The Effect Of PD-L1 On The Induction Of Peripheral Tolerance And Prolonging Allograft Survival

【Objective】Type 1 diabetes (TID) is a T-cell-mediated autoimmune disease thatresults in the destruction of the insulin-secretingβ-cells in the pancreas by CD4⁺ and CD8⁺T cells, as well as macrophages infiltrating the isletsre and destruction of the insulinsecretingβ-cells in the pancreas.There is compelling evidence that the disease is associatedwith loss of immunological tolerance to self.Autoreactive T-cells have been identified andbeen considered to play a direct role in type 1 diabetes immunopathogenesis.Pancreaticislettransplantation has long been considered as a safer alternative and a potentiallypreferable treatment other than conventional exogenous-insulin therapy for T1D.Programmed Death-1 Ligand (PD-L1) has been identified as the ligand for theimmunoinhibitory receptor PD-1, and has been demonstrated to play a role in the negativeregulation of immune responses and peripheral tolerance.In the present study, weinvestigated the negative regulation function of PD-L1 and improvement of allorejection inβ-cells transplantation.【Methods】1.Establishment of PD-L1-GFP stable transfectants in NIT-1 cellspPD-L1-EGFP or pEGFPn1 were transfected in NIT-1 cells, for establishment of pPD-L1-EGFP or pEGFPn1 stable transfectants, namely NIT-PD-L1 and NIT-EGFP.2.Preparation of primary reactive and primed splenic lymphocytesThe splenoeytes from Balb/C (H-2d) mice of 8-12 weeks were collected and used asprimary reactive lymphocytes.Balb/C from above were immunized twice at days 1 and 8with NIT-1, NIT-EGFP or NIT-PD-L1 cells by i.p.injection, respectively.On day 14,splenocytes from these immunized mice were collected and used as primed spleniclymphocytes. 3.Mixed cells reactionPrimary reactive and primed splenic lymphocytes were stimulated in vitro with NIT-1,NIT-EGFP or NIT-PD-L1 cells pretreated with mitomycin C for 7 days, respectivly.4.Lymphocyte proliferation assayProliferation of splenic lymphocytes labeled with CFSE was analyzed by FCM.5.Lymphocyte apoptosis assayApoptosis of CFSE-unlabeled splenic lymphocytes were detected by labeling AnnexinV-Cy5 and PI using FCM.6.Combined induction in vivo and in vitro extended CTL assayBalb/C mice were immunized twice at days 1 and 8 with NIT-1, NIT-EGFP or NIT-PD-L1cells by i.p.injection, respectively.On day 14, splenocytes were collected andrestimulated with NIT-1, NIT-EGFP or NIT-PD-L1 cells pretreated with mitomycinCrespectively, and used as effector cells.NIT-1 cells were used as target cells after labledwith CFSE.Target cells were cultured with effector cells and stained with PI beforeanalyzed by FCM.Determination of cytolysis is based on the number of dead target cells,which are characterized by CFSE and PI double positivity.7.Cytokine detectionThe supematants from above co-culture of primary reactive or primed lymphocyteswere harvested for determination of Cytokine by ELISA.Intracellular cytokine weredetected by flow cytometry.8.The function of PD-L1 in induction of islet transplantation toleranceThe diabetes model was established by a low dose of streptozotocin (STZ) in Balb/Cmice.NIT-1, NIT-EGFP or NIT-PD-L1 cells were transplanted into diabetic mice by i.p.injection, respectively.After transplantation, body weight, survival time, BG level weremonitored every other day.At day 7 and 24 of post-transplantation, in vivo-sfimulatedinsulin secretion assay was performed on the recipients.At 7th-day of post-transplantation,proliferation and apoptosis of splenic lymphocytes from these transplanted diabetic mice were detected by FCM, the production of cytokine was detected by FCM and ELISA.On7^th-day of post-transplantation, peritoneal cells from these transplanted diabetic mice were collected anddetected the insulin-secreting NITs.【Results】1.PD-L1 leaded to negative regulation of allogeneie lymphocyte activationPD-L1 inhibited proliferation of primary reactive lymphocytes and primarylymphocytes, and enhance apoptosis of allogeneic lymphocytes.In addition, proliferativeresponse of NIT-1 or NIT-EGFP-primed lymphocytes stimulated with NIT-PD-L1 cellswas significantly lower than these lymphocytes resfimulated with NIT-1 cells or NIT-EGFPcells.Furthermore, proliferative response of NIT-PD-L1-primed lymphocytes restimulatedwith NIT-PD-L1 cells was the lowest among all groups.The apoptosis rate of NIT-1 orNIT- EGFP- primed lymphocytes cocultured with NIT-PD-L1 cells was much higher thanthose lymphocytes cocultured with NIT-1 cells or NIT-EGFP cells.Furthermore, theapoptosis rate of NIT-PD-L1- primed lymphocytes cocultured with NIT-PD-L1 cells wasthe highest among all, groups.ELISA and intracellular cytokine assay results indicated thatPD-L1 downregulated IFN-γbut upregulated IL-4 and IL-10 production by the primedlymphocytes.The NIT-1 had a severe necrosis cultured with NIT-1 or NIT-EGFP-primedlymphocytes, however, the NIT-1 cells had moderate necrosis cultured with NIT-PD-L1-primed lymphocytes.2.PD-L1 can prolong allograft survivalStreptozotocin-induced diabetic mice received NIT-1, NIT-EGFP or NIT-PD-L1 cellsshowed decreased BG levels after 3 days of transplantation.However, BG of diabetic micethat transplanted with NIT-1 or NIT-EGFP cells gradually rebounded by day 7 posttransplantation.On the contrary, diabetic mice that received NIT-PD-L1 cells reachednormoglycemia after 3 days of transplantation, and the lowest BG was observed at day 13of post-transplantation.These mice had maintained normoglycemic for about 21 days, then their BG increased gradually and became hyperglycemic (>11.1mmol/L) again by day 24of post-transplantation.We examined body weight for all recipients further.Diabetic mice receiving NIT-1 orNIT-EGFP cells showed a continual decrease in body weight.However, mice transplantedwith NIT-PD-L1 cells gained weight on 7th-day after transplantation.To evaluate the glucose-induced insulin releasing capability on 7^th-day and 24^th-day ofpost-transplantation, insulin radioimmunoassay was performed.Because all the micetransplanted with NIT-1 or NIT-EGFP cells died before day 24 post-transplantation, onlythose mice transplanted with NIT-PD-L1 cells were included at this time-point.The insulinrelease amounts of mice were assayed at 15 min, after feeding the mice with glucose bolus.At day 7 post-transplantation, insulin contents for mice transplanted with NIT-1 or NIT-EGFPcells were significantly lower than those of non-diabetic control mice, whereas, formice transplanted with NIT-PD-L1 cells, insulin content remained similar to normal mice.More importantly, their life span was significantly longer than that of mice transplantedwith NIT-1 or NIT-EGFP cells.In order to explore the mechanisms underlying the suppressive effect of PD-L1, theproliferation and apoptosis of lymphocytes from recipients were further examined.Theseresults displayed that PD-L1 inhibited proliferation of lymphocytes and induced apoptosisof lymphocytes.The results of ELISA and intracellular cytokine assay indicated that PD-L1altered the cytokines profiles of activated lymphocytes, with increased seen in the Th2cytokines, IL-10 and IL-4, and a decreased in Th1 cytokines, IFN-γ.Immunofluorescencestaining showed that a larger member of insulin-secreting cells from NIT-PD-L1 cellstransplanted mice could be seen in peritoneal.【Conclusions】Over-expression PD-L1 on NIT-1 cells inhibted proliferation oflymphocytes and induced apoptosis of lymphocytes.In vitro extended CTL assay showedthat PD-L1 can protect NIT-1 cells from CTL-mediated lysis.PD-L1 altered the cytokineprofiles of activated lymphocytes, with increases seen in the Th2 cytokines, IL-10 and IL-4, and a decrease in Th1 cytokine, IFN-γ.Expression of PD-L1 on pancreaticβ-cells hassignificantly prolonged allograft survival, and the understanding of this will guide rationaltherapeutic strategies for the improvement of allorejection inβ-cells transplantation.