Allograft Survival In Mice Research Articles

Delivery of butyrate to the lower gut by polymeric micelles prolongs survival of distal skin allografts

The microbiota composition is known to influence the kinetics of graft rejection, but many questions remain as to whether/how microbiota-derived metabolites affect graft outcome. We investigated the effects of the short-chain fatty acid butyrate, a product of dietary fiber fermentation. Sustained intragastric administration of a micelle-based formulation of butyrate (butyrate micelle [ButM]) that releases its cargo in the lower gastrointestinal tract elevated cecal butyrate content and significantly prolonged minor-mismatched and major-mismatched skin allograft survival in mice. While ButM did not influence regulatory T cells or the adaptive alloimmune responses we tested, it modulated the myeloid cell compartment. At steady state, ButM treatment reduced the number of circulating Ly6ChiCD11b+ monocytes and other myeloid cells in secondary lymphoid organs and skin, altered their expression of genes involved in mitochondrial metabolism and key inflammatory processes, and reduced their ability to produce TNFa, likely via an indirect mechanism. ButM treatment also reduced numbers of graft-infiltrating monocytes but not T cells. Consistent with its critical effect on myeloid cells, ButM’s extension of graft survival depended on the presence of CCR2+ cells. These findings imply that cecal ButM improves distal allograft outcomes by quantitatively and qualitatively modulating myeloid cells, thereby inhibiting the innate immune cell-mediated effector phase of alloimmunity.

TIGIT-Fc Prolongs Corneal Allograft Survival in Mice by Upregulating TIGIT/CD226 Expression and the Proportion of Helios + Foxp3 + Treg Cells.

Reduction of graft rejection remains key issue for supporting long-term graft retention after corneal transplantation. The relevance of Treg in reduction of corneal allografts rejection has been demonstrated. It has been recently reported that in addition to Foxp3, Helios is also considered to be a marker of activated Treg. Helios + Foxp3 + Treg are considered to be the true immunosuppressive Treg. TIGIT is an immunosuppressive costimulatory molecule that was found to be highly expressed on the surface of Helios + Foxp3 + Treg. In this study, we aimed to explore whether supplementing TIGIT would result in an expansion and activation of Helios + Foxp3 + Treg thus to mediate an immune tolerance following corneal transplantation by administering topically and systemically TIGIT-Fc treatment in murine models. TIGIT-Fc treatment significantly improved the survival of corneal allograft compared with the control group. TIGIT-Fc treatment increased TIGIT/CD226 expression, the proportion of Helios + Foxp3 + Treg cells and an enhanced ex vivo suppressive effect from peripheral lymph nodes isolated Treg cells. Furthermore, the expression of Helios in corneal grafts was upregulated, whereas expression of CD226 and production of aqueous interferon-γ and VEGF were reduced by TIGIT-Fc treatment. TIGIT-Fc treatment could specifically upregulate Helios + Foxp3 + Treg-mediated immune response after allogeneic corneal transplantation via TIGIT/CD226-CD155 pathway which improves the survival of allografts.

Induction of Endotoxin Tolerance Delays Acute Rejection in a Hindlimb Transplantation Model in Rats.

Acute rejection is seen in 85 percent of composite vascular allogeneic transplants despite long-term immunosuppression. Recently, it was reported that the induction of endotoxin tolerance prolonged heart allograft survival in mice. However, it produced side effects in all the animals secondary to the inflammatory reaction. Galactomannan has shown endotoxin tolerance without this side effect in vitro. The authors hypothesized that galactomannan-induced endotoxin tolerance delays acute rejection in vascular allogeneic transplantation without the side effects produced by lipopolysaccharide. Twenty-four rat hindlimb transplants were divided into four groups according to the preconditioning received: control, lipopolysaccharide (0.16 ml/kg), galactomannan 72 hours before (galactomannan-72) (8 ml/kg), and galactomannan 24 hours before (galactomannan-24) (8 ml/kg). Median acute rejection time, weight loss, and diarrheal episodes were monitored. Blood samples were collected at 0, 7, 21, 30, 45, and 60 days. Plasma cytokines (i.e., tumor necrosis factor alpha, interferon gamma), peripheral chimerism, and lymphocyte percentages were analyzed. Median allograft survival was 40 days (range, 40 to 44 days) in the control group, 68 days (range, 61 to 71 days) in the lipopolysaccharide group, and 70 days (range, 69 to 73 days) in both galactomannan groups (p = 0.001). Weight loss was higher in the lipopolysaccharide group (p < 0.001), as was the 83.3 percent rate of diarrheal episodes (control, 0 percent, p = 0.015; galactomannan-72, 0 percent, p = 0.015; and galactomannan-24, 16.7 percent, p = 0.02). Preconditioned rats had higher peripheral blood chimerism (lipopolysaccharide, 2.30 ± 0.13 percent; galactomannan-72, 2.63 ±1.46 percent; and galactomannan-24, 2.47 ± 0.19 percent) compared to the control group (2.06 ± 0.36 percent) (lipopolysaccharide, p = 0.04; galactomannan-72, p = 0.002; and galactomannan-24, p = 0.002). Induction of endotoxin tolerance delays acute rejection in the rat hindlimb transplantation model. Galactomannan preconditioning has no lipopolysaccharide side effects and was equally effective in delaying acute rejection. The contributions of this experimental work are very incipient. Although the use of galactomannan in clinical practice requires more studies to assess its safety, there is no doubt that immunomodulation may be one of the responses that solve the problem of long-term immunosuppression.

Absence of TSC1 Accelerates CD8+ T cell-mediated Acute Cardiac Allograft Rejection.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by inactivating mutations in TSC1 or TSC2.Patients with TSC often require organ transplantation after organ failure. TSC1 serves as an important control node in immune cell development and responses; however, its effect on T cells in transplant immunity has not yet been explored. Here, we characterized the effect of TSC1 deficiency in T cells on acute allograft rejection using a mouse cardiac transplantation model. We observed compromised allograft survival in mice with TSC1-deficient T cells. Notably, the allografts in mice transferred with TSC1-deficient CD8+T cells showed accelerated acute allograft rejection. TSC1 deficiency triggered the increased accumulation of CD8+ T cells in allografts due to augmented infiltration caused by increased CXCR3 expression levels and elevated in-situ proliferation of TSC1-deficient CD8+ T cells. Compared to CD8+ T cells from wild-type (WT) mice, TSC1-deficient CD8+ T cells exhibited enhanced cell proliferation and increased expression levels of interferon-γ and granzyme B after alloantigen stimulation. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used to treat patients with TSC and prevent rejection after solid-organ transplantation. Although rapamycin induced most cardiac allografts to survive beyond 100 d in WT mice, rapamycin-treated cardiac allografts in TSC1-deficient mice were rejected within 60 d. These results suggest that TSC1-deficient recipients may be more resistant to rapamycin-mediated immunosuppression during organ transplantation. Collectively, TSC1 significantly accelerates acute allograft rejection by enhancing the alloreactivity of CD8+ T cells, making them more resistant to mTOR inhibitor-mediated immunosuppression.

MiR-21 antagonism reprograms macrophage metabolism and abrogates chronic allograft vasculopathy

Despite much progress in improving graft outcome during cardiac transplantation, chronic allograft vasculopathy (CAV) remains an impediment to long‐term graft survival. MicroRNAs (miRNAs) emerged as regulators of the immune response. Here, we aimed to examine the miRNA network involved in CAV. miRNA profiling of heart samples obtained from a murine model of CAV and from cardiac‐transplanted patients with CAV demonstrated that miR‐21 was most significantly expressed and was primarily localized to macrophages. Interestingly, macrophage depletion with clodronate did not significantly prolong allograft survival in mice, while conditional deletion of miR‐21 in macrophages or the use of a specific miR‐21 antagomir resulted in indefinite cardiac allograft survival and abrogated CAV. The immunophenotype, secretome, ability to phagocytose, migration, and antigen presentation of macrophages were unaffected by miR‐21 targeting, while macrophage metabolism was reprogrammed, with a shift toward oxidative phosphorylation in naïve macrophages and with an inhibition of glycolysis in pro‐inflammatory macrophages. The aforementioned effects resulted in an increase in M2‐like macrophages, which could be reverted by the addition of L‐arginine. RNA‐seq analysis confirmed alterations in arginase‐associated pathways associated with miR‐21 antagonism. In conclusion, miR‐21 is overexpressed in murine and human CAV, and its targeting delays CAV onset by reprogramming macrophages metabolism.

Galectin-9 is required for endometrial regenerative cells to induce long-term cardiac allograft survival in mice

BackgroundEndometrial regenerative cells (ERCs), a novel type of mesenchymal-like stem cells, were identified as an attractive candidate for immunoregulation and induction of cardiac allograft tolerance. However, the underlying mechanisms of ERCs in immune regulation still remain largely unclear. The present study is designed to determine whether the expression of Galectin-9 (Gal-9), a soluble tandem-repeat member of the galectin family, is crucial for ERC-based immunomodulation.MethodsIn this study, we measured Gal-9 expression on ERCs and then co-cultured Gal-9-ERCs, ERCs, and ERCs+lactose (Gal-9 blocker) with activated C57BL/6-derived splenocytes. Furthermore, we performed mouse heart transplantation between BALB/c (H-2d) donor and C57BL/6 (H-2b) recipient. ERCs were administrated 24 h after the surgery, either alone or in combination with rapamycin.ResultsOur data demonstrate that ERCs express Gal-9, and this expression is increased by IFN-γ stimulation in a dose-dependent manner. Moreover, both in vitro and in vivo results show that Gal-9-ERC-mediated therapy significantly suppressed Th1 and Th17 cell response, inhibited CD8+ T cell proliferation, abrogated B cell activation, decreased donor-specific antibody production, and enhanced the Treg population. The therapeutic effect of ERCs was further verified by their roles in prolonging cardiac allograft survival and alleviating graft pathological changes.ConclusionsTaken together, these data indicate that Gal-9 is required for ERC-mediated immunomodulation and prevention of allograft rejection.

Circular RNA circSnx5 Controls Immunogenicity of Dendritic Cells through the miR-544/SOCS1 Axis and PU.1 Activity Regulation

Dendritic cells (DCs) can orchestrate either immunogenic or tolerogenic responses to relay information on the functional state. Emerging studies indicate that circular RNAs (circRNAs) are involved in immunity; however, it remains unclear whether they govern DC development and function at the transcriptional level. In this study, we identified a central role for a novel circRNA, circSnx5, in modulating DC-driven immunity and tolerance. Ectopic circSnx5 suppresses DC activation and promotes the development of tolerogenic functions of DCs, while circSnx5 knockdown promotes their activation and inflammatory phenotype. Mechanistically, circSnx5 can act as a miR-544 sponge to attenuate miRNA-mediated target depression on suppressor of cytokine signaling 1 (SOCS1) and inhibit nuclear translocation of PU.1, regulating DC activation and function. Furthermore, the main splicing factors (SFs) were identified in DCs, of which heterogeneous nuclear ribonucleoprotein (hnRNP) C was essential for circSnx5 generation. Moreover, our data demonstrated that vaccination with circSnx5-conditioned DCs prolonged cardiac allograft survival in mice and alleviated experimental autoimmune myocarditis. Taken together, our results revealed circSnx5 as a key modulator to fine-tune DC function, suggesting that circSnx5 may serve as a potential therapeutic avenue for immune-related diseases.

Oral administration of salmon cartilage proteoglycan extends the survival of allografts in mice.

Proteoglycan (PG) is a complex glycohydrate that is widely distributed in the extracellular matrix. Oral administration of PG extracted from salmon nasal cartilage has been reported to attenuate the severity and proinflammatory cytokine responses in mouse experimental colitis, autoimmune encephalomyelitis, collagen-induced arthritis and obesity-induced inflammation. In the present study, the effects of salmon nasal cartilage PG on skin allografts were investigated in a mouse model. Oral administration of PG prolonged the survival of skin grafts within 10 days of transplantation. Although PG failed to inhibit allograft rejection at the final stage of transplantation, PG attenuated the cell infiltration in the skin under the transplanted site.

Triptolide inhibits CD4+ memory T cell-mediated acute rejection and prolongs cardiac allograft survival in mice.

There have been numerous investigations into the immunosuppressive effects of triptolide; however, its inhibitory effects on memory T cells remain to be elucidated. Using a cluster of differentiation (CD)4+ memory T-cell transfer model, the aim of the present study was to determine the inhibitory effects of triptolide on CD4+ memory T cell-mediated acute rejection and to determine the potential underlying mechanisms. At 4 weeks after skin transplantation, mouse cervical heart transplantation was performed following the transfer of CD4+ memory T cells. Mice were divided into two groups: A Control [normal saline, 30 ml/kg/day; intraperitoneal injection (ip)] and a triptolide group (triptolide, 3 mg/kg/day; ip). Graft survival, pathological examination and the corresponding International Society for Heart & Lung Transplantation (ISHLT) scores were assessed 5 days following heart transplantation, and levels of interleukin (IL)-2, interferon-γ (IFN-γ), IL-10 and transforming growth factor β1 (TGF-β1) in cardiac grafts and peripheral blood were assessed using reverse transcription-quantitative polymerase chain reaction and ELISA. The duration of cardiac graft survival in the triptolide group was significantly increased compared with the control group (14.3±0.4 vs. 5.3±0.2 days; P<0.001). Further pathological examinations revealed that the infiltration of inflammatory cells and myocardial damage in the cardiac grafts was notably reduced by triptolide, and the corresponding ISHLT scores in the triptolide group were significantly lower than those of the control group (grade 2.08±0.15 vs. 3.67±0.17; P<0.001). In addition, triptolide was able to significantly reduce IL-2 and IFN-γ secretion (P<0.01), significantly increase TGF-β1 secretion in the cardiac grafts and peripheral blood (P<0.01) and increase IL-10 secretion in the cardiac grafts. Therefore, the present study suggests that triptolide inhibits CD4+ memory T cell-mediated acute rejection and prolongs cardiac allograft survival in mice. This effect may be mediated by the inhibition of cytokine secretion by type 1 T helper cells and promotion of regulatory T cell proliferation.

Targeted deletion of Traf2 allows immunosuppression-free islet allograft survival in mice.

Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses. T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4+ T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies. Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4+ T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4+ and CD8+ T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4+ T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts. Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

Prolongation of Cardiac Allograft Survival by Endometrial Regenerative Cells: Focusing on B-Cell Responses.

Endometrial regenerative cells (ERCs) have been recently evaluated as an attractive candidate source for emerging stem cell therapies in immunosuppression, but their role in immunoregulation is not fully understood. The present study was designed to investigate their effects, especially on B‐cell responses in heart transplantation. In this study, ERCs were noninvasively obtained from menstrual blood. Heart transplantation was performed between C57BL/6 (H‐2b) donor mice and BALB/c (H‐2d) recipients. B‐cell activation and antibody levels were determined using fluorescence‐activated cell sorting, enzyme‐linked immunosorbent assay and ELISpot. In this study, we demonstrated that ERCs negatively regulated B‐cell maturation and activation in vitro without affecting their viability. ERC treatment prolonged cardiac allograft survival in mice, which was correlated with a decrease in IgM and IgG deposition and circulating antidonor antibodies, as well as with reduction in frequencies of antidonor antibody‐secreting CD19+ B cells. In addition, upon ex vivo stimulation, B cells from ERC‐treated heart transplant recipients had impaired proliferation capacity and produced less IgM and IgG antibody. Moreover, ERC treatment of mice receiving ovalbumin (OVA)‐aluminum hydroxide vaccine resulted in significant lower numbers of anti‐OVA IgG antibody‐secreting splenic B cells and lower anti‐OVA antibody titres. Our results indicate that therapeutic effects of ERCs may be attributed at least in part by their B‐cell suppression and humoral response inhibition, suggesting the potential use of ERCs for attenuating antibody‐mediated allograft rejection. Stem Cells Translational Medicine 2017;6:778–787

PD-L1 Deficiency within Islets Reduces Allograft Survival in Mice.

BackgroundIslet transplantation may potentially cure type 1 diabetes mellitus (T1DM). However, immune rejection, especially that induced by the alloreactive T-cell response, remains a restraining factor for the long-term survival of grafted islets. Programmed death ligand-1 (PD-L1) is a negative costimulatory molecule. PD-L1 deficiency within the donor heart accelerates allograft rejection. Here, we investigate whether PD-L1 deficiency in donor islets reduces allograft survival time.MethodsGlucose Stimulation Assays were performed to evaluate whether PD-L1 deficiency has detrimental effects on islet function. Islets isolated from PDL1-deficient mice or wild- type (WT) mice (C57BL/6j) were implanted beneath the renal capsule of streptozotocin (STZ)-induced diabetic BALB/c mice. Blood glucose levels and graft survival time after transplantation were monitored. Moreover, we analyzed the residual islets, infiltrating immune cells and alloreactive cells from the recipients.ResultsPD-L1 deficiency within islets does not affect islet function. However, islet PD-L1 deficiency increased allograft rejection and was associated with enhanced inflammatory cell infiltration and recipient T-cell alloreactivity.ConclusionsThis is the first report to demonstrate that PD-L1 deficiency accelerated islet allograft rejection and regulated recipient alloimmune responses.

Arsenic Trioxide Induces T Cell Apoptosis and Prolongs Islet Allograft Survival in Mice.

T cell-mediated immune rejection is a key barrier to islet transplantation. Preliminary studies have shown that arsenic trioxide (As2O3) can inhibit T cell responses and prolong heart allograft survival. Here, we sought to investigate the possibility of using As2O3 to prolong islet allograft survival in an acute rejection model of Balb/c to C57B/6 mice. Recipient mice were treated with As2O3 and/or rapamycin after islet allograft transplantation. At day 10 after transplantation, the graft, spleen, lymph nodes, and blood of the recipient mice were recovered for analysis. In vitro, to further examine the mechanism underlying As2O3 protection of islet allografts against T cell-mediated rejection, mixed lymphocyte reaction and apoptosis analyses of T cells were performed. The phosphorylation levels of IκBα and p38 were also evaluated to confirm the proliferation and apoptosis of As2O3-treated T cells. We found that As2O3 prolonged islet allograft survival by reducing inflammatory reactions, influencing cytokine synthesis and secretion and T-cell subset proportions, and inhibiting T-cell responses. Furthermore, As2O3 and rapamycin showed a synergistic effect in suppressing islet allotransplant rejection. Arsenic trioxide may prevent allograft rejection by inhibiting T-cell proliferation and inducing T-cell apoptosis.

CD27low natural killer cells prolong allograft survival in mice by controlling alloreactive CD8+ T cells in a T-bet-dependent manner.

Natural killer (NK) cells play a dichotomous role in alloimmune responses because they are known to promote both allograft survival and rejection. The aim of this study was to investigate the role of functionally distinct NK cell subsets in alloimmunity with the hypothesis that this dichotomy is explained by the functional heterogeneity of distinct NK cell subsets. Because T-bet controls thematuration of NK cells from CD27high to terminally differentiated CD27low NK cells, we used Rag−/−T-bet−/− mice that lackmature CD27low NK cells to study the distinct roles of CD27low versus CD27high NK cells in a model of Tcell–mediated skin transplant rejection under costimulatory blockade conditions. We found that T cell–reconstituted Rag1−/− recipients (possessing CD27low NK cells) show significantly prolonged allograft survival on costimulatory blockade when compared to Rag1−/−T-bet−/− mice (lacking CD27low NK cells), indicating that CD27low but not CD27high NK cells enhance allograft survival. Critically, Rag1−/−T-bet−/− recipients showed strikingly increased alloreactive memory CD8+ Tcell responses, as indicated by increased CD8+ Tcell proliferation and interferon-γ production. Therefore, we speculated that CD27low NK cells directly regulate alloreactive CD8+ Tcell responses under costimulatory blockade conditions. To test this, we adoptively transferred CD27low NK cells into Rag1−/−T-bet−/− skin transplant recipients and found that the CD27low NK cells restore better allograft survival by inhibiting the proliferation of alloreactive interferon-γ+CD8+ T cells. In summary, mature CD27low NK cells promote allograft survival under costimulatory blockade conditions by regulating alloreactive memory CD8+ T-cell responses.

The roles of IL-2 and IL-10 enhance anti-CD45RBmAb immune inhibition in allograft skin.

As a new type of immune tolerance inducer, anti-CD45RB monoclonal antibodies (anti-CD45RBmAb) can prolong the graft survival time of animal organs or cell transplantation as well as induce stable immune tolerance. Both interleukin (IL)-2 and IL-10 have important roles in the induction and maintenance of immunological tolerance. However, whether these cytokines combined with anti-CD45RBmAb can promote immune tolerance is poorly understood. Therefore, we investigated the effect of IL-2 and IL-10 in vitro and in vivo on the tolerance induction by anti-CD45RBmAb. The changes of Treg and Th17 cells and Th1/Th2 cytokines in anti-CD45RBmAb induced prolongation of skin allograft survival in mice. The finding of a role for IL-2 is novel, of interest, IL-2 promoted anti-CD45RBmAb-induced CD4(+) T cell differentiation into Treg and Th2 cells and suppressed Th17 and Th1 cells. IL-2 enhanced the induction of immune tolerance by anti-CD45RBmAb and significantly prolonged skin graft survival time in vivo. In contrast, this effect should be demonstrated experimentally by neutralizing IL-2 and inhibition of the effect of anti-CD45RBmAb, and neutralizing IL-10 showed no effect for anti-CD45RBmAb-induced tolerance. These data reveal that IL-2 significantly enhances anti-CD45RBmAb-induced immune tolerance via up-regulated T regulatory (Treg) cells and the balance of Th1/Th2 shifts. Conversely, IL-10 showed no effect on anti-CD45RBmAb-induced tolerance.

Overexpression of Jagged-1 combined with blockade of CD40 pathway prolongs allograft survival.

Dendritic cells (DCs) have the tolerogenic potential to regulate adaptive immunity and induce allografts acceptance. Here we investigated whether blockade of the CD40 pathway could enhance the immune tolerance induced by DC2.4 cells modified to express Jagged-1 (JAG1-DC) in heart transplantation. Results showed that JAG1-DC treatment combined with anti-CD40L monoclonal antibody (mAb) administration significantly prolonged cardiac allograft survival in mice, with long-term survival (>110 days) of 50% of the allografts in the recipients. The therapy specifically inhibited the immune response, induced alloantigen-specific T-cell hyporesponsiveness, upregulated transforming growth factor-β synthesis and increased the population of regulatory T cells (Tregs) driven by Jagged-1-Notch activation. These results highlight the potential application of gene therapy to induce alloantigen-specific Tregs effectively by providing the Jagged-1 stimulation.

Retraction: ‘CCR5 blockade in combination with rapamycin prolongs cardiac allograft survival in mice’ by J. Li, K. Zhang, P. Ye, S. Wang and J. Xia.

Retraction: ‘CCR5 blockade in combination with rapamycin prolongs cardiac allograft survival in mice’ by J. Li, K. Zhang, P. Ye, S. Wang and J. Xia.

Immunomodulation Induced By a Novel Immunosuppressant, NK026680 Plus Donor Specific Transfusion Permits a Long-Term Cardiac Allograft Survival in Mice.

Background: We have recently demonstrated that a novel triazolopylimidine derivative, NK026680 (NK), inhibits T cell proliferation and prolongs rat cardiac allograft survival. In this study, we examined the immunomodulatory effect of NK in combination with donor specific transfusion (DST) in mice. Methods: A fully MHC incompatible BALB/c-to-C57BL/6 mouse heart transplantation was performed on day 0. DST, 20x106 donor-splenocytes, was given intravenously on day -7. NK (40 mg/kg/day) was orally administered from day -7 to day +6. Recipient-splenocytes were cocultured with irradiated donor-splenocytes, and T cell proliferation (3H-thymidine incorporation), IFN-g production (ELISPOT) and cell phenotypes (FACS) were assessed. Results: Untreated mice promptly rejected cardiac allograft at a graft median survival time (gMST) of 6.5 days. DST or NK monotherapy prolonged gMST to 24.5 and 25.5 days, respectively. In a sharp contrast, NK plus DST treatment (NK+DST) markedly prolonged gMST to 75.5 days. (A). In the NK+DST group, both proliferation and IFN-g production against donorantigens transiently increased on day 0, while they were both significantly suppressed on day 7 (p<0.05, vs. Untreated). Following in vivo antigen-challenge by infusing 20x106 donor-splenocytes on day 0, the splenic Foxp3+CD4+ T cells increased[Figure 1](B); however, CD69+ effector T cells were abolished.Figure: No Caption available.(C and D) in the NK+DST group. Conclusion:NK together with DST induces a potent immunomodulatory effect by shifting immune-balance towards a regulatory milieu, and markedly prolongs cardiac allograft survival in mice. DISCLOSURES:Saiga, K.: Employee, salary.

Donor Specific Transfusion Combined With Anti-CD134L and Rapamycin Inhibits Accelerated Rejection Mediated By Alloantigen-Primed Memory Cells in Mice.

Background Donor-reactive memory cells threaten the survival of transplanted organs, and hamper maintenance of transplant tolerance. In this study, we investigated the inhibitory effect of donor splenocyte transfusion(DST) combined with anti-CD134L and rapamycin on memory cells-mediated accelerated rejection in an allogeneic heart transplantation model in mice. Methods Male Balb/c and C57BL/6 mice were used as the donor and recipient. After sensitization with donor skin allografts, splenocytes from the alloantigen-primed C57BL/6 mice were prepared and adoptively transferred (5×106) to a naïve recipient followed by heart transplantation (HTx). Thus, we established a memory cells-mediated accelerated rejection model of heart transplantation. Mitomycin C-treated donor splenocytes were transfused to HTx recipients seven days prior to transplantation, and short-term low dose of rapamycin and anti-CD134L were initiated after transplantation. Graft survival and pathology of rejection was examined. Memory cells proliferation, functionand the proportion of regulatory T and B cell populations were also examined. Results The mean survival time (MST) of grafts in thecontrolgroup was 5.3 days,and more than 40 days in the group treated with DST/anti-CD134L/rapamycin. The DST/anti- CD134L/rapamycinregimen prevented lymphocytic infiltration in the grafts, inhibited memory cells proliferation in the spleen, reduced donor specific antibody level, and increased proportion of CD4+Foxp3+regulatory T cells (Tregs) as well as CD1d+CD5+regulatoryB cells (Bregs). Conclusions Our data suggest that DST combined with rapamycin and anti-CD134L can synergistically inhibit accelerated rejection mediated by memory cells and thus prolong heart allograft survival in mice. Both regulatory T cells and regulatory B cells may play important roles in modulation of memory cells.

Pretreatment of Transfused Donor Splenocytes and Allografts With Mitomycin C Attenuates Acute Rejection in Heart Transplantation in Mice

ObjectiveThe aim of this study was to investigate the effect of pretreatment of donor splenocytes and grafts with mitomycin C (MMC) on heart allograft survival, as well as to demonstrate the mechanism of function. MethodsDonor splenocytes from Balb/C mice were incubated with MMC (40 μg/mL) in vitro and then transfused into recipients (C57BL/6 mice). The heart allograft was perfused with MMC before harvest. Graft survival and histopathology were examined. Lymphocyte activation, regulatory T cells, and donor splenocyte apoptosis were examined with the use of flow cytometry. ResultsMMC incubation in vitro induced apoptosis of donor splenocytes (15.5 ± 2.3% vs 23.2 ± 4.2%; P < .01). Either intravenous injection of MMC-treated donor splenocytes or transplantation of allograft pretreated with MMC prolonged heart allograft mean survival time from 7 ± 0.8 days to 20.5 ± 1.9 days or 10 ± 0.9 days, respectively (both P < .01). A combination of MMC-pretreated donor splenocyte transfusion and allografts showed the best effect on prolongation of graft survival (28.5 ± 1.8 days). Activation of CD4+ T cells in spleen and peripheral lymph nodes of recipients was significantly inhibited by either MMC-splenocyte transfusion or the combination treatment. Meanwhile, the percentage of CD4+CD25+Foxp3+ regulatory T cells in the spleen was increased in the MMC-splenocyte transfusion group (15.5 ± 1.1% vs 18.2 ± 0.9%; P < .05). ConclusionsBoth injection of MMC-conditioned donor splenocytes and MMC-conditioned allograft have effects on prolongation of heart allograft survival in mice, and MMC-conditioned donor splenocytes might play an essential role. MMC pretreatment induced regulatory T cells likely through induction of donor splenocyte apoptosis, and thus it inhibited T-cell activation.