The Study Of Tf In Triggering Ibmir And Islet Graft Protection By Human Treg In Xenotransplantation

Clinical islet transplantation is a promising treatment for typeⅠdiabetes and using pig organs could solve the significant increasing shortage of donor organs for allotransplantation. However early innate immune destruction of islets and rejection are major problems preventing clinical trials. Therefore in this study,①we used small interfering RNAs (siRNAs)-mediated gene knockdown to specifically silence TF gene expression in neonatal porcine islet cell clusters (NICC). This gene targeting approach allowed us to not only analyze directly the importance of TF in the initiation of IBMIR mediated by NICC, but also to explore the potential targets for developing strategies that can enhance islet engraftment and survival;②Using NOD-SCID IL2rγ-/- mice as recipients for pig islet transplantation, we studied whether and how immune tolerance of islet grafts to T-cell mediated cellular rejection is induced by reconstitution of these mice with human PBMCs and nTregs and produced a cell-based therapy that can be developed ex vivo that will suppress the T cell mediated response to the xeno-graft and thereby reduce or eliminate the requirement for immunosuppression.1. TF knockdown in NICCTF is expressed on newly isolated NICC and can be detected on neonatal pig pancreas tissue prior to NICC production confirming that it is not a by product of the isolation procedure. In order to test the hypothesis that specific suppression of islet TF expression would lead to a significant inhibition of IBMIR, small interfering RNA (siRNA) was used to knockdown TF gene expression in NICC. 5 pairs of siRNA were designed and transfected into NICC respectively or with different combinations under the optimal conditions. A 60%(50%-70%) and a 50% reduction in TF gene and protein expression respectively was achieved with selected 3 pairs of siRNA. FACS results showed that siRNA transfection had no significant effect on the viability of NICC used in these experiments. These data suggested that siRNA transfection was sufficient to specifically knockdown TF gene and protein expression in NICC.2. Suppressed TF expression in NICC led to inhibition of IBMIR in vitroAfter transfection, non-transfected, control siRNA transfected and TF siRNA transfected NICC were cultured with ABO compatible human blood in loops tubing respectively for 60 mins. Clot size and weight and blood cell counts were determined. Plasma was analyzed to determine levels of thrombin-antithrombin complexes (TAT) and C3a using commercially available ELISA kits. Immunohistochemical staining for neutrophil elastase and human IgG were used to determine neutrophil infiltration and antibody deposit in clots.α-Gal gene expression as assessed by real-time PCR. The results showed that TF siRNA resulted in substantially reduced not only clot size and weight but also the consumption of platelets, white blood cells and neutrophils. Consistent with these findings TAT and C3a levels were significantly less in loops containing TF siRNA transfected NICC compared to those with non-transfected or control siRNA transfected NICC. When TF siRNA transfected NICC were included in the loops with human blood, the number of infiltrating neutrophils within the clot was decreased considerably. These data showed that TF expression was an important initiator of the NICC mediated inflammatory response and this response was inhibited by TF knockdown. Meantime immunofluorescence staining of NICC containing clots taken from the loops showed strong deposition of human IgG which was not diminished when TF siRNA transfected NICC were used and a-Gal gene expression as assessed by real-time PCR was similar in all three groups. These data suggested that antibody mediated activation of the alternative pathway of thrombosis was not the major initiating event in this study.3. Islet xenograft protection by ex vivo-expanded human regulatory T cells in humanized NOD-SCID IL2rγ-/- miceCD4+CD25+CD127 dim/- T cells were isolated from PBMC by using a CD4+CD25+CD127 dim/- regulatory T cell isolation kit. Fresh Treg cells were cultured with human IL-2, CD3/CD28 Dynabeads and rapamycin for expansion. The expanded cells had the typical phenotype of Tregs and the percentage of CD4+CD25+Foxp3+ triple positive cells was over 90%; Mixed lymphocyte reaction (MLR) was used to investigate the suppression function of expanded Tregs. The results showed that expanded human Treg cells were able to suppress antiporcine proliferative responses in vitro. The concentration of IL-2 and IFN-γwere reduced and the IL-10 level was upregulated in the co-cultured cells supernatant, but the concentration of TGF-βhad no change. These data demonstrated that expanded Treg cells retained the phenotype and suppressive activity. NICC were transplanted to NOD-SCID IL2rγ-/1 mice under renal capsule.1×10'CD25+cell-depleted human PBMCs were co-transferred with or without 2×106 expanded human nTregs into the NICC recipient NOD-SCID IL2rγ-/- mice within 7 days after transplantation. Graft survival was monitored by histology. Human cell engraftment was determined by FACS. Phenotype of CD25-human cells and expanded human Tregs and their gene expression were analysed by FACS and real-time PCR. The results showed that NICC xenograft was intact with insulin, glucogan and somatostatin positive staining in NOD-SCID IL2rγ-/- recipient mice without human cell transfer for at least 100 days after transplantation. While NICC grafts were rejected in NOD-SCID IL2rγ-/- recipient mice 28 days after transfer with 1x107 human CD25+ cell-depleted PBMCs in the absence of autologous Tregs. There were lots of cells infiltrated in the graft which were CD45+ cells including CD4+ and CD8+ cells detected by immunohistology and only CD4 positive staining cells were detected by immunofluorescence double staining. NICC graft infiltrating human CD4+ cells isolated from rejecting NOD-SCID IL2rg-/- recipients did not express Foxp3 and IL-10 but IFN-γdetermined by FACS. In contract, NICC xenografts in human Treg transferred NOD-SCID IL2rγ-/- recipients still remained intact up to at least 100 days with insulin, glucogan and somatostatin positive staining after rechallenge with CD25+ cell-depleted human PBMC. CD45+ cells which included CD4+ and CD8+ cells didn't infiltrate but surround the graft detected by immunohistology and CD4 positive staining cells and CD4+Foxp3+double positive cells were detectable by immuno-fluorescence double staining. A large number of human CD4+ T cells expressed CD25 and Foxp3 (18.6±2.3%) in human Tregs transferred NICC recipient NOD-SCID IL2ryγ-/- mice at 28 day until to 100 day after rechallenging with human PBMCs. These data suggested that transfer of NOD-SCID IL2rγ-/- recipients with 1 x 107 CD25+ cell-depleted human PBMCs was sufficient to cause NICC xenograft rejection and the human PBMC-mediated xenograft rejection was significantly suppressed by human expanded Tregs which led to a long-term NICC graft survival. High level expression of Treg (Foxp3, CTLA-4, IL-10) but not T effect cell (IFN-y) associated genes was detected in NICC grafts from human Treg transferred NICC recipient mice 28 days after human PBMCs rechallenge by real-time PCR and human Tregs transferred NICC recipients demonstrated reduced level of human IFN-y and elevated level of human IL-10 in their serum 28 days after rechallenge with CD25+ cell-depleted human PBMC detected by cytometric bead array. These data suggest that the prolonged xenograft survival in the human Treg transferred and PBMC rechallenged NOD-SCID IL2ryγ-/- recipients was associated with the presence of human Tregs coexpressing IL-10 and inhibited production of effector cytokine IFN-y in these mice.Conclusions:Tissue factor expression in NICC can be reduced by siRNA mediated knockdown and TF knockdown can reduce IBMIR in vitro. Human expanded Tregs were capable of protecting NICC xenograft from rejection mediated by CD25+ cell-depleted human PBMC in NOD-SCID IL2rg-/- recipients and IL-10 was required for human Treg-mediated suppression of the human anti-pig xenogeneic response in vivo. Humanized NOD-SCID IL2rg-/- mice are a suitable mouse model to study human Treg-mediated tolerance and the mechanisms involved in islet xenotransplantation.