The Experiment Of CTLA4-Ig Induce Mixed Spleen Cells Of Adaptor And Receptor Anergic And That Mediate Renal Allograft Tolerance In Rats

Introduction and purpose:Kidney transplantation is the most effective therapy for end-stage renal diseases.Allografts have higher long-term survival rate for successful renal transplantation.The transplanted patients'long-term surviving rate and quality of life are superior to dialyzed patients',The technique of kidney transplantation has gradually been mature at present,the reject reaction after transplantation is the main factor affecting the survival rate of transplanted kidneys.Although the advent of immunosuppressive agents in renal transplantation has got milestone progress, but daily administration of non-specific immunosuppressive agents easily leads to double infection,cancer and other serious complications,the vast majority of calcineurin inhibitors have nephrotoxicity.Therefore, the induction of immune tolerance of the graft is the best way to address the rejection,and it has been a hot research in today's academia on transplantation.Rejection in kidney transplantation is due to abnormal activation of T cells,activation of T cells depends on the congenerous effect of the first signal and the second signal (costimulatory signal).The first signal is the combination of major histocompatibility molecules-antigen(MHC-Ag) presented by antigen-presenting cells (APCs) and the T cell surface receptors (T cell receptor, TCR ).The second signal is the combination of costimulatory molecules on the T cell surface and ligand on APCs, including B7-CD28/CTLA4, CD40-CD154 and so on.one of the most important from them is the combination of B7 molecules on the antigen-presenting cells and CD28/CTLA4 on the T cell .Cytotoxic T lymphocyte associated antigen 4 (CTLA4) is a transmembrane glycoprotein on cytotoxic T lymphocytes (CTL).CTLA4 can block the B7/CD28 costimulatory signal pathway, so that reactive cells received allogeneic antigen become anergic, and even death, so that cells'proliferation and differentiation into the role of lymphocyte cytotoxicity are inhibited, inducing antigen-specific lymphocytes to be anergic T cells.CTLA4-Ig is a fusion protein combined the extracellular domain of CTLA4 with amino acids on CH2,CH3 andγzone of IgFc section,which remains keep the biological characteristics of B7,it can be dissolved in the water and has the advantage of easy purification.Recently, many research have reported the suppression of anergic T cells in vivo and in vitro.Treatment with anergic T cells was used in human bone marrow transplantation with a relatively low risk of graft—versus—host disease.Therefore,it has been proposed that anergic T cells generated ex vivo may also induce solid organ allograft long-term survival in humans.In this experiment,we used the rats as the research objects,prepared the anergic T cells by blocking the B7/CD28 co-stimulatory pathway with CTLA4Ig,Then we transferred the anergic T cells to renal transplant recipients by the tail vein injection to explore that the anergic T cells can induce renal allograft tolerance and its mechanism.We have found a new safe and effective method for clinical kidney transplantation on treatment of rejection.Methods:1.The preparation of suspension of rat splenocytes.2.Mixed lymphocyte reaction:CTLA4Ig was added to the mixed lymphocyte reaction of SD rat splenocytes as responding cells and Wistar rat splenocytes as stimulus cells,testing whether or not the proliferation happened for mixed donor and recipient spleen cells, non-proliferation should be anergic T cells. Further we test that the anergic T cells can inhibit in vitro activity of allogeneic T cell response,detecting that inhabitation of anergic T cells can be reversed by rIL-2.3.Carried out the isotype orthotopic renal transplantation.4.Experimental infusion of cells and division: the 34 receptor SD rats were randomly divided into A,B,C,D four groups,group A was the experimental group,B,C,D groups were the control groups.Packet based on the type of infused cell, group A: infused(donor spleen cells+receptor spleen cells+CTLA4-Ig)the prepared 4×107 anergic T cells (10 rats);group B: infused 4×107 mixed donor and recipient spleen cells(10 rats) ; group C :infused(third-party spleen cells + receptor spleen cells + CTLA4-Ig)the prepared 4×107 anergic T cells(8 rats);group D: infused 1ml PBS (6 rats). 5.Observed the survival time of allografts and histopathological changes: recorded the survival time of allografts in 21 recipients;another 13 recipients were done section 3,11,28,60 days after renal transplantation,each day taken a body to produce renal biopsy by light microscopy and observe the histopathological changes,do system integration Banff score.Results:1.In the primary MLR of donor splenocytes and recipient splenocytes,CTLA4-Ig inhibited T cell proliferation,with the addition of concentration,the ability of inhibitory enhanced,the proliferation in CTLA4-Ig 10μg/ml was completely inhibited.And in PBS control group ,the proliferation of MLR occurred.Each comparison of proliferation values for two concentrations:p<0.05,the difference was statistically significant.2.CTLA4-Ig inhabited the MLR of the same donor splenocytes and recipient splenocytes,with the addition of anergic cells/mixed lymphocyte ratio,the ability of inhibitory enhanced.Each comparison of proliferation values for two ratios:p<0.05,the difference was statistically significant.3. Adding rIL-2 to the secondary MLR of anergic T cells and adnor spleen cells as stimulating cells,with the addition of rIL-2,the ability of reversion enhanced.And high-dose rIL-2 (100 U/ml), caused the complete reversion of the state.Each comparison of proliferation values for two concentrations:p<0.05,the difference was statistically significant.4.Compare the mean survival time (MST) of each group rats: Animals group A showed a MST of 102.0±92.0 days, which was significantly longer (all p<0.05) than MST of 10.6±2.3 days of animals group B or a MST of 18.6±2.3 days in animals group C or a MST of 8.0±1.8 days in animals group D.5. Renal pathological changes in the organization:(1) visual observation: Renal grafs in group A,the size of renal normal,the surface of red, uniform, bright;Renal grafs in the other three groups have shown renal swelling and bleeding,increased volume,quality hard and brittle,the surface is dark purple, was called"Raise the Red Kidney".(2) light microscopy:Renal grafs in group A showed only a small number of lymphocytes and mononuclear cell infiltration with renal interstitial,no infiltration of renal arterioles,glomerulus,renal tubular normal.Renal grafs in the other three groups showed acute cellular or vascular rejection.(3) system integration Banff score:total score of transplanted kidney in group A rats was lower than other three groups,histological injury was lighter than the other three groups (all p<0.05,statistically significant).Conclusion:1.The research ex vivo showed that CTLA4-Ig induced the production of anergic T cells by blocking the B7/CD28 pathway.2.Anergic T cells was transferred to the allogeneic graft,inhabited the reject reaction of rat allogeneic renal transplantation,prolonging the survival time of renal allografts.