Cardiac Transplantation Model Research Articles

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

Graft Protective and Intercellular Immunomodulatory Effects by Adoptive Transfer of an Agonistic Anti-BTLA mAb (3C10) Induced CD4+CD25+ Regulatory T Cells in Murine Cardiac Allograft Transplant Model

We demonstrated that an agonistic anti-B and T lymphocyte attenuator antibody (3C10) prolonged cardiac survival by inducing regulatory T cells (Treg). However, the mechanisms of immune tolerance in the recipients remained unclear. In this study, we investigated the graft-protective and intercellular immunomodulatory effects of adoptive transfer (AT) of 3C10-induced Tregs in a murine cardiac allograft transplant model. Thirty days after transplantation of a C57BL/6 heart into the primary 3C10-treated CBA recipients, splenic CD4+CD25+ cells from these recipients (3C10/AT group) or naïve CBA mice (no-treatment group) were adoptively transferred into secondary CBA recipients with a C57BL/6 heart. To confirm the requirement for 3C10-induced Tregs, we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to secondary CBA recipients. Additionally, histologic and fluorescent staining, cell proliferation analysis, flow cytometry, and donor-specific antibody (DSA) measurements were performed. 3C10/AT-treated CBA recipients resulted in significantly prolonged allograft survival (median survival time [MST], >50 days). Allografts displayed prolonged function with preservation of vessel structure by maintaining high numbers of splenic CD4+CD25+Foxp3+ Treg and intramyocardial CD4+Foxp3+ cells. DSA levels were suppressed in 3C10/AT-treated CBA recipients. Moreover, PC-61 administration resulted in a shorter MSTs of cardiac allograft survivals, a detrimental increase in DSA production, and enhanced expression of programmed cell death (PD)-1. AT of 3C10-induced Tregs may be a promising graft-protective strategy to prolong allograft survival and suppress DSA production, driven by the promotion of splenic and graft-infiltrating Tregs and collaboration with PD-1+ T cells and Treg.

Cardioprotective effect of Interleukin-11 against warm ischemia-reperfusion injury in a rat heart donor model

Shortage of donor organs for heart transplantation is a worldwide problem. Donation after circulatory death (DCD) has been proposed to expand the donor pool. However, in contrast to the donation after brain death that undergoes immediate cold preservation, warm ischemia and subsequent reperfusion injury are inevitable in DCD. It has been reported that interleukin-11 (IL-11) mitigates ischemia-reperfusion injury in rodent models of myocardial infarction and donation after brain death heart transplantation. We hypothesized that IL-11 also offers benefit to warm ischemia in an experimental model of cardiac transplantation that resembles DCD. The hearts of naïve male Sprague Dawley rats (n = 15/group) were procured, subjected to 25-min warm ischemia, and reperfused for 60 min using Langendorff apparatus. IL-11 or saline was administered intravenously before the procurement, added to maintenance buffer, and infused via perfusion during reperfusion. IL-11 group exhibited significantly better cardiac function post-reperfusion. Severely damaged mitochondria was found in the electron microscopic analysis of control hearts whereas the mitochondrial structure was better preserved in the IL-11 treated hearts. Immunoblot analysis using neonatal rat cardiomyocytes revealed increased signal transducer and activator of transcription 3 (STAT3) phosphorylation at Ser727 after IL-11 treatment, suggesting its role in mitochondrial protection. Consistent with expected activation of mitochondrial respiration by mitochondrial STAT3, immunohistochemical staining demonstrated a higher mitochondrial cytochrome c oxidase subunit 2 expression. In summary, IL-11 protects the heart from warm ischemia reperfusion injury by alleviating mitochondrial injury and could be a viable therapeutic option for DCD heart transplantation.

Synthesis of mizoribine prodrugs and their in vivo evaluation as immunosuppressive agents

Mizoribine is a well-known immunosuppressive drug, based on a nucleoside scaffold, that targets inosine-monophosphate dehydrogenase (IMPDH). In an effort to increase its in vivo efficacy, three different types of prodrugs (a phosphoramidate prodrug, a lipophilic ester derivative and an amino acid conjugate) were prepared. Screening of these prodrugs in a rapid whole blood assay revealed that the two ester-based mizoribine prodrugs potently inhibited interleukin 2 secretion. Moreover, these prodrugs were able to prolong graft survival, when evaluated in a mouse model of cardiac allograft transplantation. Strikingly, a combination therapy of these mizoribine prodrugs with tacrolimus had a synergistic in vivo effect.

A murine groin site cardiac transplantation model-applicable tool for studying roles of peripheral lymph nodes in transplantation.

The murine heterotopic cardiac transplantation model has been widely used to study antigen-specific immune responses or new immunosuppressive agents, which have a strong correlation with peripheral lymph nodes. Thus, a new organ transplantation model that is applicable to related studies is needed. Here, we describe a groin-site murine heart transplantation model using a cuff technique, in which the donor aorta and pulmonary artery are anastomosed to the truncated femoral vessels of the recipient. The mean survival time (MST) of the grafts in BALB/c-to-C57BL/6 allo-transplant group was 7.2 ± 0.3 days, and 1.9 ± 0.2 days in BALB/c-to-Sprague-Dawley (SD) rat xeno-transplant group. H&E results show that donor hearts from both groups demonstrate typical pathological features at the endpoint. Evans Blue tracing revealed that the popliteal lymph nodes of the grafted side hindlimb are larger than those of the contralateral side. Moreover, IHC staining for CD3, CD20 shows that the germinal center and cortex region of the grafted side of popliteal lymph nodes is apparently increased than that of the contralateral side. To sum up, this model may serve as an ideal model to study the role of peripheral lymph nodes in organ transplant rejection. In addition, extra-peritoneal grafting makes a step forward in animal welfare under the 3Rs' principle (Replacement, Reduction, Refinement).

Noninvasive quantification of granzyme B in cardiac allograft rejection using targeted ultrasound imaging.

Endomyocardial biopsy is the gold standard method for the diagnosis of cardiac allograft rejection. However, it causes damage to the heart. In this study, we developed a noninvasive method for quantification of granzyme B (GzB) in vivo by targeted ultrasound imaging, which detects and provides quantitative information for specific molecules, for acute rejection assessment in a murine cardiac transplantation model. Microbubbles bearing anti-GzB antibodies (MBGzb) or isotype antibodies (MBcon) were prepared. Hearts were transplanted from C57BL/6J (allogeneic) or C3H (syngeneic) donors to C3H recipients. Target ultrasound imaging was performed on Days 2 and 5 post-transplantations. A pathologic assessment was performed. The expression of granzyme B and IL-6 in the heart was detected by Western blotting. After MB injection, we observed and collected data at 3 and 6min before and after the flash pulse. Quantitative analysis revealed that the reduction in peak intensity was significantly higher in the allogeneic MBGzb group than in the allogeneic MBcon group and the isogeneic MBcon group at PODs 2 and 5. In the allogeneic groups, granzyme B and IL-6 expression levels were higher than those in the isogeneic group. In addition, more CD8 T cells and neutrophils were observed in the allogeneic groups. Ultrasound molecular imaging of granzyme B can be used as a noninvasive method for acute rejection detection after cardiac transplantation.

Research Highlights.

Research Highlights.

Single-Cell RNA Sequencing Maps Immune Cell Heterogeneity in Mice with Allogeneic Cardiac Transplantation

Objective: Immune cells play important roles in mediating allograft rejection and tolerance after cardiac transplantation. However, immune cell heterogeneity at the single-cell level, and how immune cell states shape transplantation immunity, remain incompletely characterized. Methods: We performed single-cell RNA sequencing (scRNA-seq) on immune cells in LNs from a mouse syngeneic and allogeneic cardiac transplantation model. Nine T cell clusters were identified through unsupervised analysis. Pathway enrichment analysis was used to explore the functional differences among cell subpopulations and to characterize the metabolic heterogeneity of T cells. Results: We comprehensively determined the transcriptional landscape of immune cells, particularly T cells, and their metabolic transcriptomes in LNs during mouse cardiac transplantation. On the basis of molecular and functional properties, we also identified T cell types associated with transplantation-associated immune processes, including cytotoxic CD8+ T cells, activated conventional CD4+ T cells, and dysfunctional Tregs. We further elucidated the contribution of JunB to the induction of Th17 cell differentiation and restriction of Treg development, and identified that HIF-1a participates in T cell metabolism and function. Conclusions: We present the first systematic single-cell analysis of transcriptional variation within the T cell population, providing new insights for the development of novel therapeutic targets for allograft rejection.

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia.

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

404.9: Oridonin Prolongs the Survival of Mouse Cardiac Allografts by Attenuating the NF-kB/NLRP3 Pathway

Introduction: Oridonin (Ori), the main bioactive ingredient of the natural anti-inflammatory herb Rabdosia rubescens, could be a covalent inhibitor of the NLRP3 inflammasome. Solid organ transplantation provides a life-saving optional therapy for patients with end- stage organ dysfunction. The long-term survival of solid organ transplantation remains restricted because of the possibility of rejection and the toxicity, infection, cardiovascular disease, and malignancy related to immunosuppressive (IS) drugs. However, the pathogenic mechanisms involved remain unclear. The ideal IS drugs to prevent allograft rejection have not been identified. Here, we investigated whether Ori could prolong the in vivo survival of completely mismatched cardiac allografts. Method: The cardiac transplantation models were conducted among three groups of mice from C57BL/6NCrSlc (B6/N) or C3H/HeNSlc (C3H) to C3H: the syngeneic and the allogeneic group, whose recipients were treated with vehicle of Ori, and the Ori treatment group, in which the recipients were transplanted hearts from MHC-I mismatched donors and treated with different dosages of Ori from post-operative day (POD) 0 to 7. Then, we investigated the effect of Ori on bone marrow-derived dendritic cell (BMDC) and allogeneic mixed lymphocyte reaction in vitro. Results: Ori with 3, 10, and 15 mg/kg Ori could prolong the survival (MST = 22.8, 49.2, and 65.3 days, respectively). We found that infiltrating CD8+ T cells and macrophages were decreased, and regulatory T cells (Tregs) were expanded in allografts on POD7. The mRNA level of IL-1b and IFN-g of allografts was downregulated. Mechanistically, Ori-treated BMDCs suppressed T-cell proliferation and IFN-g+CD4+ T-cell differentiation, along with the expansion of Tregs and IL-10+CD4+ T cells. Ori inhibited NOD, LRR-, and pyrin domain-containing protein 3 (NLRP3) expression; attenuated NF-kB and IkBa phosphorylation in LPS-activated BMDCs; downregulated NLRP3, Caspase-1, IL-1b, IL-18, and IFN-g; and upregulated IL-10 expression. Conclusion: Our findings highlight the potential of Ori as a novel and natural IS agent to improve transplant tolerance. Ori could exert IS activity through decreasing IL-1b and IL- 18 production and Th1 differentiation and proliferation and expanding Tregs via inhibiting the NF-kB/NLRP3 signaling pathway. The National Nature Science Foundation of China (81671958 and U1604282). Science and Technology Innovation Talents in Henan Universities (No. 19HASTIT003). Grants of Ministry of Education, Culture, Sports, Science and Technology of Japan (Grants-in-Aid 16K11064, 24/17H04277, and 18K08558). the National Center for Child Health and Development (29–09).

The Combination of Rhodosin and MMF Prolongs Cardiac Allograft Survival by Inhibiting DC Maturation by Promoting Mitochondrial Fusion.

Despite being the gold-standard treatment for end-stage heart disease, heart transplantation is associated with acute cardiac rejection within 1 year of transplantation. The continuous application of immunosuppressants may cause side effects such as hepatic and renal toxicity, infection, and malignancy. Developing new pharmaceutical strategies to alleviate acute rejection after heart transplantation effectively and safely is of critical importance. In this study, we performed a murine model of MHC-full mismatch cardiac transplantation and showed that the combination of Rhodosin (Rho) and mycophenolate mofetil (MMF) could prevent acute rejection and oxidative stress injury and prolong the survival time of murine heart transplants. The use of Rho plus MMF in allografts improved the balance of Tregs/Teff cells, which had a protective effect on allotransplantation. We also isolated bone marrow-derived dendritic cells (BMDCs) and determined that Rho inhibited DC maturation by promoting mitochondrial fusion mainly through the mitochondrial fusion-related protein MFN1. Herein, we demonstrated that Rho, an active ingredient isolated from the plant Rhodiola rosea with antioxidant and anti-inflammatory activities, could efficiently alleviate acute rejection and significantly prolong murine heart allograft survival when used with a low dose of MMF. More importantly, we found that Rho restrained DC maturation by promoting mitochondrial fusion and decreasing reactive oxygen species (ROS) levels, which then alleviated acute rejection in murine cardiac transplantation. Interestingly, as a novel immunosuppressant, Rho has almost no side effects compared with other traditional immunosuppressants. Taken together, these results suggest that Rho has good clinical auxiliary applications as an effective immunosuppressant and antioxidant, and this study provides an efficient strategy to overcome the side effects of immunosuppressive agents that are currently used in organ transplantation.

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Pre-transplant infusion of donor leukocytes treated with extracorporeal photochemotherapy induces immune hypo-responsiveness and long-term allograft survival in murine models

Recipients of solid organ transplantation (SOT) rely on life-long immunosuppression (IS), which is associated with significant side effects. Extracorporeal photochemotherapy (ECP) is a safe, existing cellular therapy used to treat transplant rejection by modulating the recipient’s own blood cells. We sought to induce donor-specific hypo-responsiveness of SOT recipients by infusing ECP-treated donor leukocytes prior to transplant. To this end, we utilized major histocompatibility complex mismatched rodent models of allogeneic cardiac, liver, and kidney transplantation to test this novel strategy. Leukocytes isolated from donor-matched spleens for ECP treatment (ECP-DL) were infused into transplant recipients seven days prior to SOT. Pre-transplant infusion of ECP-DL without additional IS was associated with prolonged graft survival in all models. This innovative approach promoted the production of tolerogenic dendritic cells and regulatory T-cells with subsequent inhibition of T-cell priming and differentiation, along with a significant reduction of donor-specific T-cells in the spleen and grafts of treated animals. This new application of donor-type ECP-treated leukocytes provides insight into the mechanisms behind ECP-induced immunoregulation and holds significant promise in the prevention of graft rejection and reduction in need of global immune suppressive therapy in patients following SOT.

Effects of Each Domain in Recombinant Human Soluble Thrombomodulin on Prolongation of Murine Cardiac Allograft Survival

Thrombomodulin is used to manage disseminated intravascular coagulation. In our murine heart transplantation model, the administration of recombinant human soluble thrombomodulin (rTM) could induce the prolongation of cardiac allograft survival. However, there are limited data on the graft protective effects of each r domain (D1, D2, and D3). In this study, we investigated the effects of each domain of rTM on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA recipients using microsurgical techniques. CBA mice that underwent transplantation of C57BL/6 cardiac allografts were assigned to 4 groups: no treatment and each domain-exposed group. The dosage of each domain was determined based on our previous experiments. Flow cytometry and histologic studies were performed to determine whether Foxp3+ regulatory T cells were generated. Untreated and D2-exposed CBA recipients acutely rejected C57BL/6 cardiac allografts within 9 days. Administration of D3 resulted in modest prolongation of allograft survival, and administration of D1 significantly prolonged allograft survival. Histologic studies showed that myocardial damage of allografts from D1- and D3-exposed CBA recipients was controlled compared with that of untreated recipients. In particular, the CD4+CD25+Foxp3+ cell population in the splenocytes of D1-exposed CBA recipients was increased. In conclusion, D1 in rTM could help prolong cardiac allograft survival through regulatory T cell induction and graft protective effects.

Evaluation of Cardiac Graft Function by Echocardiography in Mice

Generally, graft function in the murine cardiac allograft transplant model is assessed daily by palpating the heart for evidence of contraction. To our knowledge, few reports have investigated the correlation of cardiac graft function using echocardiography and immunohistochemical studies. In this study, we investigated the efficacy of echocardiographic and histologic evaluation of alloimmune responses in the acute phase of murine cardiac allografts. Fully vascularized heterotopic hearts from CBA (allogeneic group) or C57BL/6 (syngeneic group) donors were transplanted into C57BL/6 recipients using microsurgical techniques. Fluctuations in heart rate, left ventricular ejection fraction (LVEF), left ventricular functional shortening (LVFS), right ventricular outflow tract maximal systolic velocity (RVOT Vmax), and RVOT velocity time integral (RVOT VTI) were evaluated on postoperative days (PODs) 1, 3, 5, 7, and 9 after transplantation using an ultrasonic device. Histologic studies were also performed. The syngeneic group did not show a complete cessation of heartbeat or deterioration of cardiac function. CBA recipients in the allogeneic group rejected cardiac allografts on POD 9 after grafting. LVEF and LVFS in the allogeneic group gradually decreased on POD 9. Consistent with the time-course echocardiographic evaluation, histologic studies showed gradual atrophy of the left ventricle. In contrast, RVOT Vmax and RVOT VTI in the allogeneic group were not significantly different during the observation period. Additionally, the thickness of the right ventricular wall did not change until POD 7. The present findings suggested that echocardiography may help to evaluate time-course murine cardiac graft function through left ventricular parameters such as LVEF and LVFS.

Cytosporone B (Csn-B), an NR4A1 agonist, attenuates acute cardiac allograft rejection by inducing differential apoptosis of CD4+T cells

CD4+T cell-mediated acute rejection remains a major factor that affects the early survival of transplanted organs post-transplantation. Here, we reveal that nuclear receptor subfamily 4 Group A member 1 (Nr4A1) was upregulated during cardiac allograft rejection and that the increased Nr4A1 was primarily localized in intragraft-infiltrating CD4+T cells. Nr4A1 acts as a transcription factor with an important role in CD4+T cell apoptosis, differentiation and T cell dysfunction, which indicates that Nr4A1 may play a critical role in transplant rejection. Cytosporone B (Csn-B) is a naturally occurring agonist of Nr4A1, and the role of Csn-B in the physiological process of cardiac rejection is poorly defined. This study constructed an acute rejection model of abdominal heterotopic cardiac transplantation in mice and investigated whether Csn-B could attenuate acute transplant rejection by modulating the CD4+T lymphocyte response. The results showed that Csn-B prolonged murine cardiac allograft survival and reduced inflammation in allografts. Subsequently, it was confirmed that Csn-B functions by inducing non-Treg apoptosis and promoting Treg cell differentiation. Finally, we also confirmed that Csn-B attenuates acute rejection by directly targeting Nr4A1 in CD4+T cells. Our data suggest that Csn-B is a promising novel therapeutic approach for acute cardiac allograft rejection.

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

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

Overexpression of PD-1 on T cells promotes tolerance in cardiac transplantation via ICOS-dependent mechanisms.

The programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway is a potent inhibitory pathway involved in immune regulation and is a potential therapeutic target in transplantation. In this study, we show that overexpression of PD-1 on T cells (PD-1 Tg) promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model when combined with costimulation blockade with CTLA-4–Ig. PD-1 overexpression on T cells also protected against chronic rejection in a single MHC II–mismatched cardiac transplant model, whereas the overexpression still allowed the generation of an effective immune response against an influenza A virus. Notably, Tregs from PD-1 Tg mice were required for tolerance induction and presented greater ICOS expression than those from WT mice. The survival benefit of PD-1 Tg recipients required ICOS signaling and donor PD-L1 expression. These results indicate that modulation of PD-1 expression, in combination with a costimulation blockade, is a promising therapeutic target to promote transplant tolerance.

Functional roles of graft‐infiltrating lymphocytes during early‐phase post‐transplantation in mouse cardiac transplantation models

Immunological behavior of graft-infiltrating lymphocytes (GILs) determines the graft fate (i.e., rejection or acceptance). Nevertheless, the functional alloreactivity and the phenotype of GILs at various times during the early post-transplantation phase have not been fully elucidated. We examined the immunological activities of early-phase GILs using a murine model of cardiac transplantation. GILs from 120-h allografts, but not 72-h allografts, showed robust activation and produced proinflammatory cytokines. In particular, a significant increase in CD69+ T-bet+ Nur77+ T cells was detected in 120-h allografts. Furthermore, isolated GILs were used to reconstitute BALB/c Rag2-/- γc-/- (BRG) mice. BRG mice reconstituted with 120-h GILs displayed donor-specific immune reactivity and rejected donor strain cardiac allografts; conversely, 72-h GILs exhibited weak anti-donor reactivity and did not reject allografts. These findings were confirmed by re-transplantation of cardiac allografts into BRG mice at 72-h post-transplantation. Re-transplanted allografts continued to function for >100 days, despite the presence of CD3+ GILs. In conclusion, the immunological behavior of GILs considerably differs over time during the early post-transplantation phase. A better understanding of the functional role of early-phase GILs may clarify the fate determination process in the graft-site microenvironment.