Genetically modified pigs are being developed to address the critical shortage of human organs for transplantation. Although porcine xenografts lacking the three major carbohydrate xenoantigens (3KO) have been considered ideal for human transplantation, the optimal combination of human transgenes (HTGs) to mitigate protein incompatibility remains undefined. In the current study, we evaluate immune responses and transplant outcomes of 3KO kidney xenografts with four different combinations of HTGs in a nonhuman primate model. Here, we show that the addition of HTGs significantly reduces transcripts associated with early immune activation, resulting in markedly prolonged survival of 3KO xenografts. Notably, the inclusion of the anti-inflammatory genes TNFAIP3 and HMOX1 is associated with improved graft survival, significantly reduced T-cell and CD11b⁺ myeloid cell infiltration, and lower expression of rejection-related gene sets in protocol xenograft biopsies.

African American (AA) kidney transplant recipients exhibit a higher rate of graft loss compared with other racial and ethnic populations, highlighting the need to identify causative factors. Here, in the Genomics of Chronic Allograft Rejection cohort, pretransplant blood RNA sequencing revealed a cluster of four consecutive missense single-nucelotide polymorphisms (SNPs), within the leukocyte immunoglobulin-like receptor B3 (LILRB3) gene, strongly associated with death-censored graft loss. This SNP cluster (named LILRB3-4SNPs) encodes missense mutations at amino acids 617-618 proximal to a SHP1/2 phosphatase-binding immunoreceptor tyrosine-based inhibitory motif. The LILRB3-4SNPs cluster is specifically enriched within AA individuals and exhibited a strong association with death-censored graft loss and estimated glomerular filtration rate decline in the AA participants from multiple transplant cohorts. In two large Biobanks (BioMe and All-of-Us), the LILRB3-4SNPs cluster was associated with the early onset of end-stage renal disease and acted synergistically with the apolipoprotein L1 (APOL1) G1/G2 allele to accelerate disease progression. The SNPs were also linked to multiple immune-related diseases in AA individuals. Last, on multiomics analysis of blood and biopsies, recipients with LILRB3-4SNPs showed enhanced inflammation and monocyte ferroptosis. While larger and prospective studies are needed, our data provide insights on the genetic variation underlying kidney transplant outcomes.

INTRODUCTION:Chronic active antibody-mediated rejection (CAMR) is a leading cause of late kidney allograft dysfunction, characterized by transplant glomerulopathy involving glomerular endothelial cells (GEC), natural killer (NK) cells, and macrophages. Mechanistic understanding remains limited due to the inability of bulk and single-cell RNA techniques to capture spatial molecular interactions. METHODS:Using spatial transcriptomics at single-cell resolution (CosMx Spatial Molecular Imager), we analyzed formalin fixed paraffin embedded kidney biopsies with CAMR and compared with controls without rejection. RESULTS:Cell typing, clustering, and dimensionality reduction identified 36 reference cell types, while selectively localizing GEC subtypes, NK cells, and macrophages in glomeruli. Cellular differential gene expression (DGE), cell proximity, and cell-to-cell ligand-receptor analyses elucidated candidate CAMR molecular mechanisms. Spatially resolved single-cell transcriptomics revealed upregulation of intraglomerular NK cell and macrophage genes in CAMR related to cytotoxicity, IgG Fc receptor and non-self-recognition. GEC subtypes developed distinctive transcript phenotypes that included upregulated genes related to complement protection, the MHC target of donor specific antibodies and IFNγ pathway and downregulation of sialyltransferase involved in protective glycocalyx synthesis and vascular integrity. Proximity of NK cells and macrophages with GEC revealed several potential ligand receptor interactions previously unappreciated, including GEC IL33→NK cell IL1RL1 and GEC HLA-DQA1→Macrophage FCGR3A, implicating NK cell and macrophage activation in endothelial injury. CONCLUSIONS:High-resolution spatial transcriptomics provided novel and confirmatory insights into CAMR pathogenesis, highlighting cell-specific activation states, molecular interactions, and potential mechanistic pathways involving GECs, NK cells, and macrophages. Our findings advance understanding of CAMR and identify molecular targets for further investigation.

Long-term, immunosuppression-free allograft survival has been induced in human and nonhuman primate (NHP) kidney recipients after nonmyeloablative conditioning and donor bone marrow transplantation (DBMT), resulting in transient mixed hematopoietic chimerism. However, the same strategy has consistently failed in NHP heart transplant recipients. Here, we investigated whether long-term heart allograft survival could be achieved by cotransplanting kidneys from the same donor. Cynomolgus monkeys were transplanted with heart allografts alone or heart and kidney allografts from the same major histocompatibility complex (MHC)–mismatched donors. All animals except one received DBMT, either at the same time or after a 2- to 4-month delay, plus short-term costimulation blockade and calcineurin inhibitor treatment. Long-term, immunosuppression-free heart allograft survival was consistently achieved in heart/kidney, but not heart-alone, recipients. This was not associated with greater donor/recipient histocompatibility or altered lymphoid cell reconstitution after conditioning. The maintenance of tolerance after heart/kidney transplantation was associated with the presence of forkhead box P3 (Foxp3 + ) regulatory T cell (T reg )–rich organized lymphoid structures in kidneys but not hearts. Substituting high-dose erythropoietin treatment for kidney transplantation was unsuccessful, suggesting that it was not the sole mechanism of action. RNA sequencing analysis revealed that gene expression in hearts from tolerant recipients closely resembled that in hearts from chronically immunosuppressed recipients but differed markedly from rejecting allografts and naïve hearts. A version of this protocol may be able to induce tolerance in patients with end-stage heart and kidney failure who require combined heart and kidney transplantation.

The purpose of this white paper is to recommend the minimum reporting standards for pathologic characterization of kidney and heart xenografts in humans. This proposal is based on the current human classifications for kidney and heart allografts, with additions and caveats relevant to xenografts that are primarily based on non-human primate and a limited number of organ xenografts in decedent and living humans. Such recommendations should not be regarded as diagnostic criteria, given that many new pathologic patterns and mechanisms remain to be fully characterized. While xenograft transplantation continues to evolve, this report serves as groundwork and an initial step towards defining international standards in xenograft histopathology assessment and reporting. Note: The authors welcome comments from the international transplantation community through the AJT Banff Blog: https://amjtransplant.wixsite.com/ajtbanffblog

Kidney diseases characterized by glomerular epithelial cell proliferation are rare but often devastating, frequently leading to progressive scarring and renal failure. Ranging from autoimmune-induced crescentic glomerulonephritis to HIV infection-induced collapsing glomerulopathy, these diseases are triggered by a wide variety of insults and have generally been thought of as different entities. Here, using immunostaining and spatial transcriptomics, we profiled human kidney biopsies collected from patients with two of these diseases, collapsing glomerulopathy and antineutrophil cytoplasmic antibody (ANCA) vasculitis-induced crescentic glomerulonephritis, to identify common disease-causing molecules. Although triggered by different insults, we identified abnormal hyperactivation of the transcription cofactors Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) in podocytes as a potential common driver of these diseases. To test this hypothesis, we genetically activated podocyte YAP and TAZ in cultured human cells and in mice by deleting the YAP and TAZ inhibitory large tumor suppressor kinases (LATSs). LATS deficiency in mouse podocytes induced a phenotypic transition in vitro, characterized by a highly distorted structure and an increase in matrix gene expression, mimicking many features of the podocytopathy seen in diseases characterized by glomerular epithelial proliferation. In mice, LATS-deficient podocytes orchestrated a profibrotic and pro-proliferative response in surrounding glomerular cells, a characteristic phenomenon of glomerular epithelial proliferative diseases. This response was attenuated when we also deleted podocyte YAP or TAZ in these mice. Together, our findings point to podocyte YAP-TAZ hyperactivation as a previously unrecognized and unifying driver of glomerular epithelial proliferative diseases.

BACKGOURND:Peritransplant ischemia/reperfusion injury (IRI) plays a central pathogenic role in nondelayed or delayed kidney allograft function immediately after transplantation and increases the risk of subsequent rejection. Potential therapies targeting specific cytokines or complement proteins to limit IRI have failed in clinical trials. Monoclonal antibody 107 (mAb107), a "pure" (nonactivating) inhibitor of the archetypal innate immune receptor integrin CD11b, has been shown to extend the survival of IRI nonhuman primate native kidneys in an in situ model. METHODS:Here, we administered mAb107 before allograft revascularization to determine its efficacy for extending the survival of ischemia-damaged donor kidneys transplanted into major histocompatibility complex-mismatched nonhuman primate recipients. RESULTS:We observed a significant delay in the onset of rejection and prolongation of allograft survival in mAb107-treated versus control recipients. Early allograft biopsies suggest this is secondary to the selective suppression of infiltrating neutrophils and macrophages. CONCLUSIONS:These observations support the hypothesis that inactivating CD11b with mAb107 may provide an effective strategy for prolonging the survival of ischemia-damaged allografts and increasing the successful use of marginal donor organs.

Xenotransplantation offers a potential solution to the organ shortage crisis. A 62-year-old hemodialysis-dependent man with long-standing diabetes, advanced vasculopathy, and marked dialysis-access challenges received a gene-edited porcine kidney with 69 genomic edits, including deletion of three glycan antigens, inactivation of porcine endogenous retroviruses, and insertion of seven human transgenes. The xenograft functioned immediately. The patient's creatinine levels decreased promptly and progressively, and dialysis was no longer needed. After a T-cell-mediated rejection episode on day 8, intensified immunosuppression reversed rejection. Despite sustained kidney function, the patient died from unexpected, sudden cardiac causes on day 52; autopsy revealed severe coronary artery disease and ventricular scarring without evident xenograft rejection. (Funded by Massachusetts General Hospital and eGenesis.).

Transcriptomic analysis of kidney biopsies has demonstrated the potential to improve diagnosis of allograft rejection. Here, we developed a molecular assessment of antibody-mediated rejection (AMR) and T cell-mediated rejection (TCMR) based on the Banff Human Organ Transplant consensus gene panel. Expression assays of formalin-fixed paraffin-embedded kidney biopsies from well-phenotyped cohorts were used to develop prediction models for AMR and TCMR and an automated report of gene expression-based diagnosis. The study population consisted of 950 kidney allograft biopsies from 10 transplantation centers in Europe and North America. The development cohort included 664 renal allograft biopsies split into a training (n = 537) and test set (n = 127), and 2 external validation cohorts (n = 286). We performed gene selection using regularized regression and developed several different base models based on Banff Human Organ Transplant expression data, which were combined into a single ensemble model for each rejection diagnosis. Model performance was assessed in the test set and the 2 external validation cohorts, showing good discriminative abilities (respective areas under the precision-recall curve: AMR = 0.811, 0.891, and 0.832 and TCMR = 0.736, 0.810, and 0.782). We identified challenging biopsies with histology below diagnostic thresholds for which gene expression-based probability can refine rejection diagnosis. This automated molecular diagnostic system shows potential for improving kidney allograft rejection diagnosis in routine practice and clinical trials.

BACKGROUND:This study evaluated the efficacy of an optimized immunosuppressive regimen, including a higher dose of anti-CD154 monoclonal antibody (TNX-1500), in prolonging graft survival in pig-to-baboon kidney xenotransplantation. Despite advances in genetic engineering of organ-source pigs and immunosuppressive regimens, we report that the development of nephrotic-range proteinuria remains a significant problem. METHODS:We assessed the TNX-1500-based immunosuppressive regimen in 9 baboon recipients of gene-edited pig kidney transplants. The regimen included induction with antithymocyte globulin, anti-CD20 monoclonal antibody (rituximab), and C1 esterase inhibitor, and maintenance with TNX-1500, rapamycin, methylprednisolone, and anti-interleukin-6 receptor blockade (tocilizumab). RESULTS:Although an increased dose of TNX-1500 of 30 mg/kg (higher dose, n = 6) versus 20 mg/kg (lower dose, n = 3) improved overall survival (median 214 versus 86 d; P  < 0.05), 4 of 9 baboons (including 3 with higher dose) developed persistent nephrotic-range proteinuria. The histopathology of these 4 grafts revealed focal glomerular thrombotic microangiopathy (4/4), transplant glomerulopathy (3/4), focal segmental glomerulosclerosis (2/4), and/or membranous nephropathy (1/4) but no definitive features of rejection. Proteinuria was associated with decreased serum albumin and, very importantly, with loss of TNX-1500 in the urine. CONCLUSIONS:Despite the efficacy of the immunosuppressive regimen, we suggest that proteinuria may lead to inadequate immunosuppressive therapy through loss of the therapeutic antibody, thus increasing the risk of rejection. Further research is needed to develop strategies to prevent this complication.

Background Acute tubulointerstitial nephritis (AIN) is one of the few causes of acute kidney injury with diagnosis-specific treatment options. However, due to the need to obtain a kidney biopsy for histological confirmation, AIN diagnosis can be delayed, missed, or incorrectly assumed. Here, we identify and validate urinary CXCL9, an IFN-γ-induced chemokine involved in lymphocyte chemotaxis, as a diagnostic biomarker for AIN.Methods In a prospectively enrolled cohort with pathologist-adjudicated histological diagnoses, termed the discovery cohort, we tested the association of 180 immune proteins measured by an aptamer-based assay with AIN and validated the top protein, CXCL9, using sandwich immunoassay. We externally validated these findings in 2 cohorts with biopsy-confirmed diagnoses, termed the validation cohorts, and examined mRNA expression differences in kidney tissue from patients with AIN and individuals in the control group.Results In aptamer-based assay, urinary CXCL9 was 7.6-fold higher in patients with AIN than in individuals in the control group (P = 1.23 × 10–5). Urinary CXCL9 measured by sandwich immunoassay was associated with AIN in the discovery cohort (n = 204; 15% AIN) independently of currently available clinical tests for AIN (adjusted odds ratio for highest versus lowest quartile: 6.0 [1.8–20]). Similar findings were noted in external validation cohorts, where CXCL9 had an AUC of 0.94 (0.86–1.00) for AIN diagnosis. CXCL9 mRNA expression was 3.9-fold higher in kidney tissue from patients with AIN (n = 19) compared with individuals in the control group (n = 52; P = 5.8 × 10–6).Conclusion We identified CXCL9 as a diagnostic biomarker for AIN using aptamer-based urine proteomics, confirmed this association using sandwich immunoassays in discovery and external validation cohorts, and observed higher expression of this protein in kidney biopsies from patients with AIN.Funding This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) awards K23DK117065 (DGM), K08DK113281 (KM), R01DK128087 (DGM), R01DK126815 (DGM and LGC), R01DK126477 (KNC), UH3DK114866 (CRP, DGM, and FPW), R01DK130839 (MES), and P30DK079310 (the Yale O’Brien Center). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory tertiary lymphoid organs (rTLOs). Single-cell RNA-seq (scRNA-seq) data and adoptive transfer of alloreactive T cells after transplantation showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required because adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite the presence of intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8-KO recipients resulted in acceptance and not rejection. Analysis of scRNA-seq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call “defensive tolerance.” This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.