INTRODUCTION:Biological parental donations provide the best option for many pediatric recipients, yielding unique immunological benefits that may enable minimization of immunosuppression in transplanted children. However, living related maternal donation in the postpartum period may introduce an increased risk of donor complications due to the physiological changes of pregnancy and childbirth, and the optimal timing of postpartum living donation is unknown. METHODS:Using US national registry data, we characterized donor and recipient outcomes for pediatric living donor liver transplants performed between 2004 and 2022 where a biological mother donated to a child ≤ 24 months old. RESULTS:Our study population included 256 donor-recipient pairs, with biliary atresia representing the most common indication for transplantation (68.0%). Donors had a median [IQR] age of 30 [25, 34] years, and the median [IQR] time from birth to donation was 9.0 [6.8, 13.0] months. 6.3% of donors experienced a biliary or other complication. When stratifying by donors who donated ≤ 6 vs. > 6 months postpartum, we found no significant differences in donor complications or readmission. Stratified analyses were also comparable for recipient mortality, graft survival, and rejection-free survival. Donors ≤ 6 months postpartum (n = 64) were more likely to experience reoperation than mothers who donated > 6 months postpartum (n = 192) (6.2% vs. 1.0%, p = 0.04). CONCLUSIONS:While maternal living donor liver transplantation is safe for most donors, there is a higher risk of reoperation when donation is performed ≤ 6 months postpartum. Surgeons should be aware that these donors are a higher risk population, requiring discussion upon consent and warranting close post-operative monitoring.

Abstract Introduction Xenotransplantation of pig organs is a promising strategy to address the organ shortage; however, rejection poses a barrier to its widespread adoption. Pig-to-decedent human transplantation provides an opportunity to study immune barriers to xenotransplantation. We followed donor-reactive T cell dynamics in a long-term (61-day) pig-to-decedent human kidney Methods We developed a method to identify xeno donor-reactive T cell clones (XDRTCCs) by TCRB CDR3 sequencing and tracked them in the circulation and xenograft. scRNA and TCR sequencing of human leukocytes isolated from the kidney graft were used to investigate the functional phenotypes of XDRTCCs infiltrating the kidney as well as additional immune responses against the xenograft. Results We observed an expansion of both CD4 and CD8 XDRTCCs in the circulation. However, CD8 XDRTCCs demonstrated more striking expansions, constituting 10% of the total CD8 repertoire at POD28 and about 30% at POD33 and POD49, both of which were established rejection timepoints. Normalizing the total quantity of circulating XDRTCCs post-transplant to their pre-transplant baseline revealed clear XDRTCC enrichment, with increases up to 11-fold at POD49 for CD8 cells. scRNA sequencing data on graft-infiltrating leukocytes revealed RNA species characteristic of effector T cells and NK cells and APCs only in the POD33 rejecting kidney specimen. Interrogation of scRNA/TCR seq data for XDRTCCs revealed infiltrating XDRTCCs that expressed some effector genes such as GZMA, GZMB and PRF1 as well as the costimulatory molecule ICOS and some coinhibitory molecules, including PDCD1, LAG3, CTLA4, and TIGIT. Conclusion The findings provide insight into the clonal dynamics of xenoreactive T cells and their contribution to rejection in xenotransplantation and demonstrates the importance of tracking xenoreactive T cell repertoires to better understand immune responses in xenotransplantation and suggests that specific T cell clones might serve as biomarkers for rejection monitoring. Funding Source n/a Topic Categories Transplantation Immunology (TRAN)

Abstract Introduction Porcine thymic transplantation tolerizes xenogeneic recipients to the porcine source animal in animal models. A 54-year-old woman with complex comorbidities received a composite thymokidney transplant from an GGTA1 knockout pig, achieving initial renal function before xenograft failure for non-immunological reasons on postoperative day (POD) 47. Methods Serial peripheral blood mononuclear cell (PBMC) samples were analyzed by spectral FCM, and mixed lymphocyte reactions (MLRs) were performed on purified T cells to assess tolerance. Thymocytes were isolated from the thymic portion of the xenograft explant and analyzed using spectral FCM Results T cell levels in peripheral blood declined to 10/µL post-induction therapy and recovered to a peak of 342/µL by POD28. By POD21, 53% of the recovering CD4 T cells displayed a CD45RA+CCR7+ “naïve” phenotype and expressed CD31, consistent with recent thymic emigrants (RTEs). Pre-transplant CD4 T cells included 20.8% RTEs (Figure 1A). FCM analysis on cells from the POD47 thymic xenograft revealed human CD45+ leukocytes (0.53%), including double-positive (CD4+CD8+), single-positive, and double-negative thymocytes. The double-negative cells expressed markers such as CD5, CD7, CD1a, and CD34, consistent with human T cell progenitors in the porcine thymus (Figure 1B). MLR results demonstrated progressive hyporesponsiveness toward the source pig with preserved responses to third-party pig and allogeneic human cells, achieving full donor-specific unresponsiveness by POD47 and POD86 (Figure 1C). Conclusion These findings suggest that a porcine thymus in a thymokidney xenograft supported human thymopoiesis and the development of T cells tolerant to the pig. Thymic transplantation is a promising approach to achieving T cell tolerance in xenotransplant recipients Funding Source n/a Topic Categories Transplantation Immunology (TRAN)

Objective Little is known about the impact of prolonged brain death on myocardial function and histopathology. The purpose of this study was to determine the echocardiographic and histopathologic sequelae of prolonged brain death in a decedent kept on somatic support for two months. Methods Periodic cardiac monitoring was performed on one brain-dead decedent who underwent experimental kidney xenotransplantation. The decedent was a 57-year-old male with a history of grade 4 glioblastoma multiforme and no known cardiac history. The decedent was monitored for a total of 61 days. Hemodynamics and inotrope infusions were tracked over the study period. Serial transthoracic echocardiography (TTE) was performed to evaluate cardiac function. At the conclusion of the study, histopathologic evaluation was performed on the decedent heart and epicardial coronary arteries to evaluate pathologic sequelae of prolonged brain death. Results Throughout the study, the decedent remained hemodynamically stable in normal sinus rhythm on serial electrocardiograms without evidence of ischemia, conduction disease, and with minimal inotropic or vasopressor requirements (Table 1). Three TTEs were performed to evaluate cardiac function. The first, performed pre-operatively, showed normal septal and posterior wall thickness. In subsequent TTEs, these parameters remained stable with improved LVEDD from 4.9 to 4.1 cm and normal right ventricular function without significant valvular abnormalities (Table 2). There was diffuse subendocardial myocyte vacuolization and mild myocyte hypertrophy (Figure 1a). The coronary arteries showed significant atherosclerosis with moderate to severe luminal stenosis, and a fibroatheromatous plaque in the circumflex artery had evidence of rupture without occlusive luminal thrombus (Figure 1b). There was no evidence of changes (interstitial hemorrhage, mononuclear infiltrates, contraction band necrosis, or coagulative necrosis) that are typically seen acutely after brain death. Conclusions Prolonged brain death requiring somatic support did not adversely affect native cardiac function and was not associated with any histopathologic changes in the myocardium. These findings serve to support the decedent model in evaluating xenotransplantation therapies for prolonged periods, and may serve as a bridge to potential future clinical trials.

Organ shortage remains a major challenge in transplantation, and gene-edited pig organs offer a promising solution1-3. Despite gene editing, the immune reactions following xenotransplantation can still cause transplant failure4. To understand the immunological response of a pig-to-human kidney xenotransplantation, we conducted large-scale multi-omics profiling of the xenograft and the host's blood over a 61-day procedure in a brain-dead human (decedent) recipient. Blood plasmablasts, natural killer cells and dendritic cells increased between postoperative day (POD) 10 and 28, concordant with an expansion of IgG and IgA B cell clonotypes and subsequent biopsy-confirmed antibody-mediated rejection (AMR) at POD33. Human T cell frequencies increased from POD14 and peaked between POD33 and POD49 in the blood and xenograft, which coincided with T cell receptor diversification, expansion of a restricted TRBV2 and TRBJ1 clonotype and histological evidence of combined AMR and cell-mediated rejection at POD49. At POD33, the most abundant human immune population in the graft was CXCL9+ macrophages, which aligned with interferon-γ-driven inflammation and a T helper 1-type immune response. There was also evidence of interactions between activated pig-resident macrophages and infiltrating human immune cells. Xenograft tissue showed pro-fibrotic tubular and interstitial injury marked by S100A6 (ref. 5), SPP1 (also known as osteopontin)6 and COLEC11 (ref. 7) expression at POD21-POD33. Proteomic profiling revealed activation of human and pig complement, with a decreased human component after AMR therapy, in which complement was inhibited. Collectively, these data delineate the molecular orchestration of human immune responses to a porcine kidney and reveal potential immunomodulatory targets for improving xenograft survival.

BACKGROUND:Porcine genome editing has revolutionized xenotransplantation, recently enabling the first pig-to-human heart xenotransplants. However, the xenoimmune response in heart xenografts remains largely unexplored. This study aimed to precisely characterize the xenoimmune response and injury in 2 heart xenografts transplanted from 10-gene-edited pigs into brain-dead human recipients. METHODS:We analyzed xenograft biopsy specimens 66 hours after reperfusion using a multimodal phenotyping approach combining morphological evaluation, immunophenotyping, ultrastructural assessment, automated quantification of multiplex immunofluorescence staining, and gene expression profiling. Xenografts before implantation and wild-type pig hearts with and without ischemia/reperfusion injury and brain death were used as controls. RESULTS:Both xenografts showed evidence of endothelial activation and mild microvascular inflammation without capillary C4d deposition. Immune infiltrates were mainly composed of CD15+ and CD68+ innate immune cells. Ultrastructural assessment showed endothelial swelling with occasional intravascular leucocytes. Deep learning-based automated multiplex immunofluorescence analysis confirmed that microvascular inflammation was primarily associated with CD15+ and CD68+ innate immune cells. Both xenografts showed increased expression of genes and pathways associated with monocyte/macrophage activation, neutrophil activation, interferon-gamma response, natural killer cell burden, endothelial activation, apoptosis, and injury repair. This phenotype was absent in all control pig hearts independent of ischemia/reperfusion injury and brain death. CONCLUSIONS:Multimodal phenotyping of pig-to-human heart xenografts revealed early signs of xenoimmune response, characterized by mild innate microvascular inflammation, endothelial activation, and a molecular signature characteristic of antibody-mediated rejection. Developing such a precision diagnostic system could improve graft monitoring in future clinical settings.

Xenotransplantation of pig organs is a promising solution to the organ shortage; however, rejection remains a major obstacle. Pig-to-human decedent transplantation provides an opportunity to study immune barriers to xenotransplantation experimentally. We tracked donor-reactive T cell dynamics in a 61-day pig-to-human decedent thymokidney xenotransplant. Xenogeneic donor-reactive T cell clones (XDRTCCs) identified using high-throughput TCRB CDR3 sequencing expanded markedly in peripheral blood in association with apparent antibody-mediated rejection (AMR). Single-cell RNA and TCR sequencing of leukocytes from the xenograft showed XDRTCC infiltration and effector transcript expression during AMR. Additionally, γδ and NK cells with cytotoxic effector phenotypes were prominent in the rejecting xenograft. These data suggest that improved suppression of innate immunity and T cell responses might enhance the success of xenotransplantation.

This manuscript aims to examine key scientific milestones leading to human decedent studies and the compassionate use of cardiac xenografts in human recipients. It also evaluates the peri-operative considerations necessary before initiating large-scale clinical trials. The early functionality of cardiac xenografts in humans was initially demonstrated in two brain-dead human decedents through the transplantation of 10-gene-edited hearts from porcine donors. Subsequently, two living human patients received heart xenografts from similarly constructed pigs. Although both patients developed diastolic heart failure and subsequently lost xenograft function, significant insights were gained regarding patterns of immune rejection and strategies to prevent similar outcomes in the future. This review consolidates findings from pre-clinical (non-human primate and decedent) and initial human cardiac xenotransplant studies, emphasizing the peri-operative considerations required before proceeding with clinical trials.