Murine Cardiac Allograft Research Articles

C5aR1 regulates migration of suppressive myeloid cells required for costimulatory blockade-induced murine allograft survival.

Costimulatory blockade-induced murine cardiac allograft survival requires intragraft accumulation of CD11b+ Ly6Clo Ly6G- regulatory myeloid cells (Mregs) that expand regulatory T cells (Tregs) and suppress effector T cells (Teffs). We previously showed that C5a receptor (C5aR1) signaling on T cells activates Teffs and inhibits Tregs, but whether and/or how C5aR1 affects Mregs required for transplant survival is unknown. Although BALB/c hearts survived >60days in anti-CD154 (MR1)-treated or cytotoxic T-lymphocyte associated protein 4 (CTLA4)-Ig-treated wild-type (WT) recipients, they were rejected at ~30days in MR1-treated or CTLA4-Ig-treated recipients selectively deficient in C5aR1 restricted to myeloid cells (C5ar1fl/fl xLysM-Cre). This accelerated rejection was associated with ~2-fold more donor-reactive T cells and ~40% less expansion of donor-reactive Tregs. Analysis of graft-infiltrating mononuclear cells on posttransplant day 6 revealed fewer Ly6Clo monocytes in C5ar1fl/fl xLysM-Cre recipients. Expression profiling of intragraft Ly6Clo monocytes showed that C5aR1 deficiency downregulated genes related to migration/locomotion without changes in genes associated with suppressive function. Cotransfer of C5ar1fl/fl and C5ar1fl/fl xLysM-Cre myeloid cells into MR1-treated allograft recipients resulted in less accumulation of C5ar1-/- cells within the allografts, and in vitro assays confirmed that Ly6Chi myeloid cells migrate to C5a/C5aR1-initiated signals. Together, our results newly link myeloid cell-expressed C5aR1 to intragraft accumulation of myeloid cells required for prolongation of heart transplant survival induced by costimulatory blockade.

Minocycline Prolongs Murine Cardiac Allograft Survival through Suppression of Acute Rejection

Background Acute rejection (AR) occurs in 10-20% of renal allograft recipients despite combination immunosuppression, suggesting there are pathways that are not adequately inhibited by current therapy. Large scale gene expression profiling using microarray has enabled the characterisation of the molecular changes in AR. In recent years there has been an increased focus on identifying the most highly connected genes in various disease states, rather than the most highly expressed, as these ‘key driver’ genes are more likely to assume key regulatory roles. This has not been studied in the context of AR in renal transplantation, and may serve as potential targets for novel therapeutics. Aims 1.To derive key driver genes from microarray data of human renal allograft biopsy tissue with AR 2.Identify drugs that can inhibit the key driver genes 3. Experimental validation. Methods A meta-analysis of microarray data of 735 human kidney allograft biopsy samples was performed to identify the differentially expressed genes, and the key drivers were further derived using bioinformatics methods. Drug repurposing tools were used to identify candidate therapeutics and Minocycline was selected for further evaluation in a full allo-mismatch murine heterotopic cardiac allograft model. The impact of minocycline on the allograft when used alone, or when administered with immunosuppression (CTLA4Ig), was evaluated. Allograft histology was assessed and quantitative RT-PCR was performed to measure mRNA expression of key driver genes and cytokines/chemokines. A competing risk analysis was used to assess cumulative incidence of graft rejection, with graft loss due to other causes regarded as a competing risk. Results 14 key driver genes were identified, with Caspase 1 identified as the central key driver gene. Minocycline, a tetracycline antibiotic, is a well described inhibitor of Caspase 1, the central key driver gene. Minocycline (50mg/kg IP BD) compared with saline did not prolong allograft survival, butwas able to reduce the inflammatory profile of acute rejection as demonstrated by the reduction in the mRNA expression in the allograft tissue of a number of key drivers and inflammatory cytokines and chemokines (eg IFNγ, CXCL9) at day 4 post transplant. However, when minocycline was administered in conjunction with immunosuppression (CTLA4Ig 250ug IP Day 2 alone with minocycline 50mg/kg IP BD up to 27 days post transplant), an extension in allograft survival was observed compared with the control group (CTLA4Ig Day 2 with saline) (Hazard ratio 0.27 CI 0.099-0.736 p-value <0.01). 3 treatment animals were euthanased prior to cessation of allograft heart beat for other reasons. Conclusion Minocycline alters the inflammatory profile of AR, and prolongs murine cardiac allograft survival when given in conjunction with immunosuppressive therapy. Further evaluation as to the precise mechanisms by which this occurs is required. Jacquot Research Entry Scholarship, Royal Australasian College of Physicians. KK and ZY are co-first authors.

Gut Microbiota Determines Graft Survival Outcome Through Regulation of Anti-Inflammatory and Pro-Inflammatory Responses

Introduction The gut microbiota plays an important role on the induction and training of the host immune system. It is unknown whether microbiota from the contrasting environments of colitic or pregnant mice are pro-inflammatory or suppressive. We hypothesized that gut microbiota from different sources could influence the survival of murine cardiac allografts through regulation of immune responses. Methods C57BL/6 mice received BALB/c vascularized cardiac allografts and fecal microbiota transfer (FMT) by oral gavage. FMT samples were from normal or pregnant C57BL/6, spontaneously colitic mice, or cultured Bifidobacterium pseudolongum (Bifido) (a dominant member of the pregnant gut microbiota). Tacrolimus was administered daily. Fecal pellets were collected weekly post-transplant and analyzed by 16S rRNA gene sequencing. Grafts were assessed for survival, harvested at days 40-60 or rejection, and assessed for inflammation by H&E and fibrosis by Masson's trichrome. Macrophage (MF) and dendritic cell (DC) lines were stimulated with purified Bifido cells and cytokine responses measured. Results Pregnant FMT significantly prolonged survival, and reduced inflammation and fibrosis of cardiac allografts. Normal or colitic FMT resulted in inferior survival, along with increased fibrosis and inflammation. 16S rRNA analyses demonstrated abundant Bifido in the pregnant and normal but not the colitic FMT samples. Transfer of Bifido alone also resulted in enhanced allograft survival and reduced inflammation and fibrosis. Serial 16S rRNA analyses of gut microbiota from normal, colitic, pregnant FMT, or Bifido groups, revealed significant differences in bacterial community structure (alpha- and beta-diversity). Bifido remained abundant in pregnant FMT samples for at least 40 days. In contrast, Desulfovibrio and Deferribacteraceae were more abundant in colitic samples. Microbiota community structures of the various groups became less divergent over time, but remained distinct. DC and MF lines stimulated with Bifido expressed higher levels of the anti-inflammatory cytokine IL-10 and homeostatic chemokine CCL19, but lesser amounts of the pro-inflammatory cytokines TNFa and IL-6 compared to LPS stimulated cells. Conclusion The gut microbiota has profound effects on host immune responses with consequences for cardiac allograft outcomes. Distinct bacterial species or genera may both alter immunity and predict graft outcomes, with Bifidobacterium species inducing anti-inflammatory effects through stimulation of DC and MF. Therapeutic targeting of the microbiota or its functions could be beneficial in reducing inflammation and subsequent graft rejection.

Graft Protective Effect of HMG-CoA Reductase Inhibitor Pravastatin in Murine Cardiac Allograft Transplantation

The HMG-CoA reductase inhibitor (statin), which reduces serum cholesterol, has been demonstrated in the control of immune responses and may potentially play an important role in the regulation of acute and chronic rejection in organ transplantations. We investigated the graft-protective effect of a kind of statin, pravastatin, in the survival of fully major histocompatibility complex--mismatched murine cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients through microsurgical techniques. CBA recipients transplanted with a C57BL/6 heart received oral administration of 40, 120, or 400 μg/kg/day of pravastatin from the day of transplantation to 7 days afterward. Immunohistochemical staining studies were performed to determine whether intimal formation of coronary arteries in the transplanted cardiac allografts was preserved and also to conduct morphometric analysis. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] 7 days). CBA recipients exposed with 40 and 120 μg/kg/day of pravastatin had a small prolonged allograft survival (MSTs of 10 and 9 days, respectively). However, the MST of CBA recipients exposed to 400 μg/kg/day of pravastatin was significantly effective for allograft survival (MST 50 days). Immunohistochemical staining assessments on 4 weeks after grafting showed suppression of intimal hyperplasia in allograft coronary arteries. Pravastatin could induce the prolongation of fully major histocompatibility complex--mismatched cardiac allograft through the protection of the coronary artery.

Graft protective effect and induction of CD4+Foxp3+ cell by Thrombomodulin on allograft arteriosclerosis in mice

BackgroundThrombomodulin (TM) is a promising therapeutic natural anti-coagulant, which exerts the effects to control disseminated intravascular coagulation. However, little is known whether TM on micro-vessels could play an important role in the regulation of intimal hyperplasia. We investigated the vessel-protective effect of TM in the survival of fully major histocompatibility complex (MHC)-mismatched murine cardiac allograft transplantation.MethodsCBA recipients transplanted with a C57BL/6 heart received intraperitoneal administration of normal saline or 0.2, 2.0, and 20.0 μg/day of TM for 7 days (n = 5, 7, 11, and 11, respectively). Immunohistochemical and fluorescent staining studies were performed to determine whether CD4+Foxp3+ regulatory T cell were generated at 2 and 4 weeks after grafting. Morphometric analysis for neointimal formation in the coronary arteries of the transplanted allograft was conducted at 2 and 4 weeks after grafting.ResultsUntreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients exposed with the above doses had significantly prolonged allograft survival (MSTs, 17, 24 and 50 days, respectively). Morphometric assessment showed that intimal hyperplasia was clearly suppressed in the left and right coronary arteries or allografts from TM-exposed recipients 2 and 4 weeks. Immunohistochemical studies at 2 weeks showed more CD4+Foxp3+ cells and lower myocardial damage in the allografts from TM-exposed recipients. Notably, fluorescent staining studies demonstrated that TM-exposed recipients 4 weeks post-engraftment had strong aggregation of CD4+Foxp3+ cells in the intima of the coronary arteries of the cardiac allografts.ConclusionsTM may prolong the survival of fully MHC-mismatched cardiac allografts through suppressing intimal hyperplasia and inducing the accumulation of regulatory CD4+Foxp3+ cells within coronary arteries.

Administration of Thrombomodulin (CD141) Could Improve Cardiac Allograft Survival in Mice

Thrombomodulin (TM) is a promising natural anti-coagulant therapeutic protein that is effective in the treatment of disseminated intravascular coagulation. However, the mechanisms by which TM on micro-vessels enable the regulation of intimal hyperplasia remain elusive. We investigated the graft-protective effects of TM in a fully major histocompatibility complex-mismatched murine cardiac allograft transplantation model. CBA recipients transplanted with a C57BL/6 heart received intraperitoneal administration of 0.2, 2.0, and 20.0 μg/day of TM for 8 days. Histological staining was conducted to assess the degree of inflammation and infiltration in the transplanted cardiac grafts. Untreated CBA recipients rejected C57BL/6 cardiac grafts acutely (median survival time [MST] was 7 days). CBA recipients exposed to the above dosages had significantly prolonged allograft survival (MSTs were 16, 21, and 37.5 days, respectively). Histologic assessments from TM-exposed recipients 2 weeks after grafting showed that the myocardium and vessel structure in their allografts were clearly preserved, and that the infiltration of inflammatory cells around coronary arteries was suppressed. TM can induce the prolongation of fully major histocompatibility complex-mismatched cardiac allograft by exerting graft protective effects within the myocardium and coronary arteries.

Aquaporin 4 blockade improves survival of murine heart allografts subjected to prolonged cold ischemia.

Prolonged cold ischemia storage (CIS) is a leading risk factor for poor transplant outcome. Existing strategies strive to minimize ischemia-reperfusion injury in transplanted organs, yet there is a need for novel approaches to improve outcomes of marginal allografts and expand the pool of donor organs suitable for transplantation. Aquaporins (AQPs) are a family of water channels that facilitate homeostasis, tissue injury, and inflammation. We tested whether inhibition of AQP4 improves the survival of fully MHC-mismatched murine cardiac allografts subjected to 8hours of CIS. Administration of a small molecule AQP4 inhibitor during donor heart collection and storage and for a short-time posttransplantation improves the viability of donor graft cells, diminishes donor-reactive T cell responses, and extends allograft survival in the absence of other immunosuppression. Furthermore, AQP4 inhibition is synergistic with cytotoxic T lymphocyte-associated antigen 4-Ig in prolonging survival of 8-hour CIS heart allografts. AQP4 blockade markedly reduced T cell proliferation and cytokine production in vitro, suggesting that the improved graft survival is at least in part mediated through direct effects on donor-reactive T cells. These results identify AQPs as a promising target for diminishing donor-specific alloreactivity and improving the survival of high-risk organ transplants.

Rikkunshito (TJ-43) Improved Reduction of Food Intake in a Murine Cardiac Transplantation Model

Rikkunshito (TJ-43), an eight-component traditional Japanese herbal medicine, has been used in clinics for gastritis, vomiting, and appetite loss. We investigated the effects of TJ-43 on the amelioration of appetite loss in the surgical-exposed model of murine cardiac allograft transplantation. CBA mice underwent transplantation of a CBA (syngeneic group) or C57BL/6 heart (allogeneic group) and received oral administration of 2 g/kg/d of TJ-43 from the day of transplantation until 7 days afterward. The amount of food intake (FI) and weight change after operation were recorded from 1 to 28 postoperative days. The allogeneic group had less average amounts of FI for 1 week compared with the syngeneic group (FI was 1.90 ± 0.43 g and 2.66 ± 0.46 g, respectively). Average FIs between the syngeneic and allogeneic groups with TJ-43 for 1 week were 2.36 ± 0.44 g and 2.30 ± 0.13 g, respectively, and those with distilled water were 2.66 ± 0.46 g and 1.90 ± 0.43 g, respectively, suggesting that exposure with TJ-43 tended to ameliorate the reduction of FI. Similarly, the effect on the amelioration of average FI in syngeneic and allogeneic groups exposed for 2 weeks was confirmed. However, exposure to with TJ-43 had no effects on FI after 4 weeks. TJ-43 could prevent reduction of average FI induced by the surgical-exposed model of murine cardiac allograft transplantation.

Induction of Regulatory CD4+ Cells and Prolongation of Fully Major Histocompatibility Complex Mismatched Murine Cardiac Allograft by Shigyakusan

Shigyakusan (also known as Tsumura Japan [TJ]-35) is composed of peony, bitter orange, licorice, and Bupleuri radix is used for cholecystitis and gastritis as an anti-inflammatory agent. We investigated the effect of TJ-35 on alloimmune response in a murine heart transplantation model. CBA mice that underwent transplantation of a C57BL/6 (B6) heart were assigned to four groups: no treatment, TJ-35–exposed, each component–exposed, or each component missing–exposed. The four groups above each received oral administration of TJ-35, each component, or TJ-35 with each component missing from the day of transplantation until 7 days, respectively. Untreated CBA recipients rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). TJ-35–exposed CBA recipients had significantly prolonged B6 allograft survival (MST, 20.5 days). However, MSTs of CBA recipients that had been administered each component and TJ-35 with each component missing did not reach that of TJ-35–exposed recipients. Adoptive transfer of CD4+ splenocytes from TJ-35–exposed primary allograft recipients resulted in significant prolonged allograft survival in naïve secondary recipients (MST, 63 days). Flow cytometry studies showed that the percentage of CD4+CD25+Foxp3+ cell population was increased in TJ-35–exposed CBA recipients. In conclusion, TJ-35–induced prolongation of fully allogeneic cardiac allografts and may generate regulatory CD4+CD25+Foxp3+ cells in our model. The effect seemed to require all components of TJ-35.

Complement Dependence of Murine Costimulatory Blockade-Resistant Cellular Cardiac Allograft Rejection.

Building on studies showing that ischemia-reperfusion-(I/R)-injury is complement dependent, we tested links among complement activation, transplantation-associated I/R injury, and murine cardiac allograft rejection. We transplanted BALB/c hearts subjected to 8-h cold ischemic storage (CIS) into cytotoxic T-lymphocyte associated protein 4 (CTLA4)Ig-treated wild-type (WT) or c3-/- B6 recipients. Whereas allografts subjected to 8-h CIS rejected in WT recipients with a median survival time (MST) of 37days, identically treated hearts survived >60days in c3-/- mice (p<0.05, n=4-6/group). Mechanistic studies showed recipient C3 deficiency prevented induction of intragraft and serum chemokines/cytokines and blunted the priming, expansion, and graft infiltration of interferon-γ-producing, donor-reactive T cells. MST of hearts subjected to 8-h CIS was >60days in mannose binding lectin (mbl1-/- mbl2-/- ) recipients and 42days in factor B (cfb-/- ) recipients (n=4-6/group, p<0.05, mbl1-/- mbl2-/- vs. cfb-/- ), implicating the MBL (not alternative) pathway. To pharmacologically target MBL-initiated complement activation, we transplanted BALB/c hearts subjected to 8-h CIS into CTLA4Ig-treated WT B6 recipientswith or withoutC1 inhibitor (C1-INH). Remarkably, peritransplantation administration of C1-INH prolonged graft survival (MST >60days, p<0.05 vs. controls, n=6) and prevented CI-induced increases in donor-reactive, IFNγ-producing spleen cells (p<0.05). These new findings link donor I/R injury to T cell-mediated rejection through MBL-initiated, complement activation and support testing C1-INH administration to prevent CTLA4Ig-resistant rejection of deceased donor allografts in human transplant patients.

What's Hot, What's New From the 2016 American Transplant Congress.

From June 11-15, 2016 the American Transplant Congress, the joint meeting of the American Society of Transplantation and the American Society of Transplant Surgeons, held its annual meeting in Boston, MA. The meeting, attended by 5200 registrants, included pre-meeting conferences, focused topic sessions, and hundreds of high-quality presentations from the transplant field. This meeting report highlights key findings from specific basic science, translational, and clinical research presentations deemed to have notable impact in thematic areas. In particular, there were a number of transformative studies indicating important advances in the understanding of alloimmunity, chronic rejection, tolerance, and organ-specific outcomes. Many of these results are discussed in the context of the published literature to showcase rapid advances in the transplant field.

MicroRNAs Involved in Acute Rejection and Tolerance in Murine Cardiac Allografts.

Induction of immunologic tolerance is the ultimate goal of organ transplant. To investigate the involvement of microRNA in tolerance induction after organ transplant, murine cardiac allografts were performed and the expression of microRNA in the grafts was analyzed. Cardiac allografts were performed using C57BL/10 (H2-Kb) to CBA/N (H2-Kk) fully mismatched combination with or without eicosapentaenoic acid for tolerance induction. Ten microRNA, mir-146a, 15b, 223, 23a, 27a, 34a, 451, 101a, 101b, 148a, discovered in hepatic grafts were examined by quantitative reverse transcription polymerase chain reaction using RNA from the cardiac allografts. The administration of eicosapentaenoic acid markedly prolonged the cardiac allograft survival (median survival time > 100 days) and decreased the pathological score. Quantitative reverse transcription polymerase chain reaction revealed that mir-223 was up-regulated in accordance with pathological deterioration as compared with the expression observed in the syngeneic grafts. In contrast, the other microRNA was down-regulated. Pearson product moment correlation analysis demonstrated that the expression patterns of mir-223 and mir-146a had high or moderate positive associations between the cardiac and haptic allografts in mice. The change in the microRNA expression in the allografts suggests that microRNA plays a role in the induction and/or maintenance of tolerance after allograft transplant. Our findings suggest that mir-223 may be associated with rejection while mir-146a, -15b, -23a, -27a, -34a, -451, -101a, -101b, -148a may be involved in tolerance. A superior grasp of the mechanism for rejection and tolerance observed in the murine heart allotransplant model may provide a better curative treatment strategy to mitigate allograft rejection.

Myeloid-derived Suppressor Cells Recruit CD4+/Foxp3+ Regulatory T Cells in a Murine Cardiac Allograft

IntroductionMyeloid-derived suppressor cells (MDSCs) play an important role in regulating allograft rejection in organ transplantation. On the other hand, CD3+/CD4+/FOXP3+ regulatory T cells (Tregs) also are of vital importance in immunological tolerance. We previously revealed that adoptive transfer of MDSCs recruited Tregs in the spleen. However, it is still uncertain whether MDSCs are capable of recruiting Tregs to an allograft in vivo. ObjectivesWe conducted adoptive transfer experiments of MDSCs to clarify the effects of MDSCs on Tregs in vivo. MethodsGr-1+/CD11b+ MDSCs were isolated from rapamycin-treated cardiac transplant (CTx) recipients (3 mg/kg, intraperitoneally on postoperative days [POD] 0, 2, 4, and 6) on POD 7 by magnetic-activated cell sorting (purity >95%). In murine heterotopic cardiac transplantation, 2 × 106 MDSCs were transferred into the graft aorta 5 minutes before reperfusion. ResultsFlow cytometric analyses of a cardiac allograft on POD 7 showed that MDSCs derived from rapamycin-treated CTx mice (MDSCs-Rap) transfer led to significant recruitment of Tregs compared with a PBS-injected allograft. The level of programmed death ligand-1 (PD-L1) on MDSCs-Rap was higher than those from non-treated recipients.Furthermore, pathological findings also confirmed accumulation of Foxp3+ Tregs in an allograft. ConclusionInduced PD-L1 on MDSCs might result in recruitment of Tregs. These results suggested that functional MDSCs possessed an ability to induce Tregs in a cardiac allograft and developed a tendency to immunological tolerance.

The Limits of Linked Suppression for Regulatory T Cells.

We have previously found that CD4(+)CD25(+) regulatory T cells (Tregs) can adoptively transfer tolerance after its induction with costimulatory blockade in a mouse model of murine cardiac allograft transplantation. In these experiments, we tested an hypothesis with three components: (1) the Tregs that transfer tolerance have the capacity for linked suppression, (2) the determinants that stimulate the Tregs are expressed by the indirect pathway, and (3) the donor peptides contributing to these indirect determinants are derived from donor major histocompatibility complex (MHC) antigens (Ags). First heart transplants were performed from the indicated donor strain to B10.D2 recipients along with costimulatory blockade treatment (250 μg i.p. injection of MR1 on day 0 and 250 μg i.p. injection of CTLA-4 Ig on day 2). At least 8 weeks later, a second heart transplant was performed to a new B10.D2 recipient who had been irradiated with 450 cGy. This recipient was given 40 × 106 naive B10.D2 spleen cells + 40 × 106 B10.D2 spleen cells from the first (tolerant) recipient. We performed three different types of heart transplants using various donors. (1) Tregs suppress the graft rejection in an Ag-specific manner. (2) Tregs generated in the face of MHC disparities suppress the rejection of grafts expressing third party MHC along with tolerant MHC. The limits of linkage appear to be quantitative and not universally determined by either the indirect pathway or by peptides of donor MHC Ags.

Blockade of Extracellular HMGB1 Suppresses Xenoreactive B Cell Responses and Delays Acute Vascular Xenogeneic Rejection

Blockade of extracellular high mobility group box 1 (HMGB1) can significantly prolong murine cardiac allograft survival. Here, we determined the role of HMGB1 in xenotransplantation. Sprague-Dawley rat hearts were transplanted heterotopically into BALB/c mice. Xenografts without any treatment developed predominant acute vascular rejection within 6 days. Both passively released HMGB1 from xenografts and actively secreted HMGB1 from infiltrated immune cells were significantly increased after xenotransplantation. HMGB1-neutralizing antibody treatment significantly prolonged xenograft survival and attenuated pathologic damage, immune cell infiltration, and HMGB1 expression and release in the xenografts. Compared to control IgG treatment evaluated at study endpoint, treatment with HMGB1-neutralizing antibody markedly suppressed xenoreactive B cell responses, as evidenced by the significant inhibition of anti-rat antibody production and deposition in xenografts at Day 6 posttransplant. Furthermore, treatment with anti-HMGB1 antibody suppressed B cell activation and reduced IFN-γ and IL-17A production after xenotransplantation. These results demonstrate for the first time that HMGB1 plays an important role in mediating acute xenograft rejection. Thus, we have shown that neutralization of extracellular HMGB1 can significantly inhibit xenoreactive B cell responses and delay xenograft rejection in a rat-to-mouse model of xenotransplantation, uncovering new insights in the role of HMGB1 in transplantation.

Galectin-9 in Combination With EX-527 Prolongs the Survival of Cardiac Allografts in Mice After Cardiac Transplantation

Galectin-9 (Gal-9), a member of the galectin family, has a variety of biologic activities. However, its role in allografts is not fully clarified yet. The relationship between interleukin-17 (IL-17) and Gal-9 and the role of Gal-9 in TH17-cell differentiation also remain unclear. We built a murine cardiac transplantation model, which we treated with Gal-9 and/or EX-527, a specific Sirtuin-1 inhibitor. Afterwards, flow-cytometric analysis and reverse-transcription polymerase chain reaction were used to detect the number of TH17 cells and the expression of key factors involved in the differentiation of TH17 cells; in addition, the survival times of cardiac transplanted mice in different groups were recorded. The levels of circulating TH17 cells were found to increase in the peripheral blood of mice that exhibited acute rejection (AR) after heart transplantation, which was determined to be correlated with the rejection grade. Gal-9 or EX-527 can inhibit the activation and differentiation of TH17 cells and effectively suppress TH17-cell–mediated AR. These data provide new evidence for the potential regulatory effects of Gal-9 in alloimmune responses in a murine model of heart transplantation, and suggest the combined use of galectin-9 and EX-527 may be a powerful method to induce tolerance of fully mismatched murine cardiac allografts.

Treadmill exercise induces murine cardiac allograft survival and generates regulatory T cell.

Exercise therapy has been associated with improvement in functional capacity and quality of life. The role of exercise therapy in heart transplant recipients is of great interest for the transplant society, although concerning the effect of exercise therapy, there is little knowledge at present. We analyzed the effects of exercise on alloimmune responses in murine cardiac allograft transplantation. CBA mice (H2k) underwent transplantation of C57Bl/6 (H2b) hearts and exercised on a treadmill. Untreated CBA recipients rejected C57Bl/6 cardiac grafts acutely (median survival time [MST], 7 days). CBA recipients treated with treadmill for 1 week after transplantation, and for 1 week both before and after transplantation prolonged allograft survivals (MSTs, 35 and 18 days, respectively). However, treadmill exercise recipients for 1 week before transplantation were not effective to allograft survival (MST, 8 days). Adoptive transfer of whole splenocytes and CD4+ cells from treadmill exercise recipients significantly prolonged allograft survival in naive secondary recipients (MSTs, 30 and 52 days, respectively), suggesting that regulatory cells was generated after treadmill exercise. Moreover, flow cytometry studies showed that CD4+CD25+Foxp3+ cell population increased in treadmill exercise recipients. Therefore, postoperative but not pre-operative exercise could induce prolongation of survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory T cells.

The regulatory T cell effector molecule fibrinogen-like protein 2 is necessary for the development of rapamycin-induced tolerance to fully MHC-mismatched murine cardiac allografts.

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4(+) and CD8(+) immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-β and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

Ectopic expression of Fas Ligand on cardiomyocytes renders cardiac allografts resistant to CD4+ T-cell mediated rejection

Fas Ligand limits inflammatory injury and permits allograft survival by inducing apoptosis of Fas-bearing lymphocytes. Previous studies have shown that the CD4+ T-cell is both sufficient and required for murine cardiac allograft rejection. Here, utilizing a transgenic mouse that over-expresses Fas Ligand specifically on cardiomyocytes as heart donors, we sought to determine if Fas Ligand on graft parenchymal cells could resist CD4+ T-cell mediated rejection. When transplanted into fully immunocompetent BALB/c recipients Fas Ligand transgenic hearts were acutely rejected. However, when transplanted into CD4+ T-cell reconstituted BALB/c-rag−/− recipients, Fas Ligand hearts demonstrated long-term survival. These results indicate that Fas Ligand over-expression on cardiomyocytes can indeed resist CD4+ T-cell mediated cardiac rejection and suggests contact dependence between Fas Ligand expressing graft parenchymal cells and the effector CD4+ T-cells.

De-functioning polymorphism in the inhibitory receptor FcγRIIB does not impact upon kidney allograft survival.

De-functioning polymorphism in the inhibitory receptor FcγRIIB does not impact upon kidney allograft survival.