Graft Survival In Mice Research Articles

Human stem cell derived beta-like cells engineered to present PD-L1 improve transplant survival in NOD mice carrying human HLA class I.

There is a critical need for therapeutic approaches that combine renewable sources of replacement beta cells with localized immunomodulation to counter recurrence of autoimmunity in type 1 diabetes (T1D). However, there are few examples of animal models to study such approaches that incorporate spontaneous autoimmunity directed against human beta cells rather than allogenic rejection. Here, we address this critical limitation by demonstrating rejection and survival of transplanted human stem cell-derived beta-like cells clusters (sBCs) in a fully immune competent mouse model with matching human HLA class I and spontaneous diabetes development. We engineered localized immune tolerance toward transplanted sBCs via inducible cell surface overexpression of PD-L1 (iP-sBCs) with and without deletion of all HLA class I surface molecules via beta-2 microglobulin knockout (iP-BKO sBCs). NOD.HLA-A2.1 mice, which lack classical murine MHC I and instead express human HLA-A*02:01, underwent transplantation of 1,000 human HLA-A*02:01 sBCs under the kidney capsule and were separated into HLA-A2 positive iP-sBC and HLA-class I negative iP-BKO sBC groups, each with +/- doxycycline (DOX) induced PD-L1 expression. IVIS imaging showed significantly improved graft survival in mice transplanted with PD-L1 expressing iP-sBC at day 3 post transplantation compared to controls. However, luciferase signal dropped below in vivo detection limits by day 14 for all groups in this aggressive immune competent diabetes model. Nonetheless, histological examination revealed significant numbers of surviving insulin+/PD-L1+ sBCs cells for DOX-treated mice at day 16 post-transplant despite extensive infiltration with high numbers of CD3+ and CD45+ immune cells. These results show that T cells rapidly infiltrate and attack sBC grafts in this model but that significant numbers of PD-L1 expressing sBCs manage to survive in this harsh immunological environment. This investigation represents one of the first in vivo studies recapitulating key aspects of human autoimmune diabetes to test immune tolerance approaches with renewable sources of beta cells.

Stem Cell Therapy Improves Human Islet Graft Survival in Mice via Regulation of Macrophages.

Islet/β-cell transplantation offers great hope for patients with type 1 diabetes. We assessed the mechanisms of how intrahepatic coinfusion of human α-1 antitrypsin (hAAT)-engineered mesenchymal stromal cells (hAAT-MSCs) improves survival of human islet grafts posttransplantation (PT). Longitudinal in vivo bioluminescence imaging studies identified significantly more islets in the livers bearing islets cotransplanted with hAAT-MSCs compared with islets transplanted alone. In vitro mechanistic studies revealed that hAAT-MSCs inhibit macrophage migration and suppress IFN-γ-induced M1-like macrophages while promoting IL-4-induced M2-like macrophages. In vivo this translated to significantly reduced CD11c+ and F4/80+ cells and increased CD206+ cells around islets cotransplanted with hAAT-MSCs as identified by multiplex immunofluorescence staining. Recipient-derived F4/80+and CD11b+ macrophages were mainly present in the periphery of an islet, while CD11c+ and CD206+ cells appeared inside an islet. hAAT-MSCs inhibited macrophage migration and skewed the M1-like phenotype toward an M2 phenotype both in vitro and in vivo, which may have favored islet survival. These data provide evidence that hAAT-MSCs cotransplanted with islets remain in the liver and shift macrophages to a protective state that favors islet survival. This novel strategy may be used to enhance β-cell survival during islet/β-cell transplantation for the treatment of type 1 diabetes or other diseases.

Sedative and Immunosuppressive Effects of Dexmedetomidine in Transplantation.

Dexmedetomidine, an α2-adrenergic receptor agonist, is used as an anti-anxiety medication. It exerts a cholinergic effect, thereby reducing the release of tumor necrosis factor alpha (TNF-α). We hypothesized that the use of dexmedetomidine as a sedative agent in transplantation would also protect allografts. We examined our patients who underwent living donor liver transplantation. Subsequently, we generated a series of mouse models to investigate the effect of dexmedetomidine on sedation-based tolerance post transplantation. A total of 49 liver recipients were enrolled in this study, of which 23 (47%) were administered dexmedetomidine through 24 h infusion on postoperative day 1. A trend toward the improvement of hepatocyte injury along with better liver function was observed in the dexmedetomidine-treated group during the first postoperative week. In animal models, dexmedetomidine inhibited the proliferation of CD4+ and CD8+ T cells and TNF-α production in a dose-dependent manner. We used dexmedetomidine to treat skin-transplanted mice and observed a significantly prolonged graft survival in mice that were administered a higher dose of dexmedetomidine. Our results revealed that dexmedetomidine exerts a dual effect of sedation and immunosuppression. This light-sedation approach will not only make patients calmer in the intensive care unit but also protect allografts from injury.

Multiphase Assembly of Small Molecule Microcrystalline Peptide Hydrogel Allows Immunomodulatory Combination Therapy for Long-Term Heart Transplant Survival.

Combination therapies that target multiple pathways involved in immune rejection of transplants hold promise for patients in need of restorative surgery. Herein, a noninteracting multiphase molecular assembly approach is developed to crystallize tofacitinib, a potent JAK1/3 inhibitor, within a shear-thinning self-assembled fibrillar peptide hydrogel network. The resulting microcrystalline tofacitinib hydrogel (MTH) can be syringe-injected directly to the grafting site during surgery to locally deliver the small molecule. The rate of drug delivered from MTH is largely controlled by the dissolution of the encapsulated microcrystals. A single application of MTH, in combination with systemically delivered CTLA4-Ig, a co-stimulation inhibitor, affords significant graft survival in mice receiving heterotopic heart transplants. Locoregional studies indicate that the local delivery of tofacitinib at the graft site enabled by MTH is required for the observed enhanced graft survival.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n=18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.

Prolongation of skin graft survival in mice by an azaphenothiazine derivative

Azaphenothiazines are predominantly immunosuppressive compounds. We evaluated the efficacy of an azaphenothiazine derivative, 6-chloroethylureidoethyldiquino[3,2-b;2′,3′-e][1,4]thiazine (DQT) in prolongation of survival of skin allografts between BALB/c and C57Bl/6 mice. The mice were treated intraperitoneally (i.p.) with 100 μg of DQT on alternate days, on days 1–13 of the experiment (7 doses). The effect of DQT on a two-way mixed lymphocyte reaction (MLR) in the human model, as well as its effect on production of TNF α and IL-10 in a whole blood cell culture, stimulated by lipopolysaccharide (LPS), were evaluated. In addition, DQT effects were investigated regarding the proportion of T cell subsets in human peripheral blood lymphocytes (PBMC) by flow cytometry. Lastly, the effect of DQT on expression of signaling molecules involved in pro apoptotic pathways was determined by RT PCR. The results showed that DQT significantly extended skin graft survival. The compound also strongly suppressed two-way MLR in the human model at a concentration range of 2.5–5.0 μM. In addition, DQT inhibited LPS-inducible TNF α, but not IL-10 production. The compound preferentially caused a loss of the CD3-CD8+CD11b + PBMC cell subset, and transformed CD3+CD8+high into CD3+CD8+low cells. Lastly, we demonstrated significant increases in expression of caspases (in particular caspase 8) and of p53 in a culture of Jurkat T cells. We conclude that the immunosuppressive actions of the compound in allograft rejection may be predominantly associated with induction of cell apoptosis and inhibition of TNF α production. The apoptosis could be predominantly selective for the CD3-CD8+CD11b + cell phenotype.

Stromal vascular fraction cells plus sustained release VEGF/Ang-1-PLGA microspheres improve fat graft survival in mice.

Autologous fat transplantation is increasingly applied in plastic and reconstructive surgery. Stromal vascular fraction cells (SVFs) combined with angiogenic factors, such as VEGF (vascular endothelial growth factor A) and Ang-1 (angiogenin-1), can improve angiogenesis, which is a critical factor for graft survival. However, direct transplant with such a mixture is insufficient owing to the short half-life of angiogenic factors. In this study, we evaluated whether a double sustained release system of VEGF/ANG-1-PLGA (poly (lactic-co-glycolic acid)) microspheres plus SVFs can improve angiogenesis and graft survival after autologous fat transplantation. VEGF/ANG-1-PLGA-sustained release microspheres were fabricated by a modified double emulsion-solvent evaporation technique. Human aspirated fat was mixed with SVF suspension plus VEGF/ANG-1 sustained release microspheres (Group C), SVF suspension (Group B) alone, or Dulbecco's modified Eagle's medium as the control (Group A). Eighteen immunocompromised nude mice were injected with these three mixtures subcutaneously at random positions. After 8 weeks, the mean volume of grafts was greater in the SVFs plus VEGF/ANG-1-PLGA group than in the control and SVFs groups (1.08 ± 0.069 ml vs. 0.62 ± 0.036 ml, and 0.83 ± 0.059 ml, respectively). Histological assessments showed that lower fibrosis, but greater microvascular density in the SVFs plus VEGF/ANG-1-PLGA group than in the other groups, though the SVFs group also had an appropriate capillary density and reduced fibrosis. Our findings indicate that SVFs plus VEGF/ANG-1-PLGA-sustained release microspheres can improve angiogenesis and graft survival after autologous fat transplantation.

Gut microbes contribute to variation in solid organ transplant outcomes in mice

BackgroundSolid organ transplant recipients show heterogeneity in the occurrence and timing of acute rejection episodes. Understanding the factors responsible for such variability in patient outcomes may lead to improved diagnostic and therapeutic approaches. Rejection kinetics of transplanted organs mainly depends on the extent of genetic disparities between donor and recipient, but a role for environmental factors is emerging. We have recently shown that major alterations of the microbiota following broad-spectrum antibiotics, or use of germ-free animals, promoted longer skin graft survival in mice. Here, we tested whether spontaneous differences in microbial colonization between genetically similar individuals can contribute to variability in graft rejection kinetics.ResultsWe compared rejection kinetics of minor mismatched skin grafts in C57BL/6 mice from Jackson Laboratory (Jax) and Taconic Farms (Tac), genetically similar animals colonized by different commensal microbes. Female Tac mice rejected skin grafts from vendor-matched males more quickly than Jax mice. We observed prolonged graft survival in Tac mice when they were exposed to Jax mice microbiome through co-housing or fecal microbiota transplantation (FMT) by gastric gavage. In contrast, exposure to Tac mice did not change graft rejection kinetics in Jax mice, suggesting a dominant suppressive effect of Jax microbiota. High-throughput sequencing of 16S rRNA gene amplicons from Jax and Tac mice fecal samples confirmed a convergence of microbiota composition after cohousing or fecal transfer. Our analysis of amplicon data associated members of a single bacterial genus, Alistipes, with prolonged graft survival. Consistent with this finding, members of the genus Alistipes were absent in a separate Tac cohort, in which fecal transfer from Jax mice failed to prolong graft survival.ConclusionsThese results demonstrate that differences in resident microbiome in healthy individuals may translate into distinct kinetics of graft rejection, and contribute to interpersonal variability in graft outcomes. The association between Alistipes and prolonged skin graft survival in mice suggests that members of this genus might affect host physiology, including at sites distal to the gastrointestinal tract. Overall, these findings allude to a potential therapeutic role for specific gut microbes to promote graft survival through the administration of probiotics, or FMT.

BM-MSCs-derived microvesicles promote allogeneic kidney graft survival through enhancing micro-146a expression of dendritic cells

ObjectiveMicrovesicles (MVs) are plasmalemmal vesicles that are released from various cells and regarded as a mediator of intermolecular communication. In present study, we aimed to evaluate the therapeutic efficacy of the bone marrow mesenchymal stem cells (BM-MSCs)-derived MVs in the mice kidney transplant model and explored the underlying mechanism. MethodsBM-MSCs were isolated from C57BL/6 mice and identified using flow cytometry. In vivo allogenic kidney transplantation model of mice was performed between C57BL/6 mice (recipient) and BALB/c mice (donor). Recipient-type BM-MSC (0.1ml) or equal volume of medium as a control was injected i.v. 24h after kidney transplantation. Serum was collected for creatinine concentration detection at 14 d after transplantation. Dendritic cells (DCs) phenotype and miR-146a expression level in plant was identified. Immature DCs (iDCs) and mature DCs (mDCs) were derived from monocytes. MVs were separated from BM-MSCs. ResultsBM-MSCs positive for CD29 (95.8%) and CD44 (94.7%) were cultured and confirmed to prolong the allogenic kidney graft survival in mice. Importantly, the expression of miR-146a increased significantly in DCs of BM-MSCs-treated allogenic kidney. Moreover, both BM-MSCs and MVs derived from BM-MSCs enhanced miR-146a expression in iDCs and mDCs in vitro. Furthermore, MVs substantially reduced IL-12 mRNA expression and IL-12 production of mDCs whereas this action was reversed by miR-146a silencing. MiR-146a silencing also abrogated the MVs-induced decrease in serum creatinine, reduction of immature DCs phenotype in transplant and increase in miR-146a expression level. ConclusionIn summary, our data suggested that the BM-MSCs-derived MVs improved allogenic kidney transplantation survival through inhibiting DCs maturity by miR-146a.

An immunotherapeutic strategy for prolonging graft survival in mice

BackgroundImmunological memory is one barrier to inducing immune tolerance in alloantigen primed transplant models. Alloreactive memory cells induced by primary organ transplant are difficult to suppress and contribute to rejection following a retransplant. Here, we assess the effects of a novel immunosuppressive strategy on graft survival and host immune response. Materials and methodsAlloantigen primed splenocytes were adoptively transferred into naïve B6 mice before heart transplant. Recipient mice were then treated with mitomycin C -pretreated donor-specific transfusion, low dose rapamycin, and anti-OX40L mAb (alone and in combination). ResultsTreatment with donor-specific transfusion+rapamycin+anti-OX40L increased the mean graft survival time from 5.2days (control group) to 42.5days (p<0.05). None of the grafts in the control group survived past 10days, and only 1 in 3 grafts survived at least 50days in the combined treatment group. Compared with the control and single treatment groups, the combination of donor-specific transfusion+rapamycin + anti-OX40L inhibited infiltration of inflammatory cells; reduced the percentages of CD4+ and CD8+ memory T cells in the blood, spleen, and lymph nodes; suppressed inflammatory cytokines; reduced serum IgG levels; and enhanced the percentage of CD4+Foxp3+ T cells. The percentage of regulatory T cells was the highest in the recipients with long-term graft survival. ConclusionsThe combination of donor-specific transfusion+rapamycin+anti-OX40L inhibits graft rejection mediated by memory cells and is likely to contribute to prolonged allograft survival.

All-trans retinoid acid promotes allogeneic corneal graft survival in mice by regulating Treg-Th17 balance in the presence of TGF-β.

BackgroundAll-trans retinoid acid (ATRA) has been proven to skew Regulatory T cell-T helper 17 cell (Treg-Th17) balance toward Treg in vitro, favoring graft acceptance. However, its in vivo effect after solid organ transplantation is under investigation.ResultsBALB/c mice were given orthotopic corneal grafts from C57BL/6 donors, and recipient mice were administered with ATRA, TGF-β, and the combination of both agents for 8 weeks after surgery. We found that a mixed treatment of ATRA and TGF-β significantly promoted graft survival. Moreover, with the presence of TGF-β, ATRA upregulated CD4+CD25+Foxp3+Treg cells and suppressed Th17 cells in the blood, spleen and draining lymph nodes of recipient mice, as well as enhanced the Foxp3 expression and inhibited the RORγt expression in grafts and peripheral blood mononuclear cells (PBMCs). Simultaneously, increased number of Foxp3+ cells and decreased number of IL-17+ cells in conjunctiva were found in recipients with mixed treatment, along with reduced IL-17 level in serum and aqueous humor and increased IL-10 level in aqueous humor. Tregs isolated from recipient mice treated with ATRA + TGF-β presented the strongest suppressive activity in vitro.ConclusionsCombined application of ATRA and TGF-β may shift the Th17-Treg balance toward Tregs, hence facilitating the induction of immunological tolerance after allogenic corneal transplantation and representing a potential therapeutic approach in the treatment of posttransplant rejection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0082-3) contains supplementary material, which is available to authorized users.

Topical application of sphingosine 1-phosphate receptor 1 prolongs corneal graft survival in mice.

The present study aimed to investigate the effects of topical application of sphingosine 1‑phosphate receptor 1 (S1P1) on allogeneic corneal transplantation in mice. A total of 45 BALB/c mice received corneal grafts from C57BL/6 donors. The recipients were randomly divided into three groups and treated with eye drops containing 0.5% S1P1, 1% cyclosporine A or saline as a negative control. The serum levels of interleukin (IL)‑2, IL‑10, tumor growth factor (TGF)‑β1 and interferon (IFN)‑γ were measured by ELISA. The numbers of CD4+ T cell and T‑regulatory (Treg) cell phenotypes were measured by flow cytometry. The cytokine mRNA expression was analyzed by quantitative polymerase chain reaction. The results demonstrated that corneal graft survival was prolonged in the S1P1 group [mean survival time (MST), 24.11±1.58 days], and 1% cyclosporine A (MST, 25.0±1.91 days) compared with the controls (MST, 13.44±0.48 days; P<0.01). S1P1 and cyclosporine A decreased CD4+ T‑cell levels (P<0.05) in the peripheral blood compared with those of the controls. However, an increase of CD4+ T cells in the spleen was noted in the S1P1 group (P<0.05) and Treg cells were also increased in the cervical lymph nodes in the S1P1 group (P<0.01). TGF‑β1 mRNA transcription in the corneal grafts increased following treatment with S1P1 (P<0.05) and TGF‑β1 in the serum following treatment with S1P1 also increased (P<0.01). In conclusion, S1P1 had a significant effect in corneal allograft rejection inhibition.

Long-Term Tolerance and Skin Allograft Survival in CD200tg Mice After Autologous Marrow Transplantation.

CD200 overexpression in transgenic recipients, or by tissue allografts, increases skin and cardiac graft survival in mice receiving rapamycin, in association with increased Foxp3(+)Treg graft infiltration. However, less than 50% of skin grafts persist beyond 25 days. We investigated whether immune ablation using busulphan and cyclophosphamide, followed by autologous marrow reconstitution, would permit long-term survival in a greater percentage of recipients and induce tolerance, allowing for withdrawal of immunosuppressive drugs. C57BL/6 wild type (BL/6 WT) or CD200(tg) mice received BALB/c skin grafts with rapamycin (1 mg/kg/36 hr) for 7 days. Thereafter, subgroups received busulphan or cyclophospamide for 6 days, followed by BL/6 marrow transplantation (BMTx), whereas controls were maintained on rapamycin. Beginning 7 days after marrow transplantation, mice receiving BMTx also received rapamycin for a further 7, 14, or 21 days (to a maximum of 42 days after transplantation) after which times, all immunosuppressions were withdrawn. Graft survival was monitored throughout, and mixed leukocyte cultures (MLCs) (using peripheral blood leukocytes) were performed for all groups at 60 days after transplantation. Gene expression was performed in grafts harvested at 80 days. Although all WT mice rejected grafts by 16 days, survival in CD200(tg) was approximately 40% at 60 days, with antigen-specific decreased MLC responses to BALB/c. After BMTx, survival in WT mice was greater than 40% at 60 days, and greater than 90% at 60 days in CD200(tg), with corresponding attenuated MLC responses. Long-term surviving grafts were infiltrated by Tregs of both host and donor marrow origins, as defined using CD45 congeneic donors or recipients. Survival was increased by infusion of an antibody directed to TNFRSF25 on Tregs. Autologous BMTx improves graft survival and promotes graft tolerance.

Administration of Low Doses of IL-2 Combined to Rapamycin Promotes Allogeneic Skin Graft Survival in Mice

Human CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) prevent allogeneic graft rejection by inhibiting T cell activation, as has been shown in mouse models. Recently, low-dose IL-2 administration was shown to specifically activate Tregs but not pathogenic conventional T cells, leading to resolution of type 1 diabetes in nonobese diabetic mice. We therefore tested the ability of low-dose IL-2 to prevent allogeneic skin graft rejection. We found that while IL-2 alone was inefficient in preventing rejection, combined with rapamycin, IL-2 treatment promoted skin graft survival both in minor disparate and semi-allogeneic skin graft combinations. Tregs are activated by this combined treatment while conventional CD4(+) cell expansion and activation are markedly inhibited. Co-administration of anti-CD25 antibodies dramatically reduces the effect of the IL-2/rapamycin treatment, strongly supporting a central role for Treg activation. Thus, we provide the first preclinical data showing that low-dose IL-2 combined with rapamycin can significantly delay transplant rejection in mice. These findings may form the rational for clinical evaluation of this novel approach for the prevention of transplant rejection.

Effects of Preexisting Autoimmunity on Heart Graft Prolongation After Donor-Specific Transfusion and Anti-CD154

Alloreactive memory T cells prevent costimulatory blockade-induced heart graft survival in mice, but whether and how preexisting autoreactive T cells affect solid-organ transplants under these conditions is unknown. We tested the impact of preexisting cardiac myosin (CM)-specific immunity on murine heart transplant recipients treated with donor-specific transfusion (DST) plus anti-CD154 monoclonal antibody MR1. Preimmunization with CM but not control ovalbumin abrogated the graft prolonging effects of DST/MR1, whether administered 2 weeks or more than 6 weeks before transplantation. Adoptive transfer of spleen cells from CM-immunized mice into naïve recipients had similar effects. CM-specific immunity did not cross-react with donor antigens and preimmunization with CM had no impact on the survival or histology of DST/MR1-treated syngeneic heart grafts, the latter indicating that persistent autoimmunity is insufficient to cause rejection in the context of costimulatory blockade. We observed that the CM preimmunized mice produced higher frequencies of donor-reactive T cells with higher ratios of CD8/CD4Foxp3 cells, suggesting that the autoreactive memory T cells provide help for activation of alloreactive T cells despite the costimulatory blockade. These mechanistic insights linking autoimmunity and alloimmunity in a model of murine heart transplantation have clinical relevance to the known association between autoimmunity and an elevated risk of acute and chronic heart transplant injury in humans.

A novel immunosuppressory peptide originating from the ubiquitin sequence

Ubiquitin is a conservative polypeptide present in every eukaryotic cell. Apart from its involvement in proteasomal degradation and other intracellular signal pathways, it was suggested to play an important role as the extracellular immunomodulator and antimicrobial agent. Moreover, ubiquitin-derived peptides were shown to express significant biological activities. Our previous studies showed a high immunosuppressive potency of the ubiquitin peptic hydrolysate in which we identified over 70 different peptides. The present work focuses on synthesizing the most abundant of these peptides and investigating their immunomodulatory potency. The peptide VKTLTGKTI possessed the highest immunosuppressory activity in AFC experiments, comparable to the previously described LEDGRTLSDY sequence (a previously discovered ubiquitin-derived peptide). Moreover, some of the investigated peptides expressed immunostimulatory effects. These findings support the idea that ubiquitin, together with products of its degradation, could represent a self-regulating immunoregulatory system. Peptide VKTLTGKTI was also tested for its activity to prolong the skin graft survival in mice. The results showed that the investigated peptide significantly extended the skin transplant rejection time, therefore it could be considered as a potential supplementary medicine in the post-transplantation therapy. Moreover, we synthesized two analogs of investigated peptides, first designed to mimic the non-linear epitope consisting of ubiquitin 16–21 and ubiquitin 52–57 fragments, and second designed to mimic the ubiquitin 5–13 hairpin. We also tested their immunosuppressory activity in in vitro experiments.

Obaculactone suppresses Th1 effector cell function through down-regulation of T-bet and prolongs skin graft survival in mice

Allograft rejection is a predominantly Th1 immune response. In this study, we showed that obaculactone, a natural compound derived from citrus fruit, prolonged skin graft survival in mice when treated after but not before transplantation. Furthermore, obaculactone inhibited alloantigen-specific production of Th1 cytokine IFN-γ as well as proinflammatory cytokine IL-2, TNFα and IL-6. In parallel, IL-10 production was markedly up-regulated. Obaculactone significantly enhanced the percentage of CD4 +CD25 +Foxp3 + Treg cells in the CD4 + splenocytes without any effect on their inhibitory function. In vitro and in vivo tests showed obaculactone down-regulated T-bet expression in Th1 effector cells. Taken together, the unique immunomodulatory properties might qualify obaculactone as a putative, therapeutic compound for the treatment of Th1-driven diseases, including transplant rejection.

Tetrandrine Attenuates Dendritic Cell-mediated Alloimmune Responses and Prolongs Graft Survival in Mice

Tetrandrine, a bisbenzylisoquinoline alkaloid, has significant immunosuppressive effects; however, the effects of tetrandrine on dendritic cells (DCs) and the associated immune reactions are unclear. In this study, we investigated the effects of tetrandrine on DCs and the effects of the tetrandrine-treated DCs on alloimmune reactions in vitro and graft survival in vivo. Tetrandrine significantly downregulated the expression of CD80 and CD86 of DCs and increased their secretion of IL-10 (p = 0.0001). Mixed leukocyte reaction showed that tetrandrine inhibited dendritic-cell allo-stimulatory activity, which was reversed by the anti-IL-10 treatment. An in vivo study demonstrated that tetrandrine-treated DCs prolonged the survival time of skin grafts in mice compared to control (p = 0.005) and decreased cellular infiltration of the graft in the histopathological study. The data suggest that tetrandrine-treated DCs cause immunosuppression and protect skin grafts from rejection. The tetrandrine-induced immunosuppression seems to be partially due to increased IL-10 secretion.

ADAP deficiency combined with costimulation blockade synergistically protects intestinal allografts

Adhesion and degranulation promoting adapter protein (ADAP) plays an important role in T cell activation. ADAP deficiency was recently found to prolong heart graft survival in mice. We investigated the role of ADAP in intestinal transplantation and the synergistic effect of ADAP deficiency and Costimulation blockade (CB). T cell proliferation and cytotoxic T lymphocyte (CTL) activity were determined. MHC mismatched intestinal allografts was transplanted heterotopically. Anti-CD40L antibody was applied to the recipient. Upon stimulation with allogenic dendritic cells (DC), ADAP-deficient (ADAP-/-) T cells displayed impaired proliferative responses compared with that of wild-type (WT) T cells. In contrast, the CTL activity in ADAP-/- mice was comparable with that of WT mice. Rejection of intestinal allografts was ameliorated, but not prevented in ADAP-/- mice. Although CB alone was not sufficient to mitigate the rejection, the combination of CB and ADAP deficiency profoundly inhibited rejection. This was accompanied by less infiltration and activation of host lymphocytes in the gut-associated lymphoid tissue of intestinal allografts. ADAP deficiency combined with CB protected the intestinal allografts synergistically. ADAP could be a novel target in the induction phase of the immune responses in organ transplantation.

CTLA4-Ig-modified dendritic cells inhibit lymphocyte-mediated alloimmune responses and prolong the islet graft survival in mice

The induction of antigen specific tolerance is critical for prevention and treatment of allograft rejection. In this study, we transfected CTLA4-Ig gene into dendritic cells (DCs), and investigated their effect on inhibition of lymphocyte activity in vitro and induction of immune tolerance on pancreatic islet allograft in mice. An IDDM C57BL/6 murine model induced by streptozotocin is as model mouse. The model mice were transplanted of the islet cells isolated from the BALB/c mice to their kidney capsules, and injected of CTLA4-Ig modified DCs (mDCs). The results showed that mDCs could significantly inhibit T lymphocyte proliferation and induce its apoptosis; whereas, unmodified DCs (umDCs) promoted the murine lymphocyte proliferation. Compared with injection of umDCs and IgG1 modified DCs, the injection of mDCs prolonged IDDM mice's allograft survival, and normalized their plasma glucose (PG) levels within 3 days and maintained over 2 weeks. The level of IFN-γ was lower and the level of IL-4 was higher in mDCs treated recipient mice than that in control mice, it indicated that mDCs led to Th1/Th2 deviation. After 7 days of islet transplantation, HE stain of the renal specimens showed that the islets and kidneys were intact in structure, and islet cells numbers are increased in mDCs treated mice. Our studies suggest that DCs expressing CTLA4-Ig fusion protein can induce the immune tolerance to islet graft and prolong the allograft survival through the inhibition of T cell proliferation in allogeneic mice.