Murine Heart Transplantation Research Articles

Tolerogenic Donor-Derived Dendritic Cells Risk Sensitization In Vivo owing to Processing and Presentation by Recipient APCs

Modification of allogeneic dendritic cells (DCs) through drug treatment results in DCs with in vitro hallmarks of tolerogenicity. Despite these observations, using murine MHC-mismatched skin and heart transplant models, donor-derived drug-modified DCs not only failed to induce tolerance but also accelerated graft rejection. The latter was inhibited by injecting the recipient with anti-CD8 Ab, which removed both CD8(+) T cells and CD8(+) DCs. The discrepancy between in vitro and in vivo data could be explained, partly, by the presentation of drug-modified donor DC MHC alloantigens by recipient APCs and activation of recipient T cells with indirect allospecificity, leading to the induction of alloantibodies. Furthermore, allogeneic MHC molecules expressed by drug-treated DCs were rapidly processed and presented in peptide form by recipient APCs in vivo within hours of DC injection. Using TCR-transgenic T cells, Ag presentation of injected OVA-pulsed DCs was detectable for ≤ 3 d, whereas indirect presentation of MHC alloantigen by recipient APCs led to activation of T cells within 14 h and was partially inhibited by reducing the numbers of CD8(+) DCs in vivo. In support of this observation when mice lacking CD8(+) DCs were pretreated with drug-modified DCs prior to transplantation, skin graft rejection kinetics were similar to those in non-DC-treated controls. Of interest, when the same mice were treated with anti-CD40L blockade plus drug-modified DCs, skin graft survival was prolonged, suggesting endogenous DCs were responsible for T cell priming. Altogether, these findings highlight the risks and limitations of negative vaccination using alloantigen-bearing "tolerogenic" DCs.

CD4+CD25−Nrp1+ T Cells Synergize with Rapamycin to Prevent Murine Cardiac Allorejection in Immunocompetent Recipients

Besides CD4+CD25+Foxp3+ regulatory T cells (Tregs), other immunosuppressive T cells also participated in the regulation of immune tolerance. Reportedly, neuropilin-1 (Nrp1) might be one of the molecules by which regulatory cells exert their suppressive effects. Indeed, CD4+CD25−Nrp1+ T cells exhibit potent suppressive function in autoimmune inflammatory responses. Here we investigated the specific role of CD4+CD25−Nrp1+ T cells in the setting of the transplant immune response. Through MLR assays, we found that CD4+CD25−Nrp1+ T cells suppressed the proliferation of naive CD4+CD25− T cells activated by allogeneic antigen-stimulation. Adoptive transfer of CD4+CD25−Nrp1+ T cells synergized with rapamycin to induce long-term graft survival in fully MHC-mismatched murine heart transplantation, which was associated with decreased IFN-γ, IL-17 and increased IL-10, TGF-β, Foxp3 and Nrp1 expression in the grafts. Importantly, our data indicated that CD4+CD25−Nrp1+ T cell transfer augments the accumulation of Tregs in the recipient, and creates conditions that favored induction of hyporesponsiveness of the T effector cells. In conclusion, this translational study indicates the possible therapeutic potential of CD4+CD25−Nrp1+ T cells in preventing allorejection. CD4+Nrp1+ T cells might therefore be used in bulk as a population of immunosuppressive cells with more beneficial properties concerning ex vivo isolation as compared to Foxp3+ Tregs.

A Novel Pathway for CD8+ T Cell Activation by Donor Derived Non-Haematopoietic Cells Leading to Acute Allograft Rejection

Introduction: Although seminal studies have shown that CD8+ T cells can reject cardiac allografts that lack donor haematopoietic cells (HPCs) through a direct cognate interaction with graft parenchymal cells, the observation from other studies that secondary lymphoid tissue (SLT) is essential for graft rejection creates a paradox yet to be resolved. Here we examine the hypothesis that CD8+ T cell activation by graft parenchymal cells requires acquisition and re-presentation of intact MHC class I alloantigen by recipient antigen presenting cells (APCs) within SLT. If proven, this hypothesis would not only resolve this paradox, but would also represent the first definitive demonstration of the contribution of the semi-direct pathway to graft rejection. Methods: CD8+ T cell-mediated allograft rejection following activation by donor parenchymal cells was examined using a transgenic heterotopic murine heart transplant model. Balb/c cardiac donors were lethally irradiated and treated with depleting antibodies ensuring complete eradication of HPCs, such that parenchymal cells in these grafts were the only source of alloantigen presentation. These HPC-depleted Balb/c (Balb/cHPC-) cardiac allografts were transplanted into: 1) 2C transgenic mice (monoclonal population of CD8+ T cells against Ld MHC class I); 2) Splenectomised aly/aly (aly/alyspl) mice (complete absence of SLT) with adoptive transfer of 2C CD8+ T cells, to investigate the importance of SLT in this pathway; 3) CD11c-DTR transgenic mice, in which host dendritic cells (DCs) are selectively depleted by diphtheria toxin, allowing delineation of the role of these cells in parenchymal alloantigen presentation to CD8+ T cells. Results: 2C transgenic mice rejected Balb/cHPC- grafts as rapidly as non-depleted Balb/c grafts (MST = 5d vs. 4d respectively; p=0.4) suggesting an effective mechanism for parenchymal cell driven CD8+ T cell mediated rejection. Balb/cHPC- allografts showed prolonged survival (>50d) in aly/alyspl mice given 2C CD8+ T cells, whereas in non-splenectomised controls all grafts rejected (MST = 17d; p=0.01) suggesting an essential role for SLT in this pathway. In addition, when aly/alyspl mice were given activated 2C CD8+ T cells (from a 2C recipient of a Balb/c cardiac graft) they rapidly rejected Balb/cHPC- allografts (MST = 7d; p=0.01) indicating that SLT plays a fundamental role in the activation of CD8+ T cells. To examine, therefore, whether recipient DCs were required to present to CD8+ T cells within SLT, CD4+-depleted CD11c-DTR mice given 2C CD8+ T cells were transplanted with Balb/cHPC- allografts. When these mice were also treated with diphtheria toxin to deplete host DCs they demonstrated significantly delayed rejection kinetics when compared with untreated controls (MST = 26d vs. 16d; p=0.02) highlighting a key role for these cells. Conclusion: These results indicate that allorecognition of graft parenchymal cells represents an important mechanism of CD8+ T cell mediated rejection. They support the role of a novel pathway in which recipient DCs acquire intact MHC class I from donor parenchymal cells and present to CD8+ T cells in SLT leading to their activation.

Music Exposure Induced Prolongation of Cardiac Allograft Survival and Generated Regulatory CD4+ Cells in Mice

In clinical practice, music has been used to decrease stress, heart rate, and blood pressure and to provide a distraction from disease symptoms. We investigated sound effects on alloimmune responses in murine heart transplantation. Naïve and eardrum-ruptured CBA/N (CBA, H2K) underwent transplantation of a C57BL/6 (B6, H2b) heart and were exposed to 1 of 3 types of music-opera (La Traviata), classical (Mozart), and New Age (Enya)-or 1 of 6 different single sound frequencies for 7 days. An adoptive transfer study was performed to determine whether regulatory cells were generated in allograft recipients. Cell-proliferation, cytokine, and flow cytometry assessments were also performed. CBA recipients of a B6 graft exposed to opera and classical music had significantly prolonged allograft survival (median survival times [MSTs], 26.5 and 20 days, respectively), whereas those exposed to 6 single sound frequencies and New Age did not (MSTs, 7, 8, 9, 8, 8, 8, and 11 days, respectively). Untreated and eardrum-ruptured CBA rejected B6 grafts acutely (MSTs, 7 and 8.5 days, respectively). Adoptive transfer of whole splenocytes, CD4+ cells, and CD4+CD25+ cells from opera-exposed primary recipients resulted in significantly prolonged allograft survival in naive secondary recipients (MSTs, 36, 68, and >50 days, respectively). Cell-proliferation, interleukin (IL)-2 and interferon-γ were suppressed in opera-exposed mice, whereas IL-4 and IL-10 from opera-exposed recipients were up-regulated. Flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population in splenocytes from opera-exposed mice. In conclusion, exposure to some types of music may induce prolonged survival of fully allogeneic cardiac allografts and generate CD4+CD25+Foxp3+ regulatory cells.

Induction of Regulatory CD4+ Cells and Prolongation of Survival of Fully Allogeneic Murine Cardiac Grafts by Danazol

Danazol, a modified testosterone, has been used to treat endometriosis and pretreatment before in vitro fertilization and embryo transfer, although its reproductive mechanisms remain unclear. We investigated the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2k) that underwent transplantation of C57BL/6 (B6, H2b) hearts received danazol (0.4 and 4 mg/kg/d) by intraperitoneal injection from the day of transplantation to days 6. We performed an adoptive transfer study to determine regulatory cells as well as cell proliferation, cytokine, and flow cytometry assessments. Danazol-treated (4 mg/kg/d) CBA mice showed prolonged allograft survival (median survival time [MST], 63 days). Moreover, secondary CBA recipients of whole splenocytes and CD4+ cells from primary danazol-treated (4 mg/kg/d) CBA recipients at 30 days after transplantation displayed prolonged allograft survival (MSTs, 29 and 60 days, respectively). Cell proliferation, interleukin (IL)-2, and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed alloproliferation in mixed leukocyte cultures. Flow cytometry studies showed an increased CD4+CD25+Foxp3+ cell population among splenocytes from danazol-treated mice. In conclusion, danazol induced prolonged cardiac allograft survival and generation of regulatory CD4+ cells.

Introduction of Modified Cervical Cardiac Transplant Model in Mice

The murine cervical heterotopic heart transplant model was initially designed to test the immune response to third-party allografts, modified by cuff techniques. While cuff techniques simplify the execution of this procedure, cutting of the carotid artery and the external jugular vein alters the blood supply to central nervous system and makes it difficult to achieve long-term graft survival. In the present study, we describe modified techniques that preserve the continuity and function of blood vessels and improve transplant integrity. The modified techniques in this study comprise the following aspects: (1) Preservation of the sternal head of the right sternocleidomastoid muscle, (2) use of the donor's intrathoracic inferior vena cava for anastomosis and (3) preservation of the function of the recipient's carotid artery and external jugular vein and thus, continuity of blood flow to the central nervous system. Stable, long-term, disease-free allograft survival has been achieved with syngeneic transplants (> 200 days), whereas allografts from fully major histocompatibility complex-mismatched donors were acutely rejected in a time similar to the traditional abdominal heterotopic heart transplant model (8.2 ± 1.3 vs 8.4 ± 1.4 days; P = .73 in the Mantel Cox test, and P = .61 in the Gehan-Breslow-Wilcoxon test). Similar alloresponses could be induced in these 2 models. It is possible and feasible to achieve long-term graft survival in the mouse cervical heart transplant model using the modified procedures described in the present study.

Reduction of chronic graft injury with a new HTK-based preservation solution in a murine heart transplantation model

ObjectiveAim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution. MethodsHearts of C57BL/6J (H-2b) mice were stored for 15h in 0–4°C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60days of observation (24h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis. ResultsThe re-beating time was significantly shorter in hearts stored in the new solution (10.3±2.6min vs. HTK 14.2±4.1min; p<0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3±0.4 vs. HTK 1.6±0.5; p<0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8±0.2 vs. HTK 2.2±0.7), less interstitial fibrosis (4.8±1.9% vs. HTK 8.5±3.8%, p<0.05), less vasculopathy (14.7±6.9% vs. 22.0±23.2%; p=0.06) and lower TGF-β1-expression (6.6±1.4% vs. HTK 12.0±4.6%). ConclusionThe new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-l-histidine, glycine, alanine, arginine and sucrose.

Mechanism of Accommodation in a Sensitized Human Leukocyte Antigen Transgenic Murine Cardiac Transplant Model

Presence of donor-specific antibodies (Abs) is detrimental to posttransplant allograft function. Some sensitized recipients have successfully undergone transplantation after pretransplant conditioning regimen using plasmapheresis and/or intravenous immunoglobulin therapy, but underlying mechanisms that confer such allograft protection are undefined. We developed a single human leukocyte antigen (HLA)-mismatched heterotopic murine heart transplant model (HLA-A2 into HLA-A2-sensitized-C57BL/6) to determine whether pretreatment of donors with low concentration of HLA class I (W6/32) or control Ab (C1.18.4) will confer protection. Expression levels of survival genes, Bcl-2 and heme oxygenase-1, were analyzed by gene array analysis and quantitative real-time polymerase chain reaction. Expression levels of cytokine panel were analyzed by Luminex. Role of Bcl-2 in the induction of allograft protection was analyzed by silencing the Bcl-2 expression in the donor hearts using a small hairpin (shRNA) specific for Bcl-2. Control Ab-pretreated hearts were rejected in less than 5 days demonstrating hemorrhage, Ab, and C4 deposition. In contrast, W6/32-pretreated hearts were rejected at 15 days (P<0.05) that was prolonged to 25 days with antilymphocyte serum treatment. W6/32-pretreated hearts on day 5 exhibited increased expression of Bcl-2 (5.5-folds), Bcl-xl (5.5-folds), and heme oxygenase-1 (4.4-folds); decreased expression of ICAM-1, VCAM-1 (3.2-fold), along with reduced levels of cytokines interleukin (IL)-1β (4.4-folds), tumor necrosis factor α (3.7-folds), IL-6 (7.5-folds), IL-12 (2.3-folds) and chemokines monocyte chemotactic protein 1 (4.5-folds), MIG (4.4-folds), MIP-1α (3.4-folds), and IL-8 (3.1-folds). Silencing of Bcl-2 in accommodated hearts before transplant resulted in loss of protection with rejection (9±3 vs. 15±2days, P<0.05). Pretreatment of hearts with low levels of anti-HLA Abs increases expression of antiapoptotic genes that inhibits caspases, leading to decreased inflammatory cytokines and chemokines, which promote allograft survival.

Danazol induces prolonged survival of fully allogeneic cardiac grafts and maintains the generation of regulatory CD4+ cells in mice

Danazol, a derivative of testosterone, is useful for treatment of endometriosis as well as pretreatment for in vitro fertilization and embryo transfer, although its mechanisms of action are unclear. The aim of this study was to investigate the effect of danazol on alloimmune responses in murine heart transplantation. CBA male mice (H2(k) ) underwent transplantation of C57BL/6 male (H2(b) ) hearts and received a single dose of danazol (0.4, 1.2 or 4mg/kg/day) by intraperitoneal injection on the day of transplantation and for 6days thereafter. An adoptive transfer study was performed to determine whether regulatory cells were generated. The median survival time (MST) of allografts in danazol-treated (1.2 and 4mg/kg/day) mice was 28 and 63days, respectively, compared with 7days in untreated mice. Moreover, secondary CBA recipients given whole splenocytes or CD4(+) cells from primary danazol-treated (4mg/kg/day) CBA recipients 30days after transplantation had prolonged allograft survival (MSTs, 29 and 60days, respectively). Cell proliferation, interleukin (IL)-2 and interferon-γ were suppressed in danazol-treated mice, whereas IL-4 and IL-10 were up-regulated. Moreover, danazol directly suppressed allo-proliferation in a mixed leukocyte culture. Flow cytometry showed an increased CD4(+) CD25(+) Foxp3(+) cell population in splenocytes from danazol-treated mice. Danazol prolongs cardiac allograft survival and generates regulatory CD4(+) cells.

Tolerogenic dendritic cells generated by RelB silencing using shRNA prevent acute rejection

It is well known that adoptive transfer of donor-derived tolerogenic dendritic cells (DCs) helps to induce immune tolerance. RelB, one of NF-κB subunits, is a critical element involved in DC maturation. In the present study, our results showed tolerogenic DCs could be acquired via silencing RelB using small interfering RNA. Compared with imDCs, the tolerogenic DCs had more potent ability to inhibit mixed lymphocyte reaction (MLR) and down-regulate Th1 cytokines and prompt the production of Th2 cytokines. They both mediated immune tolerance via the increased of T cell apoptosis and generation of regulatory T cells. Administration of donor-derived tolerogenic DCs significantly prevented the allograft rejection and prolonged the survival time in a murine heart transplantation model. Our results demonstrate donor-derived, RelB-shRNA induced tolerogenic DCs can significantly induce immune tolerance in vitro and in vivo.

Mizoribine—An inosine monophosphate dehydrogenase inhibitor—acts synergistically with cyclosporine A in prolonging survival of murine islet cell and heart transplants across major histocompatibility barrier

IntroductionMizoribine (MZR) is an inosine monophosphate dehydrogenase inhibitor. It has been widely used in Japan in the treatment of autoimmune diseases and is known to inhibit T and B cell proliferation. The aim of this study was to evaluate the efficacy of MZR as an immunosuppressive agent and determine its ability to synergize with a commonly used calcineurin inhibitor Cyclosporine A (CsA) in prolonging survival of murine islet cells and heart transplanted across major histocompatibility barrier. MethodsMurine allogeneic islet cell transplantation between Balb/c donor mice and C57BL/6 recipient mice and heterotopic heart transplantation was done between C3H/He donor mice and Balb/c recipient mice. Recipients were divided into groups based on immunosuppression: Group 1—No immunosuppression, Group 2—MZR alone (20mg/kg/day), Group 3—CsA alone (20mg/kg/day), Group 4—MZR+CsA (20mg/kg/day). Donor specific IFN-γ, IL-10, IL-2, IL-4 secreting cells were enumerated by ELISpot. Serum cytokine and chemokine concentration was measured by Luminex. ResultsIslet cell allograft recipients treated with CsA and MZR had prolonged islet function compared to other groups [normoglycemia (blood glucose <200mg/dL) up to 32±4days, p<0.05]. Similarly, heart allograft survival was significantly improved in mice treated with CsA and MZR compared to other groups (50% 30-day survival, p=0.04). Donor specific IFN-γ, IL-4, IL-2 secreting cells were significantly decreased in recipients treated with CsA and MZR with marked increase in IL-10 secreting cells (p<0.05). There was also an increase in serum IL-10 with decrease in IFN-γ, IL-4, IL-2, MCP-1, and IL-6 in mice treated with CsA and MZR ConclusionMZR and CsA when used in combination are potent immunosuppressive agents in murine islet cell and heart transplantation models. These agents lead to a decrease in donor specific IFN-γ with increase in IL-10 secreting cells leading to improved allograft survival and function.

Features of synergism between mesenchymal stem cells and immunosuppressive drugs in a murine heart transplantation model

BackgroundMesenchymal stem cells (MSCs) can be used for immunomodulation therapy after solid organ transplantation. Here, we focus on the immunoregulatory potential of combination therapies of MSCs and classic pharmacotherapy to mediate acceptance of solid organ grafts. MethodsTo determine which drugs influence the immunosuppressive effect of MSCs, we assessed the interaction of MSCs and common clinical immunosuppresants (MMF, sirolimus [Srl], and ciclosporin A [CiA]) in a parent-into-F1 cell transfer model. In this model, the transfer of parental strain T cells into semi-allogeneic F1 recipients induces a graft-versus-host reaction (GvHR). Re-isolated CFSE-labelled T lymphocytes were analyzed by flow cytometry. These findings were compared to a fully allogeneic heart transplantation model. ResultsWe found that MSC treatment alone had no significant effect on allograft survival of heterotopic heart grafts. However, MSCs combined with short-term mycophenolate mofetil (MMF) significantly prolonged graft survival. Quantitative analysis of three different MSC – drug combinations in the F1 model revealed, that only the MSC–MMF combination led to a super-additive immunosuppressive effect. We also investigated the effect of MMF and CiA on IFNγ production of stimulated lymphocytes and found that MMF left the expression of IFNγ unaffected, whereas CiA completely abolished the production of IFNγ. ConclusionOur data show that the type of concurrent immunosuppression strongly influences the immunosuppressive effect of MSC, most likely through differential secretion of IFNγ. A regimen combining MSCs and MMF was most immunosuppressive.

Prevention of transplant coronary artery disease by prenylation inhibitors

In this study we systematically dissect the prenylation pathway to better define the mechanism behind statin inhibition in chronic allograft rejection in heart transplants, or transplant coronary artery disease (TCAD). Utilizing a murine heterotopic heart transplant model, animals received daily treatments of either statin or selective isoprenoid blockade inhibitors to block the four major downstream branches of the mevalonate pathway. TCAD was assessed by morphometric analysis at Day 52. Graft-infiltrating cells, cytokine production, smooth muscle cell proliferation and migration and endothelial cell MHC II expression were detected on Day 7. Atorvastatin and two prenylation inhibitors, NE-10790 and manumycin A, significantly reduced TCAD lesions compared with untreated animals. Perillyl alcohol treatment resulted in a trend toward decreased luminal narrowing. Finally, zaragozic acid (cholesterol blockade only) did not alter TCAD severity. Statins and prenylation inhibitors reduced inflammatory cell allograft recruitment, but did not always correlate with TCAD reduction. Cytokine production was decreased in recipient spleens in all treatment groups. Both in vitro and in vivo IFN-γ-stimulated MHC II expression was decreased in a dose-dependent manner in the atorvastatin, perillyl alcohol and NE-10790 groups. In vitro smooth muscle cell proliferation was decreased in all treatment groups. Finally, in vitro smooth muscle cell migration was decreased in the atorvastatin, NE-10790 and manumycin A groups only. FPT and GGPT-2 (inhibition) are the key enzymes in the HGM-CoA reductase pathway and most influential in TCAD prevention. TCAD reduction is most closely related to smooth muscle cell migration, but not its anti-inflammatory properties.

94 Gamma Delta T Cells Contribute to Acute and Chronic Allograft Rejection Via IL-17 Induced Inflammation in a Murine Heart Transplantation Model

<h3>Purpose</h3> The role of gamma delta (GD) T cells in allograft rejection remains poorly understood. We have reported that IL-17 contributes to allograft rejection in a murine heart transplant model. Because intragraft GD T cells are the predominant source of IL-17, we hypothesized GD T cells may play a key role in allograft rejection. <h3>Methods and Materials</h3> Acute rejection: FVB donor hearts heterotopically transplanted into (a) GD T cell-deficient C57BL/6 (TCR G^−/−) or (b) C57BL/6 (WT) recipients. Graft survival measured. Graft infiltrating cells detected with FACS analysis. Neutrophil activity accessed via myeloperoxidase (MPO) assay. Cytokine mRNA expression detected with Taqman PCR. Chronic rejection: C-H-2^bm12KhEg (H-2^bm12) donor hearts transplanted into (a) TCR G^−/− or (b) WT recipients. Graft survival assessed with graft beating scores (0 to 4). Grafts harvested day 52 and graft coronary artery disease (GCAD) evaluated with morphometric analysis (luminal occlusion, intima-to-media ratio, and percentage of diseased vessels). Graft infiltrating cells assessed by FACS. <h3>Results</h3> Acute model: Graft survival was prolonged in TCR G^−/− recipients (n = 18) compared to WT recipients (n = 14) (14.4±8.2 days vs. 8.6±1.2 days, p < 0.01). Graft infiltrating CD45⁺, CD4⁺, CD8⁺ cells, neutrophils, and MPO activity were decreased in TCR G^−/− recipients (p < 0.05). FACS analysis revealed GD T cells were key IL-17 producers (WT); IL-17 producing cells were decreased in TCR G^−/− recipients (p < 0.05). Supporting these findings, IL-17 and IL-23 mRNA expression was decreased in TCR G^−/− recipients (p < 0.05). Chronic model: Graft survival was improved in TCR G^−/− recipients, and GCAD development was reduced in TCR G^−/− recipients. Graft infiltrating CD45⁺cells were attenuated in TCR G^−/− recipients (p < 0.05). <h3>Conclusions</h3> GD T cells are (1) crucial for the development of acute and chronic allograft rejection during heart transplants, (2) key producers of IL-17, and (3) important in inflammatory cell recruitment.

Deficiency of Toll-Like Receptor on Only the Donor or Recipient Side Does Not Affect Graft Survival in Murine Heart Transplantation

Deficiency of Toll-Like Receptor on Only the Donor or Recipient Side Does Not Affect Graft Survival in Murine Heart Transplantation

IL-17 Contributes to the Development of Chronic Rejection in a Murine Heart Transplant Model

Although interleukin-17 (IL-17) has been reported to participate in the pathogenesis of infectious, autoimmune and allergic disorders, the precise role in allograft rejection remains uncertain. This study illustrates that IL-17 contributes to the pathogenesis of chronic allograft rejection. Utilizing a murine heterotopic heart transplant model system, IL-17-deficient recipient mice had decreased allograft inflammatory cell recruitment, decreased IL-6, MCP-1, and KC production, and reduced graft coronary artery disease (GCAD). Intragraft gamma delta (gammadelta) T cells appear to be the predominant source of IL-17 production. Therefore, IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.

Intracellular signaling pathways control mitochondrial events associated with the development of ischemia/ reperfusion-associated damage

Ischemia (I) and reperfusion (R) trigger a series of events, which culminate in severe injury to the transplanted organ. Cell death resulting from the formation of mitochondrial reactive oxygen species (ROS) coupled with the perturbation of mitochondrial Ca2+ homeostasis is central to the development of IR-associated tissue damage. We and others have shown recently that intracellular signaling pathways critically control these mitochondrial changes, making them potential targets for therapeutic intervention. Using a heterotopic murine heart transplant model as well as primary and immortalized cardiomyocyte cells we established the activity patterns of mitogen-activated protein kinases (MAPKs) ERK, JNK, and p38 during IR, and probed into their role in the perturbation of mitochondrial ROS and Ca2+ homeostasis, which are necessary for cardiomyocyte death. Our results showed a strong activation of all three MAPKs as well as a rise in mitochondrial ROS and Ca2+ during early reoxygenation. Inhibiting p38 kinase most efficiently prevented ROS production, Ca2+ overload and cell death, suggesting that targeting this signaling molecule may provide a possible strategy to limit the effects of IR.

Memory Alloreactive B Cells and Alloantibodies Prevent Anti-CD154-Mediated Allograft Acceptance

The impact of memory B cells and alloantibodies on the ability to induce transplantation tolerance has not been elucidated. We have developed a murine heart transplant model that isolates the contributions of functional memory B cells from memory T cells in allograft rejection. Memory 3-83 B cells with dual specificity for H-2K(k) and H-2K(b) were generated in 3-83 Igi BCR knockin (BALB/c background) mice by the transplantation of C3H (H-2K(k)) hearts in the absence of immunosuppression. To test the effect of functional memory 3-83 B cells, C3H-primed 3-83 Igi recipients were challenged with C57BL/6 hearts (H-2K(b)) at 60-90 days post-C3H heart transplant and treated with anti-CD154 mAbs. Despite immunosuppression, the C57BL/6 hearts were acutely rejected within 10-13 days and graft rejection was associated with increased frequencies of C57BL/6-specific IFN-gamma-producing T cells. Histology revealed significant numbers of infiltrating T cells, consistent with acute T cell-mediated rejection. The resistance to tolerance induction was dependent on the synergistic effects of memory 3-83 B cells and alloantibodies, whereas memory T cells are not necessary. We conclude that the combined effects of functional memory B cells and alloantibodies prevent anti-CD154-mediated graft acceptance by facilitating the CD40-CD154-independent activation of alloreactive T cells. This study provides insight into the potential ability of memory B cells and alloantibodies to prevent anti-CD154-mediated graft acceptance.

Abstract 5849: The Effects of Statins and Prenylation Inhibitors on Chronic Rejection in Heart Transplantation

HMG-CoA Reductase Inhibitors (statins) increase patient survival and decrease Graft Coronary Artery Disease (GCAD) in heart transplants. Although the exact mechanism is unknown, the protective role of statins may be attributed to two factors: modifications in cholesterol metabolism or small G protein prenylation, and/or immunosuppression or changes on graft endothelial cell (EC) and smooth muscle cell (SMC) function. We hypothesized that statins attenuate GCAD development through alterations in G protein prenylation primarily in graft SMC and EC. Using a murine heterotopic heart transplant model (B6 BM12 to C57BL/6), animals received no treatment (control); HMG-CoA Reductase inhibitor (Atorvastatin, AT); or drugs that selectively block the four major downstream branches of the mevalonate pathway (squalene synthase (ZA), farnesyl protein transferase (MA), geranylgeranyl protein transferase (GGPT-2) (NE), GGPT-1 and -2 (PA), and both FPT and GGPT-2 (NE+MA)). Intimal proliferation was measured with morphometric analysis. In vitro assays were utilized to determine SMC activity, T-cell function, and EC MHC-2 expression. AT, MA, and NE significantly decreased GCAD development, while ZA and NE+MA had no effect (control 63.2± 38%, AT 29.3± 37.5%, MA 43.0± 39.3%, PA 47.5+/−42.5%, NE 24.7± 37.1%, ZA 65.4± 32.5%; p&lt; 0.01). After 5 days of therapy, ELISPOT analysis revealed that all selective inhibitors decreased the Th2 response, and all except AT decreased the Th1 response, with NE levels equivalent to syngeneic controls (p&lt; 0.001). In vitro, inhibition with AT, PA, or NE reduced SMC proliferation and migration compared to controls (p&lt; 0.01). IFNγ-stimulated MHC-2 expression on EC decreased with AT, PA, or NE treatment in a dose dependent manner (p&lt; 0.01). This is the first study to evaluate the effects of prenylation inhibitors on both recipient and graft responses to heart transplantation. Our data shows that inhibition of GGPT-2, and not cholesterol synthesis, is the molecular target of statins’ attenuation of GCAD. Our data suggests that the beneficial effects seen with statin therapy after heart transplant are due to decreased T-cell function and decreased graft SMC and EC function.

Stopping the Primal RAGE Reaction in Myocardial Infarction

The report of Andrassy and colleagues1 in the current issue of Circulation adds to the growing body of evidence that the receptor for advanced glycation end products (RAGE) and its ligands, particularly high mobility group box-1 (HMGB1), are central mediators of ischemia/reperfusion (I/R) injury in the heart.1–4 A major cause of injury, especially in the reperfusion phase, is the influx of inflammatory cells into the stressed heart. Andrassy and colleagues show that infiltrating leukocytes express proinflammatory HMGB1 and that HMGB1 plays fundamental roles in injury responses in the I/R heart. In homeostasis, HMGB1 is largely a nuclear protein. In stress conditions, HMGB1 may be released from injured cells, particularly on necrosis. The chief receptor for HMGB1, RAGE, is expressed in multiple cell types in the I/R heart, such as inflammatory cells, and also in cardiomyocytes and vascular cells (endothelial cells and smooth muscle cells).2 Although HMGB1 may interact with distinct receptors beyond RAGE (for example, toll receptors),5 the current study of Andrassy and colleagues reveals major roles for RAGE in transducing the effects of HMGB1 in the heart, as administration of recombinant HMGB1 or antagonists of this molecule had significant proinjury effects in the wild-type mouse heart, but no additive effects were noted in mice devoid of RAGE. The biology of RAGE, however, is complex and extends beyond HMGB1 in the injured heart. Article p 3216 In addition to HMGB1, other ligands of RAGE likely play pivotal roles in the response to ischemia or I/R in the heart. For example, rapid generation of pre–advanced glycation end products (AGE) species (methylglyoxal) and AGEs in transient occlusion/reperfusion of the LAD coronary artery has been illustrated.2 In hypoxia, in both the murine heart and in isolated primary murine endothelial cells, AGE-immunoreactive species are central mediators of regulation of …