Effects Of Donor-derived Spleen Cells Combined With Anti CD154 Monoclonal Antibody On Bone Marrow Transplantation In Non-myeloablative Preconditioning Regimen For The Induction Of Allogeneic Mixed Chimerism

Objective To explore the role and possible mechanisms of donor-derived spleen cells(Scs) and it's transfusion pathway combined with anti CD154 monoclonal antibody(mAb) on bone marrow transplantation in non-myeloablative preconditioning regimen for induction of allogeneic mixed chimerism and donor-specific immune tolerance.Methods We adopt Lewis(Rtl¹) as donor,Wistar-Furth(Wistar,Rtl^u) as recipient and Sprague-Dawley(SD) as the third party donor.Scs and bone marrow cells(BMCs) were harvested from donor Lewis rats.Recipient Wistar rats were randomly divided into 4 groups,20 rats in each group. Group Scs_i.v.,penis dorsal vein(i.v.) injection of Lewis rats 2×10⁸ Scs on day 0 alone. Group Scs_P.V.,portal vein(P.V.) injection of Lewis rats 2×10⁸ Scs on day 0 alone. Group Scs_P.V.+BMCs_i.v.,P.V.injection of Lewis rats 2×10⁸ Scs on day 0,in combination with i.v.injection of Lewis rats 1×10⁸ BMCs on day 3. Group Scs_P.V.+ AH.F5(hamster anti-rat CD154 mAb)+BMCs_i.v.,P.V.injection of Lewis rats 2×10⁸ Scs on day 0,intraperitoneal injection 3 mg AH.F5 2 hours later,in combination with i.v.injection of Lewis rats 1×10⁸ BMCs on day 3.On day 20 and 40, enzyme linked immunosorbent assay(ELISA) to determine the levels of the serum interleukin(IL)-10 and interferon(IFN)-γ,were performed.Tolerance was assessed by stimulation index(SI) of one-way mixed lymphocyte reaction(MLR),rat recombined IL-2 reverse assay of one-way MLR on day 20,40 and foodpad increment(FI) of delayed type hypersensitivity(DTH) on day 50.Rtl¹ positive chimersim in recipients spleen and thymus were followed by fluorescenceactivited cell sortor(FACS) on day 20,40.Group SCS_P.V.+AH.F5+BMCs_i.v was additionally carried on chimerism detection on day 100.Moreover,tolerance mechanism was investigated.Results The levels of cytokine were invariable in group Scs_i.v. on day 20,40.While,the levels of IL-10 and IFN-γin group Scs_P.V.+AH.F5+BMCs_i.v. were individual(42.01±4.87) pg/mL and(28.97±3.83) pg/mL on day 20,(40.19±5.30) pg/mL and(25.89±4.30) pg/mL on day 40.UP-regulation of IL-10 was significant higher than other groups, down-regulation of IFN-γwas significant difference from groups Scs_P.V. and Scs_P.V.+BMCs_i.v. The balance of Th1/Th2 alter to Th2 in group SCS_P.V.+AH.F5+BMCs_i.v.,to Th1 in groups Scs_P.V. and Scs_P.V.+BMCs_i.v. SI values were(4.43±0.19) and(4.97±0.25) in group Scs_P.V.+AH.F5+BMCs_i.v. on day 20,40. There were significant lower than other groups(P<0.01).IL-2 added to one-way MLR can partly reverse immue hyporesponsiveness to allogeneic antibody. Meanwhile,Scs derived from this group had strong activity of proliferation to Scs from the third party SD rats.In DTH test,only group Scs_P.V.+AH.F5+BMCs_i.v. had suppressive activity.FI values of other groups had no significant difference from normal control group(P>0.05).The results of Rtl¹⁺ assay reveal that there was no chimersim in group Scs_i.v..While chimersim was transient and low in groups Scs_P.V. and Scs_P.V.+BMCs_i.v. on day 20,and rapidly descend,even disappeared on day 40. Only group SCS_P.V.+AH.F5+BMCs_i.v. had stable and higher level chimerism in spleen and thymus on day 20,40.Furthermore,long-term chimerism(>100 days) was observed in the group.Conclusion I.V.injection of 2×10⁸ Scs alone can't induce chimerism and tolerance.Transfusion pathway of Scs via P.V.can only induce gentle tolerance and establish short-course(< 20 days) and low level(< 20%) peripheral chimerism.Combined with 1×10⁸ BMCs can partly enhance tolerance and prolong the existence of chimerism to 40 days,but not stable.However,only combined with anti CD 154 mAb can significantly promote BMCs engraftment.High level of chimerism (>30%in peripheral spleen and >20%in central thymus) provide the foundation to BMCs to play a critical role in the induction or maintenance of tolerance.Our studies demonstrate that P.V.injection of Scs and anti CD154 mAb,plus i.v.injection of BMCs have synergistic effect to induce donor-specific tolerance and establish stable allogeneic mixed chimersim,without participation of megadose BMCs, myleosuppressive drugs and irradiation.Multiple mechanisms,including altering the balance in Th1/Th2,T cells clonal anergy and clonal deletion are involved in the tolerance. Objective To provide a tool for research grafts rejection and to explore the key technique of this operation,we establish the model of allogeneic whole pancreaticoduodenal transplantation(WPDT) in rats.Methods Wistar-Furth rats with type 1 diabetes mellitus(DM) were induced by intraperitoneal administration of streptozotocin(STZ) at a single dose of 60mg/Kg.On the basis of our primal model established by dual cuff combined with abdominal aorta connected with Y-tube,we improved the way of arterial anastomosis.End to side anastomosis was performed for abdominal aorta of donors and recipients.The portal vein of the graft was anastomosed with the recipients left renal vein by cuff technique.And side to side anastomosis was made between the graft duodenum and the host jejunum.According to the technique mentioned above,we successfully established WPDT model between Lewis rats as donors and Wistar rats as recipients.To monitor the levels of blood glucose 3 times weekly and record the survival time(ST) of each rats.And grafts of 3 rats were collected to observe pathohistologieal changes on day 7 posttransplantation. Results The successful rate of diabetes rats induced by STZ was 85.7%.The incidence rate of diabetic ketoacidosis was 5.6%and the mortality was 7.4%.44 rats were successfully performed WPDT of 50 rats.The mean levels of blood glucose were decreased from(22.83±4.37) mmol/L preoperative to(6.36±2.18) mmol/L postoperative in 44 rats.Among of them,8 rats died in 3 days postoperative,the ST of residual 36 rats was 6-16 days,the mean of ST was(10.45±3.3) d.Three was a progressive increase of blood glucose on day 4,7,10 and 13.The peak of death appeared on day 7-10.The typical acute rejection in pathological changes was observed on day 7.Conclusion Skilled microsurgical techniques and emphasis on details are important to establish WPDT model.The endocrine function of grafts and typical acute rejection can be observed in this model.It is a stable and reliable model, which might be used in research of the theoretical problems involved in clinical pancreas transplantation rejection.