary MLR and cytotoxicity assay were used. RESULTS: CTLA4Ig significantly inhibited primary MLR(P<0.05) and the maximal inhibition was achieved at 72 h of incubation with CTLA4Ig. Secondary MLR experiment showed cells primed in the presence of CT-LA4Ig had a specific hyporesponsiveness when challenged with PBMC from the original donor, yet responded normally to PBMC from the third party donor. CONCLUSIONS: CTLA4Ig can inhibit T cell proliferation and induce T cell anergy in vitro by blocking B7/CD28 co-stimulatory pathway.
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Objective:To study the inhibited function of CTLA4 Ig on T cell activation and its mechanism Methods:After separating fresh PBMC,the effects of CTLA4 Ig on T lymphocyte transformation and mixed lymphocyte reaction were analyzed,and its inhibitory effect on the CD25 expression on T cell surface was observed At last,the changes of the nuclear protein in active T cell were analyzed by the EMSA method.Results:CTLA4 Ig has an inhibitory effect on the lymphocyte transformation,MLR and CD25 expression on T cell surface,and it can inhibite the activation of RE/AP binding factor related to IL 2 expression. Conclusion:CTLA4 Ig inhibit the activation of T cell by multipathway,one of the mechanism might be its inhibitory effect on the activation of RE/AP binding factor.
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Objective To study CTLA4Ig gene expressed in dermal papilla cells and to provide evidence for immune tolerance after dermal papilla cells transplantation. Methods CTLA4Ig cDNA was transferred into dermal papilla cells by recombinant adenovirus vector, and the dermal papilla cells containing CTLA4Ig gene were transplanted into mice skin. The target gene expression was detected by histological and immunohistochemistry technique. Results CTLA4Ig protein was expressed in plasma 6 hours after gene transfection and increased gradually. When the transferred papilla cells were transplanted into mice skin the gene began to express in 24 hours and lasted for 2 weeks. No rejection was observed. Conclusion Dermal papilla cells containing CTLA4Ig gene can survive in vitro and in vivo and express CTLA4Ig for a long time.
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Objective To study the effects of CTLA4-Ig on mu rine allergic contact dermatitis.Methods Mice were exposed to DNFB to induce allergic contact dermatitis and were i njected with CTLA4-Ig.Ear swelling was measured 24h after antigen challenge.Splenocytes from treated mice were assayed for their ability to prolif erate in response to DNFB or FITC stimulation in vitro.Results Profound inhibition of contact hypersensitivity response(CHS )was shown by 69.7%in mice treated with CTLA4-Ig compared with mice treate d with PBS control.CT-LA4-Ig-treated mice displayed DNFB-specific tolerance,but exhibited a vigorous immune response to FITC when re-sensitizing 14days after the fir st challenge.Adoptive transfer of l ymphocytes from CTLA4-Ig-treated mice could induce inhibition of CHS in recipien t mice.Conclusions CTLA4-Ig can inhibit CHS by blocking B7/CD28co-stimulatory pathway,which provides a new way to suppress typeⅣallergic reaction.[
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AIM: To investigate the feasibility and infection efficiency of MSCs as the target cells of gene delivery mediated by adenoviral vector carrying CTLA4Ig gene, and to study the mechanism of transgenic MSC to inhibit immune response ex vivo. METHODS: The recombinant adenovirus containing CTLA4Ig gene was constructed, by which rat MSCs with various multiplicity of infection (MOI) were conducted. The infection efficiency was analyzed with FACS and fluorescence microscope. The expression of CTLA4Ig protein in transgenic MSCs was detected by FACS and western blot. Co-culturing the transgenic MSCs with mixed lymphocytes, the inhibitory effect of transgenic MSCs on lymphocyte proliferation was also observed. RESULTS: The adenoviral vector delivered CTLA4Ig gene with high efficiency to MSCs. The expression of CTLA4Ig protein was detected in transgenic MSCs. The gene modified MSCs inhibited the proliferation of mixed lymphocytes and maximal inhibition rate was observed on day 4 of MLR. The inhibition induced by CTLA4Ig was donor-specific. CONCLUSION: MSCs is a promising target cell for gene delivery. The expressed CTLA4Ig specifically inhibits the lymphocyte proliferation ex vivo.
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Objective: To determine the anti rejection effects of CTLA 4 Ig fusion protein on cardiac allografts in mice and to discuss its mechanism in vivo . Methods: BALB/c recipients were performed cervical heterotopic heart transplantation to receive C57BL/6 donor hearts with a cuff technique. BALB/c recipients were intraperitoneally injected with CTLA 4 Ig [100 ?g/d?15 times], control immunoglobins and PBS to observe the survival time of allografts with ECG. The hyporesponsiveness of splenic T cell, the polarization of the T subsets were analyzed after the recipients treated with CTLA 4 Ig. Results: After treated with CTLA 4 Ig, the survival of cardiac grafts was significantly prolonged compared with the control groups, and more than 40% cardiac grafts survived over 2 months. The splenic T cells isolated from recipients did not respond to restimulation of donor splenocytes in MLR, but did exhibit the capacity to proliferate in response to C3H splenocytes(third party).The levels of IL 2 and IFN ? decreased and the level of IL 10 increased in CTLA 4 Ig treated mice. Conclusion: Administration of CTLA 4 Ig can induce donor specific tolerance, which induce T subsets to polarize toward Th2 subset and hyporesponsiveness to alloantigen, and prolong the survival time of the cardiac grafts effectively. [
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Objective:To investigate the effect of anti-CD134 mAb or CTLA4Ig on ConA induced splenic cell proliferation,Th cytokine secretion and production of anti-dsDNA antibody from splenic lymphtocyte in vitro in lupus-prone BXSB mice. Methods:Eighteen male lupus-prone BXSB mice model and 6 syngeneic normal C57BL/6 male mice were used in the experiment. The model mice were divided into three groups:un-treated group,Lupus recipe(LR) treated group and prednisone(pred. ) treated group. The mice's splenic cell suspension from above groups was culture stimulated by ConA respectively. The splenic cells from un-treated model mice were further divided into Anti-GD134L mAb,CTLA4Ig or Anti-CD134L mAb + CTLA4Ig treated subgroups. The ConA induced splenic cell proliferation was measured by MTT colorimetric assay. The levels of IFN-?, IL-6 and anti-dsDNA antibody in cell supernatant were measured by ELISA. Results; (1 )The splenic cell proliferative reaction and contents of IFN-?,IL-6 and anti-dsDNA antibody in cell supernatant of either spontaneous or ConA induced culture in the un-treated model group were obviously higher than that of the normal control or other groups. (2) The splenic cell proliferative reaction and production of IFN-?,IL-6 and anti-dsDNA antibody in the CD134L/CTLA4Ig treated group,LR treated goup or pred. treated group was not different from the normal control significantly. (3)To compared with CD134L treated group or CTLA4Ig treated gruop,the CD134L/CTLA4Ig and prednisone reduced significantly the splenic cell proliferative reaction and production of IFN-?,IL-6 and anti-dsDNA antibody in cell supernatant of either spontaneous or ConA induced culture,while no difference was found between CD134L treated group and CTLA4Ig treated proup. Conclusion:The lupus-prone BXSB mice might present abnormal lymphocyte proliferation,spontaneously express cytokines and secrete high level of autoantibody during the SLE development. LR and corticosteroids could obviously inhibit the abnormal lymphocyte proliferation;reduce the Th cytokine formation and antoantibody production Blockade of CD134-CD134L or B7-CD28 costimulatory pathway by Anti-CD134L rnAb or CT-LA4Ig could inhibit the activation of T cells and B cells like LR and corticosteroids. Furthermore, by blockade of both CD134-CD134L and CD28-B7 pathways,the frequency of alloreactive T cell was markedly reduced and was maintained at low levels so as to treat SLE effectively.