CTLA4Ig Modified DCS Induce Immune Tolerance To Oxidized-Low Density Lipoprotein

PartⅠCTLA4Ig induces immune anergy of T cells to oxidized-low density lipoprotein in vitro.Aim:Recently it is accepted that atherosclerosis is a disease of inflammation,and immune reaction is the most important element in inflammation.The immune elements related to atherosclerosis include dendritic cells(DC),T cells and oxidized-low density lipoprotein(ox-LDL).In this study,CTLA4Ig was used to block the co-stimulation signals between DCs and T cells to induce anergy of T cells to ox-LDL.Methods:Monocytes were separated from peripheral blood by adhension methods and cultured with rhGM-CSF 500IU/ml and rhIL-4 500IU/ml to get DCs.After 6 days culture,cells were treated with PBS,LPS(20ng/ml),LDL(10μg/ml)and ox-LDL(10μg/ml)for 48 hours.Phenotype(CD14,CD86 and HLA-DR)were analysed by flow cytometry(FCM).IL-12 secreted by DCs was assayed by ELISA method.The treated DC were mixed with allogeneic T lymphocytes for further 4 days.CTLA4Ig of different concentration(0,0.31,0.62,1.25μg/ml)were added at the beginning of the mix.The proliferation of Tlymphocytes was analysed by MTT method.Results:1.Ox-LDL stimulated DCs to secret IL-12 more than PBS and LDL significantly.Expression of CD86 and HLA-DR of DCs treated with LPS and ox-LDL were significantly higher than that with PBS and LDL(P＜0.05, respectively).2.DCs treated with LPS and ox-LDL stimulated the allogenic lymphocyte mix reaction significantly than that treated with PBS and LDL(P＜0.05, respectively).3.Ox-LDL and LPS treated DCs stimulated T cells in MLR to secret IL-2,IFNγand IL-4 significantly higher than those of PBS and LDL(P＜0.05, respectively).4.CTLA4Ig suppressed the allogenic lymphocyte mix reaction significantly with significant dose-dependent effect(P＜0.05,P＜0.01,respectively).5. CTLA4Ig inhibited CD25 expression in MLR stimulated by ox-LDL treated DCs significantly compared with that in absence of CTLA4Ig(P＜0.05,P＜0.01, respectively).6.CTLA4Ig induced T cell apoptosis in MLR significantly compared with that in absence of CTLA4Ig(P＜0.01,respectively).7.CTLA4Ig inhibited ELISpot counts of IL-2 and IFNγsignificantly compared with that in absence of CTLA4Ig,while increased ELISpot counts of IL-4(P＜0.05,P＜0.01,respectively).Conclusions:1.Ox-LDL is an antigen.2.Ox-LDL can stimulates both Thl immune reaction and Th2 immune reaction.3.CTLA4Ig can induced T cell anergy to ox-LDL in vitro.4.CTLA4Ig can induce Th1/Th2 deviation in MLR.5.CTLA4Ig can inhibit T cell activation in MLR.6.CTLA4Ig can induce T cell apoptosis in MLR. PartⅡInfluence of CTLA4Ig gene transfection on immune function of DCs to oxidized-low density lipoprotein.Aim:Now it is accepted that atherosclerosis is one kind of inflammation disease, and DCs are concerned with atherosclerosis.CTLA4Ig can combine with B7 on DCs to block costimulation signals conducted between DCs and T cells.In this study,DCs derived from peripheral mononuclear cells were transfected with CTLA4Ig gene by adenovirus,to detect the influence on immune function of DCs to oxidized-low density lipoprotein.Methods:Monocytes were separated from peripheral blood by adhension methods and cultured with rhGM-CSF 500IU/ml and rhlL-4 500IU/ml to get DCs.After 4 days culture,DCs were transfected with adenovirus with CTLA4Ig gene(Ad-CTLA4Ig)at different multiplicity of infection(MOI)(MOI=100,MOI=200 and MOI=300 respectively)and cultured for further 4 days.DCs transinfected with Ad-EGFP and non-transfected DCs were used as controls.CTLA4Ig expression was detected by ELISA,fluorescence microscope,flow cytometry and Western blot respectively. DCs transfected for 3 days were treated with ox-LDL(10ug/ml)for further 48 hours. IL-12 secretion was assayed by ELISA,and phenotypes(CD86,HLA-DR)were tested by flow cytometry.DCs(Ad-CTLA4Ig DC,Ad-EGFP DC,un-transfected DC, un-transfected DC with 10ug/ml CTLA4Ig)treated with ox-LDL were mixed with allogeniec T cells for further 4 days.CD25 expression of T cells in MLR and apoptosis of T cells in MLR were assayed by flow cytometry.Cytokine secretion (IL-2,IL-4 and IFNγ)were assayed by ELISpot.Results:1.The climax of CTLA4Ig expression is at the time after 3 days transfection at MOI=200.2.Secretion of IL-12 of DCs transfected with Ad-CTLA4Ig were lower than those of DCs transfected with Ad-EGFP and un-tranfected significantly(P＜0.05,respectively).3.CTLA4Ig transfection inhibited expression of CD86 and HLA-DR significantly(P＜0.05,respectively).4.SI of MLR stimulated with DC transfected with Ad-CTLA4Ig was inhibited significantly than those of DCs transfected with Ad-EGFP and un-tranfected(P＜0.05,respectively).5.CD25 expression of T cells in MLR stimulated with DCs transfected with Ad-CTLA4Ig was lower than than those of DCs transfected with Ad-EGFP and un-tranfected significantly(P＜0.05,respectively).6.Apoptosis of T cells in MLR stimulated with DCs transfected with Ad-CTLA4Ig was higher than than those of DCs transfected with Ad-EGFP and un-tranfected significantly(P＜0.05,respectively).7.DCs transfected with Ad-CTLA4Ig inhibited seretion of IL-2 and IFNγsignificantly compared with DCs transfected with Ad-EGFP and un-tranfected significantly (P＜0.05,respectively),while stimulated IL-4 secretion significantly(P＜0.05, respectively).Conclusions:1.The appropriate transfection time of Ad-CTLA4Ig on DCs was 3 days,and the appropriate transfection MOI=200.2.Ad-CTLA4Ig transfection decreases expression of CD86 and HLA-DR on DCs,and attenuates function of DCs to stimulate MLR.So Ad-CTLA4Ig transfection retards maturation of DCs.3.DCs tranfected with Ad-CTLA4Ig can induce immune anergy ofT cells to ox-LDU 4.DCs tranfected with Ad-CTLA4Ig induce immune anergy to ox-LDL through mechanisms of inhibiting T cells activation,stimulation of T cells apoptosis and induction of Th1/Th2 deviation.