The Preliminary Study Of Immunological Mechanisms For The Induction Of Donor Specific Tolerance By Ethylenecarbodiimide-treated Human Allogeneic Antigen Presenting Cells

ECDI(Ethylene carbodiimide)is a kind of dehydrating agent and activator used in industry.In 1966,H.M.Johnson et al.first found that ECDI was able to link bovine serum albumin(BSA)to rabbit red blood cells.In 1979,Miller.D first described that ECDI-fixed syngeneic splenocytes and self-antigen peptides could induce autoimmune tolerance.Recently,ECDI-treated donor splenocytes(ECDI-SPs)was used for transplant tolerance induction by Xunrong Luo et al.They found that ECDI-treated donor splenocytes(ECDI-SPs)were able to induce long-term allograft tolerance in full MHC-mismatched allogeneic transplantation mice models.It suggested that ECDI-SPs has a clear and efficient role to induce transplant tolerance in rodent transplantation models.In this study,mixed lymphocyte reaction(MLR)system including PBMCs and ECDI-treated antigen presenting cells(ECDI-APCs)from three health volunteers,whose HLA-A,-B and-DR fully mismatched,were set up to mimic transplant circumstances in vitro.It was proposed to investigate the effect and mechanism of ECDI-APCs to induce human donor-specific tolerance for T cells,which might hold promise for induction of clinical transplant tolerance in the future.PART 1 ECDI-APCs induced the responder T cells tolerance to allogenic antigen stimulationIn this part,ECDI-APCs and PBMCs from three HLA fully mismatched individuals were established a system to mimic transplant circumstances in vitro.On day-6(day 0 being the time for allogenic antigen simulation),ECDI-treated donor APCs from donor and peripheral blood mononuclear cells(PBMCs)from responder were co-cultured in MLR system.On day 0,freshly isolated APCs from priming donor(A-APCs)were added as stimulating cells.On day 8,the dilution of CFSE in responder T cells was detected by flow cytometry to understand the proliferate status of responder T cells.In order to further confirm whether the inhibition effect induced by ECDI-APCs is donor antigen specific,HLA-A,-B and-DR fully mismatched indifferent donor APCs(I-APCs)was used as stimulating cells in the same MLRs system on day 0.Furthermore,the effects of ECDI treated APCs in different concentrations(75 mg/ml,150 mg/ml,300mg/ml and 500 mg/ml),and the ratios for donor ECDI-APCs to responder PBMCs at 1:1,0.5:1,0.2:1,0.1:1 and 0.05:1)in co-cultured system,were also investigated for responder T cells proliferation on day 8 in MLRs system.Data showed that,ECDI-treated donor APCs treatment does not have the ability to stimulate the activation and proliferation of allogeneic T cells from responder.When they were restimulated with APCs from original donor,responder T cells primed with ECDI-APCs showed a certain degree of tolerance.The tolerance of responder CD8+T cells was specific to the donor antigen,while the tolerance of responder CD4+T cells was non-antigen-specific.When donor APCs were treated with ECDI at 150mg/ml,the ratio of ECDI-APCs to responder PBMCs was 0.1:1 in prime co-culture system,the proliferation rate of responder CD8+T cells was significantly lower than other groups and also showed donor specific inhibitory effect.This part of work established a mimic transplant system in vitro and determined the optimal condition for their experiments of ECDI-APCs could induce donor specific tolerance.PART2 A pilot study of the mechanism for ECDI-APCs induced donor specific toleranceBased on the established mimic transplant system in vitro,in this part,the apoptosis of donor cells induced by ECDI was observed for the first time.And,on day 8,we intend to explore the proportion of CD4+CD25+Foxp3+regulatory T cells(Tregs),the expression of negative regulatory molecules on responder CD4+T cells,and cytokines in cultural supernatant.It has been reported that the apoptosis induced by ECDI is one of the mechanisms for its induction tolerance.In this part of the experiment,we observed the apoptosis effects of different ECDI concentrations(75mg/ml,150mg/ml and 500mg/ml)in culture system in vitro.Results showed that donor APCs treated with ECDI(0h)at 150mg/ml,the proportion of early apoptotic cells was increased while the proportion of necrotic cells was decreased significantly.However,the proportion of apoptosis induced by different concentrations of ECDI would increase gradually and reach a similar level at 120h.In the further analysis of the effects of apoptotic cell components(early apoptotic cells or early necrotic cells)on tolerance induction,we found that early apoptotic cells are the main factors to induce the proliferation inhibition of responder CD8+T cells.In addition,we also found that responders APCs is necessary for transplant tolerance induced by donor ECDI-APCs,indicating the importance of the indirect pathway in tolerance induction.Tregs play an important role in inducing immune tolerance.The detection of Tregs in this system revealed that ECDI-APCs pretreatment could specifically induce the production of Tregs.Moreover,the detection of cytokines in culture supernatants showed that the expression of IL-10 was significantly increased,while the expression of IFN-y,IL-6 and IL-17A significantly decreased under the ECDI-APCs primary incubation.And,the effects of different concentrations of ECDI on tolerance induction is associated with expression of cytokines.ECDI-APCs treatment could obviously regulate the expression of negative regulatory molecules(CTLA-4、Tim 3 and PD-1)on responder CD4+T cells in the context of ECDI-APCs pretreated with responder PBMCs for 6 days.In conclusion,this part of work explored the potential mechanisms of ECDI-APCs induced tolerance in mimic transplant circumstances in vitro.