CD80mAb And CD40LmAb Synergistically Enhance Immature Dendritic Cells To Induce Donor-specific Immune Tolerance In Rat Pancreaticoduodenal Transplantation

Objective:To observe the effection of co-stimulation blockade CD80mAb and CD40LmAb synergistically enhance immature dendritic cells to induce donor-specific immune tolerance in rat pancreaticoduodenal transplantation . Methods:After injection iv with three different does of Alloxan (45mg/kg ,50mg/kg ,55mg/kg),the changes of blood sugar and rat survival time of each group were observed.These diabetic rats served as recipients. The donor pancreaticodudodenal is excised with en bloc technique while a fine needle with a infusion was introduced into the aorta distal and perfused continiously until bloodless with 7-8 ml of cold (4℃)saline,then the graft including stomach and part colon was clipped . The portal venous of the graft was anastomosed with the recipent's left renal vein by a cuff technique.End to side anastomosis was performed for artery employing 8-0 nylon sutures,a side to side anastonosis was made between the graft duodenum and the host jejunum.2×10~6 1B1 and 4E5 hybridoma cell strain was injected ip to BABL/C mice,then,CD80mAb and CD40LmAb from ascites produced in BABL/C mice were purified through protein G affinity colum.The pre-DC was dissociated from donor marrow, and stimulated with GM-CSF and IL-4 in vitro.After co-cultured with IL-10 for 8 days,we detected the expression of co-stimulation molecular and MHC-Ⅱ.7 days before transplantation, 2×10~6 imDC was injected iv to all recipitients.1ml NS,5mg of CD80mAb,CD40LmAb,and CD80mAb+CD40LmAb were injected ip on the day imDC infused and 0.5mg per day for the following consecutive 14 days.We observed: 1.Survival times of recipients after transplantation.2. The pathologic changes of pancreas and duodenm 14 days of transplantation.3. Donor spleen T cell were used as stimulor cells,and recipient spleen T cells obtained 7,14 days after transplantation were used as responders.[3H]TdR incorporation of cells were used to assess the T cell hyporesponsiveness of recipient rat against donor-derived cells after MLR. 4.The supernatants from MLR cultured on day 3 in vitro were harvested and subjected to the detection of IL-2,IFN-γ,IL-4,IL-10 concentrations with ELISA kits according to the manafacture's instructions,respectively. 5. Level of IgG and IgM alloantibody in the recipients sera 7 and 21 days after transplantation. Results:It was a effective method to obtain diabetic rat model by vein injection with a dose of 50mg/kg Alloxan,and the blood surgar concentration was over 16.6mmol/L in 93.3% rats.The 24h survival rate was 92% after allotransplantation of pancreaticoduodenal.The donor harvesting time was 40±3min,and warm ischemia time was 1±1min;The recipient's operation time was 55±4min,and the cold ischemia time was 50±2min.The concentrationof blood surgar before transplantation was 28.7±1.7mmol/L,whereas,it decent to normal level after allotransplantation;The titer in ascitic fluid extrated by hybridoma technique of 4E5 and 1B1 was 1:1000,and the concentration was 2.13mg/ml and 1.87mg/ml respectively.The expression of co-stimulatory molecules and MHC-Ⅱafter co-cultured with IL-10 in pre-DC was 31%,28%,28% .The difference was statistically significient compared with controls. When combined treated of CD80mAb and CD40LmAb,the median survival time was improved from 12.69 days of the contral group (imDC+NS infusion) to 92.03 days.Histological examination of pancreaticoduodenal graft 14 days after transplantation in experimental group indicated that:the structure of pancreas glandular lobule exist, and a small quantity of pancreas gland was destructed .The allograft infiltrated by numerous of leukocytes,and a few of fibrous tissue proliferated .There were no obvious changes of mucosa and gland in duodenum. Whereas, there were severe destruction of graft in contral group: mass of fibrous tissue hyperplasia could be seen everywhere, but pancreas gland cells were seldom seen.7 and 21 days after transplantation ,when MRL responses at 1:4 stimulator: responder cell ratios, [3H]incorporation (cpm)in imDC+NS group was 39870±1252 and 41250±1458; whereas in imDC+CD80mAb+CD40LmAb group,the cpm was 9908±542 and 9873±173,the difference was statistically significient .But with the gradual increase of stimulator : respondense ratio, the difference of four groups was no significient. Incontrol group, cytokine profile of IL-2,INF-γin the supernatants from MLR cultrues from recipients 7 days after transplantation was 307.3±21.1 pg/ml and 147.6±9.2 pg/ml. But in imDC+CD80mAb+CD40LmAb group, the concentration was 21.9±1.7pg/ml and 23.5±2.1pg/ml.In imDC+NS group, the level of IL-4,IL-10 was 19.2±2.7pg/ml and 82.3±6.9pg/ml.But in imDC+CD80mAb+CD40LmAb group,it was 17.4±1.2pg/ml and 164.1±9.3pg/ml. The difference of IL-10 was statistically significant. 7 days after transplantation, IgM level of recipient sera in imDC+NS group was higher,but 21 days after transplantation, it decreased obviously.Whereas there was no different change in imDC+ CD80mAb+CD40LmAb group.7 days after transplantation,IgG level of recipient sera in imDC+NS group was lower,but it increased obviously 21 days after transplantation. Conclusions: 1.It was a effective method that injection iv Alloxan with a does of 50mg/kg to build a diabetic rat model. 2.It was a accessible procedure to build rat pancreaticoduodenal transplantation model based on microsurgical technique and a improved method in obtaining donor. 3.The co-stimulation blockade CD80mAb,CD40LmAb can synergistically enhance imDC to induce donor-specific T cell tolerance with donor graft,and suppressed acute and chronic rejection by host versus graft resulted in indefinite survival of pancreaticoduodenal allograft.