Genetically Modified Porcine-to-Human Cardiac Xenotransplantation.

A 57-year-old man with nonischemic cardiomyopathy who was dependent on venoarterial extracorporeal membrane oxygenation (ECMO) and was not a candidate for standard therapeutics, including a traditional allograft, received a heart from a genetically modified pig source animal that had 10 individual gene edits. Immunosuppression was based on CD40 blockade. The patient was weaned from ECMO, and the xenograft functioned normally without apparent rejection. Sudden diastolic thickening and failure of the xenograft occurred on day 49 after transplantation, and life support was withdrawn on day 60. On autopsy, the xenograft was found to be edematous, having nearly doubled in weight. Histologic examination revealed scattered myocyte necrosis, interstitial edema, and red-cell extravasation, without evidence of microvascular thrombosis - findings that were not consistent with typical rejection. Studies are under way to identify the mechanisms responsible for these changes. (Funded by the University of Maryland Medical Center and School of Medicine.).

Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report.

BACKGROUND: A genetically engineered pig cardiac xenotransplantation was done on Jan 7, 2022, in a non-ambulatory male patient, aged 57 years, with end-stage heart failure, and on veno-arterial extracorporeal membrane oxygenation support, who was ineligible for an allograft. This report details our current understanding of factors important to the xenotransplantation outcome. METHODS: Physiological and biochemical parameters critical for the care of all heart transplant recipients were collected in extensive clinical monitoring in an intensive care unit. To ascertain the cause of xenograft dysfunction, we did extensive immunological and histopathological studies, including electron microscopy and quantification of porcine cytomegalovirus or porcine roseolovirus (PCMV/PRV) in the xenograft, recipient cells, and tissue by DNA PCR and RNA transcription. We performed intravenous immunoglobulin (IVIG) binding to donor cells and single-cell RNA sequencing of peripheral blood mononuclear cells. FINDINGS: After successful xenotransplantation, the graft functioned well on echocardiography and sustained cardiovascular and other organ systems functions until postoperative day 47 when diastolic heart failure occurred. At postoperative day 50, the endomyocardial biopsy revealed damaged capillaries with interstitial oedema, red cell extravasation, rare thrombotic microangiopathy, and complement deposition. Increased anti-pig xenoantibodies, mainly IgG, were detected after IVIG administration for hypogammaglobulinaemia and during the first plasma exchange. Endomyocardial biopsy on postoperative day 56 showed fibrotic changes consistent with progressive myocardial stiffness. Microbial cell-free DNA testing indicated increasing titres of PCMV/PRV cell-free DNA. Post-mortem single-cell RNA sequencing showed overlapping causes. INTERPRETATION: Hyperacute rejection was avoided. We identified potential mediators of the observed endothelial injury. First, widespread endothelial injury indicates antibody-mediated rejection. Second, IVIG bound strongly to donor endothelium, possibly causing immune activation. Finally, reactivation and replication of latent PCMV/PRV in the xenograft possibly initiated a damaging inflammatory response. The findings point to specific measures to improve xenotransplant outcomes in the future. FUNDING: The University of Maryland School of Medicine, and the University of Maryland Medical Center.

The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

Genetic Engineering of Donor Pig for the First Human Cardiac Xenotransplantation: Combatting Rejection, Coagulopathy, Inflammation, and Excessive Growth.

PURPOSE OF REVIEW: The first successful pig to human cardiac xenotransplantation in January 2022 represented a major step forward in the fields of heart failure, immunology, and applied genetic engineering, using a 10-gene edited (GE) pig. This review summarizes the evolution of preclinical modelling data which informed the use of each of the 10 genes modified in the 10-GE pig: GGTA1, Β4GalNT2, CMAH, CD46, CD55, TBM, EPCR, CD47, HO-1, and growth hormone receptor. RECENT FINDINGS: The translation of the 10-GE pig from preclinical modelling to clinical compassionate xenotransplant use was the culmination of decades of research combating rejection, coagulopathy, inflammation, and excessive xenograft growth. Understanding these 10 genes with a view to their combinatorial effects will be useful in anticipated xenotransplant clinical trials.

An intrinsic link to an extrinsic cause of cardiac xenograft growth after xenotransplantation: Commentary (in response to): Zaman, R. et al. Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress. Immunity 54, 2057-2071.e6 (2021).: Commentary (in response to): Zaman, R. et al. Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress. Immunity 54, 2057-2071.e6 (2021).

Post-transplantation cardiac xenograft growth in an orthotopic pig to baboon model is a life-limiting phenomenon that is poorly understood. Possible causes of growth include both intrinsic and extrinsic etiologies. Extrinsic causes are thought to be attributed to maladaptive hypertrophy as a result of increased mean arterial pressure experienced by the cardiac xenograft after transplantation. Intrinsic causes are thought to be a result of discordant growth between pig xenografts and recipients. This results in intrinsic xenograft growth that parallels the donor and continues in a recipient in which growth is relatively minimal, controlled in part by the growth hormone receptor, IGF-1 axis. Recently, Zaman, et al. published a study titled, "Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress," in Immunity, Volume 54; Issue 9, pp. 2057-2071. They demonstrated that insulin growth factor-secreting resident macrophages that sense hypertensive stress are a mechanistic link to hypertension and maladaptive hypertrophy in the setting of hypertension. While notable in its own right, we comment on how this work may shed light on a new underlying mechanism for the use of growth hormone receptor knockout (GHRKO) pig donors and its role in addressing post-transplantation xenograft growth. We hypothesize that GHRKO pig donors contain syngeneic resident cardiac macrophages that abrogate IGF-1 mediated maladaptive hypertrophy from hypertension. Futures studies in post-transplantation cardiac xenotransplantation growth should examine this mechanism as a potential contributor.

The road to the first FDA-approved genetically engineered pig heart transplantation into human.

We have been testing genetically engineered (GE) pig hearts and optimizing immunosuppression (IS) in non-human primates (NHPs) since 2005. We demonstrate how we translated this preclinical investigation into a US Food and Drug Administration (FDA)-approved clinical cardiac xenotransplantation. First, genetically engineered (GE) pig hearts were transplanted into the abdomen of NHP along with IS, which included anti-CD20 and anti-CD40-based co-stimulation blockade antibodies. We reported 945 days of survival of three gene GE pig hearts in NHPs. Building on this proof-of-concept, we tested 3-10 gene-modified GE pig hearts (in order to improve the immunocompatibility of the xenograft further) in a life-supporting orthotopic model, but had limited success due to perioperative cardiac xenograft dysfunction (PCXD). With novel non-ischemic continuous perfusion preservation (NICP), using the XVIVO Heart solution (XHS), life-supporting survival was extended to 9 months. We approached the FDA under an application for "Expanded Access" (EA), to transplant a GE pig heart in a patient with end-stage non-ischemic cardiomyopathy. He was without other therapeutic options and dependent on VA-ECMO. A team of FDA reviewers reviewed our preclinical research experience and data and allowed us to proceed. This clinical cardiac xenotransplantation was performed, and the patient survived for 60 days, demonstrating the translational preclinical investigation of cardiac xenotransplantation from bench to bedside. The ultimate etiology of graft failure is currently a topic of investigation and lessons learned will progress the field forward.

Progressive genetic modifications of porcine cardiac xenografts extend survival to 9 months.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

Cardiac Xenotransplantation: Progress in Preclinical Models and Prospects for Clinical Translation.

Survival of pig cardiac xenografts in a non-human primate (NHP) model has improved significantly over the last 4 years with the introduction of costimulation blockade based immunosuppression (IS) and genetically engineered (GE) pig donors. The longest survival of a cardiac xenograft in the heterotopic (HHTx) position was almost 3 years and only rejected when IS was stopped. Recent reports of cardiac xenograft survival in a life-sustaining orthotopic (OHTx) position for 6 months is a significant step forward. Despite these achievements, there are still several barriers to the clinical success of xenotransplantation (XTx). This includes the possible transmission of porcine pathogens with pig donors and continued xenograft growth after XTx. Both these concerns, and issues with additional incompatibilities, have been addressed recently with the genetic modification of pigs. This review discusses the spectrum of issues related to cardiac xenotransplantation, recent progress in preclinical models, and its feasibility for clinical translation.

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs.

A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen.

A rare case of diffuse intracranial aspergillosis masquerading as skull base meningioma in an immunocompetent patient.

Intracranial aspergillosis is rare in immunocompetent patients. Its presentation is subtle, often without any diagnostic characteristics, and is frequently mistaken for tuberculous meningitis, pyogenic abscess, or a space-occupying lesion. The authors report a case of diffuse intracranial aspergillosis, in an immunocompetent 34-year-old male, that mimicked a meningioma on preoperative imaging. The origin, clinical course, radiological features, histopathological findings, and surgical treatment are discussed based on review of literature.
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CD4+CD25Hi FoxP3+ regulatory T cells in long-term cardiac xenotransplantation.

BACKGROUND: CD4+CD25Hi FoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long-term baboon recipients who have received genetically modified cardiac xenografts from pig donors. METHODS: Heterotopic cardiac xenotransplantation was performed on baboons using hearts obtained from GTKO.hCD46 (n = 8) and GTKO.hCD46.TBM (n = 5) genetically modified pigs. Modified immunosuppression regimen included antithymocyte globulin (ATG), anti-CD20, mycophenolate mofetil (MMF), cobra venom factor (CVF), and costimulation blockade (anti-CD154/anti-CD40 monoclonal antibody). FACS analysis was performed on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies (mAb) to analyze the percentage of Treg cells in six baboons that survived longer than 2 months (range: 42-945 days) after receiving a pig cardiac xenograft. RESULTS: Total WBC count was low due to immunosuppression in baboons who received cardiac xenograft from GTKO.hCD46 and GTKO.hCD46.hTBM donor pigs. However, absolute numbers of CD4+CD25Hi FoxP3 Treg cells in PBLs of long-term xenograft cardiac xenograft surviving baboon recipients were found to be increased (15.13 ± 1.50 vs 7.38 ± 2.92; P < .018) as compared to naïve or pre-transplant baboons. Xenograft rejection in these animals was correlated with decreased numbers of regulatory T cells. CONCLUSION: Our results suggest that regulatory T (Treg) cells may contribute to preventing or delaying xenograft rejection by controlling the activation and expansion of donor-reactive T cells, thereby masking the antidonor immune response, leading to long-term survival of cardiac xenografts.

Consideration of appropriate clinical applications for cardiac xenotransplantation.

The field of cardiac xenotransplantation has entered an exciting era due to recent advances in the field. Although several hurdles remain, the use of rapidly evolving transgenic technology has the potential to address current allogeneic donor pool constraints and mechanical circulatory system device limitations. The success of xenotransplantation will undoubtedly be dependent on specific patient selection criteria. Defining these particular indications for xenotransplantation is important as we approach the possibility of clinical applications.

Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model.

BACKGROUND: Innovations in transgenic technology have facilitated improved xenograft survival. Additional gene expression appears to be necessary to overcome the remaining immune and biologic incompatibilities. We report for the first time the novel use of six-gene modifications within a pig-to-baboon cardiac xenotransplantation model. METHODS: Baboons (8-15 kg) underwent heterotopic cardiac transplantation using xenografts obtained from genetically engineered pigs. Along with previously described modifications (GTKO, hCD46), additional expression of human transgenes for thromboregulation (endothelial protein C receptor, tissue factor pathway inhibitor, thrombomodulin), complement inhibition (decay accelerating factor), and cellular immune suppression (hCD39, hCD47) was used. Immunosuppression consisted of targeted T-cell and B-cell depletion and conventional anti-rejection agents. RESULTS: Heterotopic cardiac transplantations were performed without complication. Flow cytometry and immunohistochemistry on donor biopsies confirmed transgenic phenotype. In contrast to the prior three-gene generation, significant coagulopathy or consumptive thrombocytopenia has not been observed in the six-gene cohort. As a result, these recipients have experienced decreased bleeding-related complications. Pro-inflammatory responses also appear to be mitigated based on cytokine analysis. Baboons survived the critical 30-day post-operative period when mortality has historically been highest, with no evidence of graft rejection. CONCLUSIONS: The inclusion of additional human genes in genetically engineered pigs appears to confer superior xenograft outcomes. Introduction of these genes has not been associated with adverse outcomes. This multifactorial approach to genetic engineering furthers the prospect of long-term cardiac xenograft survival and subsequent clinical application.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein.

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway-regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life-supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri-transplant platelet activation (as ß-thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

Overexpression of FABP3 inhibits human bone marrow derived mesenchymal stem cell proliferation but enhances their survival in hypoxia.

Studying the proliferative ability of human bone marrow derived mesenchymal stem cells in hypoxic conditions can help us achieve the effective regeneration of ischemic injured myocardium. Cardiac-type fatty acid binding protein (FABP3) is a specific biomarker of muscle and heart tissue injury. This protein is purported to be involved in early myocardial development, adult myocardial tissue repair and responsible for the modulation of cell growth and proliferation. We have investigated the role of FABP3 in human bone marrow derived mesenchymal stem cells under ischemic conditions. MSCs from 12 donors were cultured either in standard normoxic or modified hypoxic conditions, and the differential expression of FABP3 was tested by quantitative (RT)PCR and western blot. We also established stable FABP3 expression in MSCs and searched for variation in cellular proliferation and differentiation bioprocesses affected by hypoxic conditions. We identified: (1) the FABP3 differential expression pattern in the MSCs under hypoxic conditions; (2) over-expression of FABP3 inhibited the growth and proliferation of the MSCs; however, improved their survival in low oxygen environments; (3) the cell growth factors and positive cell cycle regulation genes, such as PCNA, APC, CCNB1, CCNB2 and CDC6 were all down-regulated; while the key negative cell cycle regulation genes TP53, BRCA1, CASP3 and CDKN1A were significantly up-regulated in the cells with FABP3 overexpression. Our data suggested that FABP3 was up-regulated under hypoxia; also negatively regulated the cell metabolic process and the mitotic cell cycle. Overexpression of FABP3 inhibited cell growth and proliferation via negative regulation of the cell cycle and down-regulation of cell growth factors, but enhances cell survival in hypoxic or ischemic conditions.

Role of anti-CD40 antibody-mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model.

BACKGROUND: Recently, we have shown that an immunosuppression regimen including costimulation blockade via anti-CD154 antibody significantly prolongs the cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon heterotopic xenotransplantation model. Unfortunately, many coagulation disorders were observed with the use of anti-CD154 antibody, and recipient survival was markedly reduced by these complications. MATERIAL AND METHODS: In this experiment, we replaced anti-CD154 antibody with a more clinically acceptable anti-CD40 antibody while keeping the rest of the immunosuppressive regimen and the donor pig genetics the same. This was carried out to evaluate the antibody's role in xenograft survival and prevention of coagulopathies. Two available clones of anti-CD40 antibody were tested. One mouse anti-human CD40 antibody, (clone 3A8), activated B lymphocytes in vitro and only modestly suppressed antibody production in vivo. Whereas a recombinant mouse non-human primate chimeric raised against macaque CD40, (clone 2C10R4), blocked B-cell activation in vitro and completely blocked antibody production in vivo. RESULTS: The thrombotic complications seen with anti-CD154 antibody were effectively avoided but the graft survival, although extended, was not as prolonged as observed with anti-CD154 antibody treatment. The longest survival for the 3A8 antibody group was 27 days, and the longest graft survival in the 2C10R4 antibody group was 146 days. All of the grafts except two rejected and were explanted. Only two recipient baboons had to be euthanized due to unrelated complications, and the rest of the baboons remained healthy throughout the graft survival period or after graft explantation. In contrast to our anti-CD 154 antibody-treated baboons, the non-Gal antibody levels started to rise after B cells made their appearance around 8 weeks post-transplantation. CONCLUSIONS: Anti-CD40 antibody at the current dose does not induce any coagulopathies but while effective, had reduced efficacy to induce similar long-term graft survival as with anti-CD154 antibody perhaps due to ineffective control of B-cell function and antibody production at the present dose. More experiments are required to determine antibody affinity and effective dose for inducing long-term cardiac xenograft survival.

Preclinical rationale and current pathways to support the first human clinical trials in cardiac xenotransplantation.

Recent initiation of the first FDA-approved cardiac xenotransplantation suggests xenotransplantation could soon become a therapeutic option for patients unable to undergo allotransplantation. Until xenotransplantation is widely applied in clinical practice, consideration of benefit versus risk and approaches to management of clinical xenografts will based at least in part on observations made in experimental xenotransplantation in non-human primates. Indeed, the decision to proceed with clinical trials reflects significant progress in last few years in experimental solid organ and cellular xenotransplantation. Our laboratory at the NIH and now at University of Maryland contributed to this progress, with heterotopic cardiac xenografts surviving more than two years and life-supporting cardiac xenografts survival up to 9 months. Here we describe our contributions to the understanding of the mechanism of cardiac xenograft rejection and development of methods to overcome past hurdles, and finally we share our opinion on the remaining barriers to clinical translation. We also discuss how the first in human xenotransplants might be performed, recipients managed, and graft function monitored.

The growth of xenotransplanted hearts can be reduced with growth hormone receptor knockout pig donors.

OBJECTIVE: Genetically engineered pigs are thought to be an alternative organ source for patients in end-stage heart failure unable to receive a timely allograft. However, cardiac xenografts exhibit growth and diastolic heart failure within 1 month after transplantation. Grafts function for up to 6 months, but only after administration of temsirolimus and afterload-reducing agents to reduce this growth. In this study we investigated the growth and hemodynamics of growth hormone receptor (GHR) knockout xenografts, without the use of adjuncts to prevent intrinsic graft growth after transplantation. METHODS: Genetically engineered pig hearts were transplanted orthotopically into weight-matched baboons between 15 and 30 kg, using continuous perfusion preservation before implantation (n = 5). Xenografts included knockout of carbohydrate antigens and knockin of human transgenes for thromboregulation, complement regulation, and inflammation reduction (grafts with intact growth hormone, n = 2). Three grafts contained the additional knockout of GHR (GHR knockout grafts; n = 3). Transthoracic echocardiograms were obtained twice monthly and comprehensively analyzed by a blinded cardiologist. Hemodynamics were measured longitudinally after transplantation. RESULTS: All xenografts demonstrated life-supporting function after transplantation. There was no difference in intrinsic growth, measured using septal and posterior wall thickness and left ventricular mass, on transthoracic echocardiogram out to 1 month in either GHR knockout or GHR intact grafts. However, hypertrophy of the septal and posterior wall was markedly elevated by 2 months post transplantation. There was minimal hypertrophy out to 6 months in GHR knockout grafts. Physiologic mismatch was present in all grafts after transplantation, which is largely independent of growth. CONCLUSIONS: Xenografts with GHR knockout show reduced post-transplantation xenograft growth using echocardiography >6 months after transplantation, without the need for other adjuncts.

CD40-CD40L Blockade: Update on Novel Investigational Therapeutics for Transplantation.

Effective immune responses require antigen presentation by major histocompatibility complexes with cognate T-cell receptor and antigen-independent costimulatory signaling for T-cell activation, proliferation, and differentiation. Among several costimulatory signals, CD40-CD40L is of special interest to the transplantation community because it plays a vital role in controlling or regulating humoral and cellular immunity. Blockade of this pathway has demonstrated inhibition of donor-reactive T-cell responses and prolonged the survival of transplanted organs. Several anti-CD154 and anti-CD40 antibodies have been used in the transplantation model and demonstrated the potential of extending allograft and xenograft rejection-free survival. The wide use of anti-CD154 antibodies was hampered because of thromboembolic complications in transplant recipients. These antibodies have been modified to overcome the thromboembolic complications by altering the antibody binding fragment (Fab) and Fc (fragment, crystallizable) receptor region for therapeutic purposes. Here, we review recent preclinical advances to target the CD40-CD40L pair in transplantation.