Donor‐Derived Exosomes With Lung Self‐Antigens in Human Lung Allograft Rejection

The immunological role of exosomes in allograft rejection remains unknown. We sought to determine whether exosomes are induced during lung allograft rejection and to define the antigenic compositions of HLA, lung‐associated self‐antigens (SAgs) and microRNAs (miRNAs). Exosomes were isolated from sera and bronchoalveolar lavage fluid from 30 lung transplant recipients (LTxRs) who were stable or who had acute rejection (AR) or bronchiolitis obliterans syndrome (BOS). Exosomes were defined by flow cytometry for CD63 and western blotting for annexin V SAgs, collagen V (Col‐V) and Kα1 tubulin were examined by electron microscopy; miRNAs were profiled by a miRNA array. Donor HLA and SAgs were detected on exosomes from LTxRs with AR and BOS but not from stable LTxRs. Exosomes expressing Col‐V were isolated from sera from LTxRs 3 mo before AR and 6 mo before BOS diagnosis, suggesting that exosomes with SAgs may be a noninvasive rejection biomarker. Exosomes isolated from LTxRs with AR or BOS also contained immunoregulatory miRNAs. We concluded that exosomes expressing donor HLA, SAgs and immunoregulatory miRNAs are present in the circulation and local site after human lung transplantation and play an important role in the immune pathogenesis of acute allograft rejection and BOS.     

Diagnosis of cardiac allograft rejection by the detection of circulating plasma cardiac myosin light chains

Twenty adult male Japanese monkeys of the species Macaca fuscata were randomly paired and subjected to heterotopic cardiac transplantation performed by the Ono-Lyndsey method. Without immunosuppression, graft survival ranged between 8 and 27 days, with a mean survival of 14 days. Plasma cardiac myosin light chains were measured by radio-immunoassay, which showed transient increases in myosin levels just following transplantation. Three hearts showed high values at this period and stopped beating when the myosin levels decreased (type 1). The other 7 hearts showed low myosin values after transient increases and 5 of them were rejected with a preceding reincrease in the myosin levels (type 2). Pathological study revealed myocardial necrosis, perivascular cuffing of mononuclear cells and/or neutrophils and/or plasma cells in the type 1 hearts. Measurement of the plasma myosin light chain level was therefore revealed to be of great value in the monitoring of cardiac allograft rejection.     

Diagnosis of cardiac allograft rejection by the detection of circulating plasma cardiac myosin light chains

Twenty adult male Japanese monkeys of the species Macaca fuscata were randomly paired and subjected to heterotopic cardiac transplantation performed by the Ono-Lyndsey method. Without immunosuppression, graft survival ranged between 8 and 27 days, with a mean survival of 14 days. Plasma cardiac myosin light chains were measured by radioimmunoassay, which showed transient increases in myosin levels just following transplantation. Three hearts showed high values at this period and stopped beating when the myosin levels decreased (type 1). The other 7 hearts showed low myosin values after transient increases and 5 of them were rejected with a preceding reincrease in the myosin levels (type 2). Pathological study revealed myocardial necrosis, perivascular cuffing of mononuclear cells and/or neutrophils and/or plasma cells in the type 1 hearts. Measurement of the plasma myosin light chain level was therefore revealed to be of great value in the monitoring of cardiac allograft rejection.     

Immunomodulation of kidney and heart transplants by anti-idiotypic antibodies.

To explore the possibility that circulating HLA antigens from the graft and anti-anti-HLA (anti-idiotypic) antibodies influence the long-term survival of renal and cardiac allografts, analysis of 330 renal allograft recipients and 174 recipients of cardiac allografts was conducted. Anti-donor-HLA antibodies (Ab1) present before or after transplantation are associated with graft failure, whereas irrelevant anti-HLA antibodies had no impact on actuarial graft survival. Ab1 may be uncovered by dissociation of immune complexes and depletion of soluble antigens with monoclonal antibody-coated magnetic beads. Of the 421 sera tested from 65 heart recipients, 97 showed Ab1 before depletion and 178 after depletion; similar rise in positive sera was seen in 39 renal transplant recipients. Three distinct patterns of appearance of Ab1 and Ab2 (anti-Ab1 antibody) were recognized. Patients with cyclic variations of Ab1 in association with Ab2 had 100% graft survival, whereas patients with cyclic variations of Ab1 but no detectable Ab2 had 2-year graft survival of 36% for kidneys and 71% for hearts. Presence of Ab1 in all sera after transplantation led to 47% and 56% 2-year renal and heart allograft survival, respectively.     

Pancreatic islets engineered with a FasL protein induce systemic tolerance at the induction phase that evolves into long‐term graft‐localized immune privilege

We have previously shown that pancreatic islets engineered to transiently display a modified form of FasL protein (SA‐FasL) on their surface survive indefinitely in allogeneic recipients without a need for chronic immunosuppression. Mechanisms that confer long‐term protection to allograft are yet to be elucidated. We herein demonstrated that immune protection evolves in two distinct phases; induction and maintenance. SA‐FasL‐engineered allogeneic islets survived indefinitely and conferred protection to a second set of donor‐matched, but not third‐party, unmanipulated islet grafts simultaneously transplanted under the contralateral kidney capsule. Protection at the induction phase involved a reduction in the frequency of proliferating alloreactive T cells in the graft‐draining lymph nodes, and required phagocytes and TGF‐β. At the maintenance phase, immune protection evolved into graft site‐restricted immune privilege as the destruction of long‐surviving SA‐FasL‐islet grafts by streptozotocin followed by the transplantation of a second set of unmanipulated islet grafts into the same site from the donor, but not third party, resulted in indefinite survival. The induced immune privilege required both CD4⁺CD25⁺Foxp3⁺ Treg cells and persistent presence of donor antigens. Engineering cell and tissue surfaces with SA‐FasL protein provides a practical, efficient, and safe means of localized immunomodulation with important implications for autoimmunity and transplantation.     

Discovery of non‐HLA antibodies associated with cardiac allograft rejection and development and validation of a non‐HLA antigen multiplex panel: From bench to bedside

We analyzed humoral immune responses to nonhuman leukocyte antigen (HLA) after cardiac transplantation to identify antibodies associated with allograft rejection. Protein microarray identified 366 non‐HLA antibodies (>1.5 fold, P < .5) from a discovery cohort of HLA antibody–negative, endothelial cell crossmatch–positive sera obtained from 12 cardiac allograft recipients at the time of biopsy‐proven rejection. From these, 19 plasma membrane proteins and 10 autoantigens identified from gene ontology analysis were combined with 48 proteins identified through literature search to generate a multiplex bead array. Longitudinal sera from a multicenter cohort of adult cardiac allograft recipients (samples: n = 477 no rejection; n = 69 rejection) identified 18 non‐HLA antibodies associated with rejection (P < .1) including 4 newly identified non‐HLA antigenic targets (DEXI, EMCN, LPHN1, and SSB). CART analysis showed 5/18 non‐HLA antibodies distinguished rejection vs nonrejection. Antibodies to 4/18 non‐HLA antigens synergize with HLA donor‐specific antibodies and significantly increase the odds of rejection (P < .1). The non‐HLA panel was validated using an independent adult cardiac transplant cohort (n = 21 no rejection; n = 42 rejection, >1R) with an area under the curve of 0.87 (P < .05) with 92.86% sensitivity and 66.67% specificity. We conclude that multiplex bead array assessment of non‐HLA antibodies identifies cardiac transplant recipients at risk of rejection.     

Allelic and Epitopic Characterization of Intra–Kidney Allograft Anti‐HLA Antibodies at Allograft Nephrectomy

The reasons for the increased incidence of de novo anti–human leukocyte antibody (HLA) donor‐specific antibodies (DSAs) observed after kidney allograft nephrectomy are not fully understood. One advocated mechanism suggests that at graft loss, DSAs are not detected in the serum because they are fixed on the nonfunctional transplant; removal of the kidney allows DSAs to then appear in the blood circulation. The aim of our study was to compare anti‐HLA antibodies present in the serum and in the graft at the time of an allograft nephrectomy. Using solid‐phase assays, anti‐HLA antibodies were searched for in the sera of 17 kidney transplant patients undergoing allograft nephrectomy. No anti‐HLA antibodies were detected in the graft if they were not also detected in the serum. Eleven of the 12 patients who had DSAs detected in their sera also had DSAs detected in the grafts. Epitopic analysis revealed that most anti‐HLA antibodies detected in removed grafts were directed against the donor. In summary, our data show that all anti‐HLA antibodies that were detected in grafts were also detected in the sera. These intragraft anti‐HLA antibodies are mostly directed against the donor at an epitopic level but not always at an antigenic level.     

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.     

Supplementing preservation solution with mitochondria‐targeted H2S donor AP39 protects cardiac grafts from prolonged cold ischemia–reperfusion injury in heart transplantation

Heart transplant has been accepted as the standard treatment for end‐stage heart failure. Because of its susceptibility to ischemia–reperfusion injury, the heart can be preserved for only 4 to 6 hours in cold static preservation solutions. Prolonged ischemia time is adversely associated with primary graft function and long‐term survival. New strategies to preserve donor hearts are urgently needed. We demonstrate that AP39, a mitochondria‐targeting hydrogen sulfide donor, significantly increases cardiomyocyte viability and reduces cell apoptosis/death after cold hypoxia/reoxygenation in vitro. It also decreases gene expression of proinflammatory cytokines and preserves mitochondria function. Using an in vivo murine heart transplant model, we show that preserving donor hearts with AP39‐supplemented University of Wisconsin solution (n = 7) significantly protects heart graft function, measured by quantitative ultrasound scan, against prolonged cold ischemia–reperfusion injury (24 hours at 4°C), along with reducing tissue injury and fibrosis. Our study demonstrates that supplementing preservation solution with AP39 protects cardiac grafts from prolonged ischemia, highlighting its therapeutic potential in preventing ischemia–reperfusion injury in heart transplant.     

Modified CD4(+) T-cell response in recipients of old cardiac allografts

With an increasing demand, organs from elderly donors are more frequently utilized for transplantation. Herein, we analyzed the impact of donor age on CD4(+) T-cell responses with regard to regulatory and effector mechanisms. Young (3months) BM12 recipients were engrafted with young or old (18months) B6 cardiac allografts. Systemic CD4(+) T-cell responses and intragraft changes were monitored and compared to age-matched syngenic transplant controls. While elderly, nonmanipulated hearts contained significantly elevated frequencies of donor-derived leukocytes prior to transplantation, allograft survival was age-independent. T-cell activation, however, was delayed and associated with a compromised immune response in mixed lymphocyte cultures (MLR; P=0.0002) early after transplantation (day 14). During the time course after transplantation, recipients of old grafts demonstrated an augmented immune response as shown by significantly higher frequencies of activated CD4(+) T-cells and a stronger in vitro alloreactivity (MLR; ELISPOT; P<0.01). In parallel, frequencies of regulatory T-cells had increased systemically and overall fewer CD4(+) T-cells were detected intragraft. Interestingly, changes in the CD4(+) T-cell response were not reflected by graft morphology. Of note, transplantation of young and old syngenic hearts did not show age-related differences of the CD4(+) T-cells response suggesting that old grafts can recover from a period of short cold ischemia time. Our data suggest that donor age is associated with an augmented CD4(+) T-cells response which did not affect graft survival in our model. These findings contribute to a better understanding of the immune response following the engraftment of older donor organs.     

Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV‐Naïve Recipients of Infected Donor Allograft Hearts

Cytomegalovirus accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in solid organ transplants; however, the mechanisms involved are unclear. We determined the efficacy of a CMV vaccine in preventing CMV‐accelerated rat cardiac allograft rejection in naïve recipients of CMV+ donor hearts. F344 donor rats were infected with RCMV 5 days prior to heterotopic cardiac transplantation into CMV‐naïve or H₂O₂‐inactivated RCMV‐vaccinated Lewis recipients. Recipients of RCMV‐infected donor hearts rejected at POD59, whereas vaccinated recipients exhibited a significantly prolonged time to rejection‐POD97, similar to recipients of uninfected donor hearts (POD108). Although all of the donor hearts were preinfected, the vaccinated recipients had lower graft and PBMC viral loads at POD 7 compared to unvaccinated controls. Adoptive T cell and passive antibody transfers from vaccinated Lewis rats into naïve recipients demonstrate that both T‐cell and B‐cell arms of the adaptive immune response provide protection against CMV‐accelerated rejection. Similar findings were obtained when testing three different adjuvants in passive transfer experiments. We have determined that the timing of the vaccine prior to transplantation and the specific adjuvant play critical roles in mediating anti‐viral responses and promoting graft survival. CMV vaccination prior to transplantation may effectively increase graft survival.     

T-cell response to cardiac myosin persists in the absence of an alloimmune response in recipients with chronic cardiac allograft rejection

BACKGROUND: Immune-mediated injury to the graft has been implicated in the pathogenesis of chronic rejection. However, little is known regarding the nature of the antigen(s) involved in this immune process. We demonstrated that cardiac transplantation in mice induces an autoimmune T-cell response to a heart tissue-specific protein, cardiac myosin (CM). This response contributes to transplant rejection in that its modulation affects cardiac graft survival. This study investigates whether anti-CM T cells undergo activation and expansion in mice with chronic cardiac allograft rejection. METHODS: The frequency of CM- and donor major histocompatibility complex (MHC)-specific interferon (IFN)-gamma-producing T cells were assessed by ELISPOT in BALB/c mice, which were injected with anti-CD40L (MR1) mAb (chronic rejection group) or CTLA4Ig fusion protein (tolerant group) and transplanted with C57BL/6 cardiac allografts. RESULTS AND CONCLUSIONS: MR1-treated BALB/c recipients of C57BL/6 hearts with chronic rejection displayed a high frequency of activated CM-specific T cells, whereas the frequency of activated alloreactive T cells were similar to na?ve, nontransplanted mice. In contrast, no activation of CM-reactive T cells was detected in tolerant recipients after CTLA4Ig treatment. Therefore, in the absence of alloimmunity, chronic rejection is associated with persistence of a T-cell response against CM. Our data indicate that anti-CM autoimmunity may be involved in the immune mechanisms of chronic rejection and suggest that tolerance strategies should target both allo- and autoimmune responses to prevent this process.     

Antibodies directed against antigens on the endothelium of peritubular capillaries in patients with rejecting renal allografts.

This study was undertaken to examine the humoral immune response against endothelial antigens of the donor kidney in human renal allograft recipients. Sera from 61 transplant recipients who received 62 grafts were studied for the presence of circulating endothelial antibodies (CEAb) using an indirect immunofluorescence technique with a pretransplant biopsy of the graft as a substrate. IgG antibodies directed against the endothelium of peritubular capillaries were found in the sera of 6 of the 10 patients with graft rejection within 7 weeks after transplantation, whereas these antibodies were not found in the absence of rejection (P less than 0.001). Immunofluorescence studies of post-transplant biopsies showed IgG along the endothelium of peritubular capillaries only in the grafts of patients with CEAb. Eluates from these grafts contained IgG antibodies that bound to the endothelium of the donor as shown by the indirect immunofluorescence technique. Absorption of endothelial antibody (EAb)-positive sera with human platelets or Wistar strain rat erythrocytes showed that the EAb were not directed against serologically defined HLA antigens or against heterophile antigens on rat erythrocytes. We conclude from this study that the presence of antibodies directed against endothelial antigens is associated with poor graft prognosis and that these antibodies may be responsible for the rejection process.     

Profiling non‐HLA antibody responses in antibody‐mediated rejection following heart transplantation

Antibody‐mediated rejection (AMR) driven by the development of donor‐specific antibodies (DSA) directed against mismatched donor human leukocyte antigen (HLA) is a major risk factor for graft loss in cardiac transplantation. Recently, the relevance of non‐HLA antibodies has become more prominent as AMR can be diagnosed in the absence of circulating DSA. Here, we assessed a single‐center cohort of 64 orthotopic heart transplant recipients transplanted between 1994 and 2014. Serum collected from patients with ≥ pAMR1 (n = 43) and non‐AMR (n = 21) were tested for reactivity against a panel of 44 non‐HLA autoantigens. The AMR group had a significantly greater percentage of patients with elevated reactivity to autoantigens compared to non‐AMR (P = .002) and healthy controls (n = 94, P < .0001). DSA‐positive AMR patients exhibited greater reactivity to autoantigens compared to DSA‐negative (P < .0001) and AMR patients with DSA and PRA > 10% were identified as the subgroup with significantly elevated responses. Reactivity to 4 antigens, vimentin, beta‐tubulin, lamin A/C, and apolipoprotein L2, was significantly different between AMR and non‐AMR patients. Moreover, increased reactivity to these antigens was associated with graft failure. These results suggest that antibodies to non‐HLA are associated with DSA‐positive AMR although their specific role in mediating allograft injury is not yet understood.     

Long-term acceptance of fully allogeneic cardiac grafts by cotransplantation of vascularized thymus in miniature swine

BACKGROUND: We have previously reported the ability of both thymokidney and vascularized thymic lobe (VTL) allografts to induce transplantation tolerance to renal allografts across a full major histocompatibility complex (MHC) mismatch in thymectomized miniature swine. However, whether vascularized thymus is capable of inducing tolerance to less tolerogeneic organs when it is transplanted simultaneously is not yet known. The present study investigates cardiac allograft survival and the mechanism of long-term acceptance in recipient swine following cotransplantation of VTL and cardiac grafts from fully MHC-mismatched donors. METHODS: Animals received a heart graft, a heart graft and a VTL, or a heart graft and a donor thymocyte infusion. Immunosuppressive regimens consisted of 12 or 28 days of tacrolimus. RESULTS: All animals that received a VTL maintained their grafts significantly longer than their counterparts that received only a heart graft, and those receiving 28 days of tacrolimus maintained their heart grafts long-term. Recipients of a donor thymocyte infusion demonstrated slightly prolonged cardiac graft survival but all rejected their grafts, highlighting the importance of thymic stroma. Cytotoxic T-lymphocyte responses against third-party antigens by cells from tolerant animals showed restriction by both self and donor MHC, whereas responses of controls were restricted to self MHC only. The presence of donor dendritic cells in the VTL grafts and results of co-culture assays suggest that both central and regulatory mechanisms were involved in achieving long-term acceptance. CONCLUSION: This is the first demonstration of the long-term acceptance of fully MHC-mismatched cardiac allografts in large animals.     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Donor kidney injury molecule‐1 promotes graft recovery by regulating systemic necroinflammation

Ischemia‐reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage‐associated molecular patterns (eg, high‐mobility group box‐1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule‐1 (KIM‐1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM‐1 expression in KIM‐1^+/+ kidney grafts posttransplantation. Compared to recipients of KIM‐1^+/+ kidneys, recipients of KIM‐1^?/? kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM‐1^?/? grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM‐1^+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM‐1^?/? grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.     

Mitochondrial permeability regulates cardiac endothelial cell necroptosis and cardiac allograft rejection

Transplantation is invariably associated with programmed cell death including apoptosis and necrosis, resulting in delayed graft function and organ rejection. We have demonstrated the contribution of necroptosis to mouse microvascular endothelial cell (MVEC) death and transplant rejection. Organ injury results in the opening of mitochondrial permeability transition pores (mPTPs), which can trigger apoptotic molecules release that ultimately results in cell death. The effect of mPTPs in the necroptotic pathway remains controversial; importantly, their role in transplant rejection is not clear. In this study, tumor necrosis factor‐α triggered MVECs to undergo receptor‐interacting protein kinase family (RIPK1/3)‐dependent necroptosis. Interestingly, inhibition of mPTP opening could also inhibit necroptotic cell death. Cyclophilin‐D (Cyp‐D) is a key regulator of the mPTPs. Both inhibition and deficiency of Cyp‐D protected MVECs from necroptosis (n = 3, P < .00001). Additionally, inhibition of Cyp‐D attenuated RIPK3‐downstream mixed‐lineage kinase domain–like protein phosphorylation. In vivo, Cyp‐D–deficient cardiac grafts showed prolonged survival in allogeneic BALB/c mice posttransplant compared with wild‐type grafts (n = 7, P < .0001). Our study results suggest that the mPTPs may be important mechanistic mediators of necroptosis in cardiac grafts. There is therapeutic potential in targeting cell death via inhibition of the mPTP‐regulating molecule Cyp‐D to prevent cardiac graft rejection.     

Myeloid‐derived suppressor cells expand after transplantation and their augmentation increases graft survival

Myeloid‐derived suppressor cells (MDSCs) expand in an inflammatory microenvironment such as cancer and autoimmunity. To study if transplantation induces MDSCs and these cells regulate allograft survival, C57BL/6 donor hearts were transplanted into BALB/c recipients and endogenous MDSCs were characterized. The effects of adoptive transfer of transplant (tx), tumor (tm), and granulocyte‐colony stimulating factor (g‐csf)–expanded MDSCs or depletion of MDSC were assessed. MDSCs expanded after transplantation (1.7‐4.6‐fold) in the absence of immunosuppression, homed to allografts, and suppressed proliferation of CD4 T cells in vitro. Tx‐MDSCs differed phenotypically from tm‐MDSCs and g‐csf‐MDSCs. Among various surface markers, Rae‐1 expression was notably low and TGF‐β receptor II was high in tx‐MDSCs when compared to tm‐MDSCs and g‐csf‐MDSCs. Adoptive transfer of these three MDSCs led to differential graft survival: control (6 days), tx‐MDSCs (7.5 days), tm‐MDSCs (9.5 days), and g‐csf‐MDSCs (19.5 days). In combination with anti‐CD154 mAb, MDSCs synergistically extended graft survival from 40 days (anti‐CD154 alone) to 86 days with tm‐MDSCs and 132 days with g‐csf‐MDSCs. Early MDSC depletion (day 0 or 20), however, abrogated graft survival, but late depletion (day 25) did not. In conclusion, MDSCs expanded following transplantation, migrated to cardiac allografts, prolonged graft survival, and were synergistic with anti‐CD154 mAb.     

3-Deazaadenosine prevents leukocyte invasion by suppression of adhesion molecule expression during acute cardiac allograft rejection: involvement of apoptotic cell death.

BACKGROUND: In the initial phase after cardiac transplantation, mononuclear cells infiltrate the graft, initiating a relevant impulse for rejection. 3-Deazaadenosine (c3Ado), an analog of adenosine, has proven anti-inflammatory properties both in vitro and in vivo. We hypothesized that c3Ado can serve as a therapeutic tool to reduce cellular infiltration in cardiac allograft transplantation. METHODS: Using the Wistar-Furth-to-Lewis rat cardiac allograft model, animals were treated with 5 mg c3Ado subcutaneously twice per day. Allografts of untreated animals served as controls. Grafts were harvested on Days 1, 3 and 6 after transplantation for further examination (n = 4 per group and timepoint). RESULTS: Immunohistochemical examination of c3Ado-treated grafts revealed up to 80% reduction of infiltrating major histocompatability complex (MHC) II-positive cells and T-cell-receptor-positive cells (R73) as well as ED1-positive monocytes and macrophages at Days 3 and 6 after transplantation. Adhesion molecule (ICAM-1 and VCAM-1) expression at Days 1 and 3 was almost completely abolished in c3Ado-treated grafts. However, c3Ado treatment did not prevent apoptotic cell death (TUNEL assay, DNA laddering) at Day 6, nor did it prolong allograft survival. As in controls, grafts were rejected at Day 7. CONCLUSION: c3Ado significantly reduces graft infiltration by preventing leukocyte invasion, most likely through suppression of adhesion molecule expression. Although graft survival was not prolonged, treatment with c3Ado may still serve as a strategy to protect hearts from early damage after transplantation. Further studies will show whether peri-operative use of c3Ado can bridge the critical phase after transplantation when standard immunosuppression is not yet completely efficacious.