Natural Killer Cell Presence in Antibody-Mediated Rejection.

Transcript analyses highlight an important contribution of natural killer (NK) cells to microvascular inflammation (MVI) in antibody-mediated rejection (ABMR), but only few immunohistologic studies have quantified their spatial distribution within graft tissue. This study included 86 kidney transplant recipients who underwent allograft biopsies for a positive donor-specific antibody (DSA) result. NK cells were visualized and quantified within glomeruli and peritubular capillaries (PTC), using immunohistochemistry for CD34 alongside CD16/T-bet double-staining. Staining results were analyzed in relation to histomorphology, microarray analysis utilizing the Molecular Microscope Diagnostic System, functional NK cell genetics, and clinical outcomes. The number of NK cells in glomeruli per mm2 glomerular area (NKglom) and PTC per mm2 cortical area (NKPTC) was substantially higher in biopsies with ABMR compared to those without rejection, and correlated with MVI scores (NKglom Spearman's correlation coefficient [SCC] = 0.55, p < 0.001, NKPTC 0.69, p < 0.001). In parallel, NK cell counts correlated with molecular classifiers reflecting ABMR activity (ABMRprob: NKglom 0.59, NKPTC 0.75) and showed a trend towards higher levels in association with high functional FCGR3A and KLRC2 gene variants. Only NKPTC showed a marginally significant association with allograft function and survival. Our immunohistochemical results support the abundance of NK cells in DSA-positive ABMR.

Glomerular endothelial cell-podocyte stresses and crosstalk in structurally normal kidney transplants.

Increased podocyte detachment begins immediately after kidney transplantation and is associated with long-term allograft failure. We hypothesized that cell-specific transcriptional changes in podocytes and glomerular endothelial cells after transplantation would offer mechanistic insights into the podocyte detachment process. To test this, we evaluated cell-specific transcriptional profiles of glomerular endothelial cells and podocytes from 14 patients of their first-year surveillance biopsies with normal histology from low immune risk recipients with no post-transplant complications and compared these to biopsies of 20 healthy living donor controls. Glomerular endothelial cells from these surveillance biopsies were enriched for genes related to fluid shear stress, angiogenesis, and interferon signaling. In podocytes, pathways were enriched for genes in response to growth factor signaling and actin cytoskeletal reorganization but also showed evidence of podocyte stress as indicated by reduced nephrin (adhesion protein) gene expression. In parallel, transcripts coding for proteins required to maintain podocyte adherence to the underlying glomerular basement membrane were downregulated, including the major glomerular podocyte integrin α3 and the actin cytoskeleton-related gene synaptopodin. The reduction in integrin α3 protein expression in surveillance biopsies was confirmed by immunoperoxidase staining. The combined growth and stress response of patient allografts post-transplantation paralleled similar changes in a rodent model of nephrectomy-induced glomerular hypertrophic stress that progress to develop proteinuria and glomerulosclerosis with shortened kidney life span. Thus, even among patients with apparently healthy allografts with no detectable histologic abnormality including alloimmune injury, transcriptomic changes reflecting cell stresses are already set in motion that could drive hypertrophy-associated glomerular disease progression.

Novel intragraft regulatory lymphoid structures in kidney allograft tolerance.

Intragraft events thought to be relevant to the development of tolerance are here subjected to a comprehensive mechanistic study during long-term spontaneous tolerance that occurs in C57BL/6 mice that receive life sustaining DBA/2 kidneys. These allografts rapidly develop periarterial Treg-rich organized lymphoid structures (TOLS) that form in response to class II but not to class I MHC disparity and form independently of lymphotoxin α and lymphotoxin ß receptor pathways. TOLS form in situ in the absence of lymph nodes, spleen, and thymus. Distinctive transcript patterns are maintained over time in TOLS including transcripts associated with Treg differentiation, T cell checkpoint signaling, and Th2 differentiation. Pathway transcripts related to inflammation are expressed in early stages of accepted grafts but diminish with time, while B cell transcripts increase. Intragraft transcript patterns at one week posttransplant distinguish those from kidneys destined to be rejected, that is, C57BL/6 allografts into DBA/2 recipients, from those that will be accepted. In contrast to inflammatory tertiary lymphoid organs (iTLOs) that form in response to chronic viral infection and transgenic Lta expression, TOLS lack high endothelial venules and germinal centers. TOLS represent a novel, pathogenetically important type of TLO that are in situ markers of regulatory tolerance.

Cutaneous leukocyte lineages in tolerant large animal and immunosuppressed clinical vascularized composite allograft recipients.

Vascularized composite allografts (VCAs) can restore fully functional anatomic units in patients with limb amputations or severe facial tissue loss. However, acute rejection of the skin is frequently observed and underscores the importance of developing tolerance induction protocols. In this study, we have characterized the skin immune system in VCAs. We demonstrate infiltration of recipient leukocytes, regardless of rejection status, and in tolerant mixed hematopoietic chimeras, the co-existence of these cells with donor leukocytes in the absence of rejection. Here we characterize the dermal T cell and epidermal Langerhans cell components of the skin immune system in our porcine model of VCA tolerance, and the kinetics of cutaneous chimerism in both of these populations in VCAs transplanted to tolerant and nontolerant recipients, as well as in host skin. Furthermore, in biopsies from the first patient to receive a hand transplant in our program, we demonstrate the presence of recipient T cells in the skin of the transplanted limb in the absence of clinical or histological evidence of rejection.

Ultrastructural Evidence for Direct Renal Infection with SARS-CoV-2.

BACKGROUND: A significant fraction of patients with coronavirus disease 2019 (COVID-19) display abnormalities in renal function. Retrospective studies of patients hospitalized with COVID-19 in Wuhan, China, report an incidence of 3%-7% progressing to ARF, a marker of poor prognosis. The cause of the renal failure in COVID-19 is unknown, but one hypothesized mechanism is direct renal infection by the causative virus, SARS-CoV-2. METHODS: We performed an autopsy on a single patient who died of COVID-19 after open repair of an aortic dissection, complicated by hypoxic respiratory failure and oliguric renal failure. We used light and electron microscopy to examine renal tissue for evidence of SARS-CoV-2 within renal cells. RESULTS: Light microscopy of proximal tubules showed geographic isometric vacuolization, corresponding to a focus of tubules with abundant intracellular viral arrays. Individual viruses averaged 76 µm in diameter and had an envelope studded with crown-like, electron-dense spikes. Vacuoles contained double-membrane vesicles suggestive of partially assembled virus. CONCLUSIONS: The presence of viral particles in the renal tubular epithelium that were morphologically identical to SARS-CoV-2, and with viral arrays and other features of virus assembly, provide evidence of a productive direct infection of the kidney by SARS-CoV-2. This finding offers confirmatory evidence that direct renal infection occurs in the setting of AKI in COVID-19. However, the frequency and clinical significance of direct infection in COVID-19 is unclear. Tubular isometric vacuolization observed with light microscopy, which correlates with double-membrane vesicles containing vacuoles observed with electronic microscopy, may be a useful histologic marker for active SARS-CoV-2 infection in kidney biopsy or autopsy specimens.

Intragraft gene expression in native kidney BK virus nephropathy versus T cell-mediated rejection: Prospects for molecular diagnosis and risk prediction.

Novel tools are needed to improve diagnostic accuracy and risk prediction in BK virus nephropathy (BKVN). We assessed the utility of intragraft gene expression testing for these purposes. Eight hundred genes were measured in 110 archival samples, including a discovery cohort of native kidney BKVN (n = 5) vs pure T cell-mediated rejection (TCMR; n = 10). Five polyomavirus genes and seven immune-related genes (five associated with BKVN and two associated with TCMR) were significantly differentially expressed between these entities (FDR < 0.05). These three sets of genes were further evaluated in samples representing a spectrum of BK infection (n = 25), followed by a multicenter validation cohort of allograft BKVN (n = 60) vs TCMR (n = 10). Polyomavirus 5-gene set expression reliably distinguished BKVN from TCMR (validation cohort AUC = 0.992), but the immune gene sets demonstrated suboptimal diagnostic performance (AUC ≤ 0.720). Within the validation cohort, no significant differences in index biopsy gene expression were identified between BKVN patients demonstrating resolution (n = 35), persistent infection (n = 14) or de novo rejection (n = 11) 6 months following a standardized reduction in immunosuppression. These results suggest that, while intragraft polyomavirus gene expression may be useful as an ancillary diagnostic for BKVN, assessment for concurrent TCMR and prediction of clinical outcome may not be feasible with current molecular tools.

Fatal Cryocrystalglobulinemia With Intravascular and Renal Tubular Crystalline Deposits.

Cryocrystalglobulinemia is a rare variant of cryoglobulinemia in which monoclonal immunoglobulins self-assemble into crystalline arrays. We report a case of a 53-year-old man who presented with systemic thrombotic microangiopathy causing multiorgan failure, including decreased kidney, lung, and gastrointestinal function; skin necrosis; and mental status changes. Skin and kidney biopsy specimens showed intravascular thrombi, along with intravascular, intratubular, and periglomerular crystalline deposits. Typical morphologic features of cryoglobulinemia, such as a leukocytoclastic vasculitis and pseudothrombi, were absent. Spindled crystals precipitated in the cryoglobulin assay, and immunofixation showed them to be composed of monoclonal immunoglobulin G κ light chains. Ultrastructural analysis demonstrated deposits to have an array-like substructure. The patient was successfully treated with a combination of plasmapheresis, steroids, and bortezomib, but experienced a relapse and died 12 months after his initial diagnosis. Cryocrystalglobulinemia causes significant morbidity and mortality and should be classified as a monoclonal gammopathy of renal significance when it occurs in patients not meeting diagnostic criteria for multiple myeloma.

Increased transfusion-free survival following auxiliary pig liver xenotransplantation.

BACKGROUND: Pig to baboon liver xenotransplantation typically results in severe thrombocytopenia and coagulation disturbances, culminating in death from hemorrhage within 9 days, in spite of continuous transfusions. We studied the contribution of anticoagulant production and clotting pathway deficiencies to fatal bleeding in baboon recipients of porcine livers. METHODS: By transplanting liver xenografts from α1,3-galactosyltransferase gene-knockout (GalT-KO) miniature swine donors into baboons as auxiliary organs, leaving the native liver in place, we provided the full spectrum of primate clotting factors and allowed in vivo mixing of porcine and primate coagulation systems. RESULTS: Recipients of auxiliary liver xenografts develop severe thrombocytopenia, comparable to recipients of conventional orthotopic liver xenografts and consistent with hepatic xenograft sequestration. However, baboons with both pig and native livers do not exhibit clinical signs of bleeding and maintain stable blood counts without transfusion for up to 8 consecutive days post-transplantation. Instead, recipients of auxiliary liver xenografts undergo graft failure or die of sepsis, associated with thrombotic microangiopathy in the xenograft, but not the native liver. CONCLUSION: Our data indicate that massive hemorrhage in the setting of liver xenotransplantation might be avoided by supplementation with primate clotting components. However, coagulation competent hepatic xenograft recipients may be predisposed to graft loss related to small vessel thrombosis and ischemic necrosis.

Early acceptance of renal allografts in mice is dependent on foxp3(+) cells.

Mouse renal allografts have a remarkable ability to promote acceptance across full major histocompatibility complex incompatibilities in certain strain combinations without immunosuppression. The mechanism is unknown but is believed to involve immunoregulation. This study tests whether Foxp3(+) T-regulatory cells are responsible in the early phase of graft acceptance, using B6.Foxp3(DTR) mice that express diphtheria toxin receptor (DTR) in Foxp3(+) cells. The administration of DT to B6.Foxp3(DTR) recipients with accepted DBA/2 kidneys, 3 weeks to 3 months after transplantation, caused a marked depletion of Foxp3 cells and triggered acute cellular rejection, manifested by a sudden increase in blood urea nitrogen within a week. None of the controls showed an increase in blood urea nitrogen, including DT-treated B6 wild-type recipients of DBA/2 kidneys or B6.Foxp3(DTR) recipients of isografts. Accepted DBA/2 allografts showed prominent lymphoid sheaths around arteries containing numerous CD3(+)Foxp3(+) cells, CD4(+) cells, dedritic cells, and B cells, which was independent of CCR4. The lymphoid sheaths disintegrate after Foxp3 depletion, accompanied by widespread CD8 interstitial mononuclear inflammation, tubulitis, and endarteritis. The Foxp3 depletion caused an increased frequency of donor-reactive cells in the spleen by interferon (IFN) γ enzyme-linked immunosorbent spot (ELISPOT) assays and increased expression of the maturation markers, CD86 and IA(b), on dendritic cells in the spleen and kidney. We conclude that Foxp3(+) cells are needed to maintain acceptance of major histocompatibility complex-incompatible renal allografts in the first 3 months after transplantation and may act by inhibiting DC maturation.

Disseminated Intravascular Coagulation During Induction Treatment With Rabbit-Derived Antithymocyte Globulin For Kidney Transplant.

Rabbit antithymocyte globulin is a lymphocytedepleting agent commonly used as induction therapy in kidney transplants. Although its use is generally safe and well tolerated, serious side effects can occur. Here, we describe a case of a severe immune complex hypersensitivity reaction with disseminated intravascular coagulation in response to rabbit antithymocyte globulin infusion. Immediate treatment required return to the operating room, massive transfusion of blood products, and plasmapheresis. The patient's posttransplant course was significant for volume overload, prolonged respiratory failure, and delayed graft function that required hemodialysis, but within 10 weeks the patient had made a full recovery and kidney allograft function had returned to normal.

The lupus susceptibility allele DRB1*03:01 encodes a disease-driving epitope.

The HLA-DRB1*03:01 allele is a major genetic risk factor in systemic lupus erythematosus (SLE), but the mechanistic basis of the association is unclear. Here we show that in the presence of interferon gamma (IFN-γ), a short DRB1*03:01-encoded allelic epitope activates a characteristic lupus transcriptome in mouse and human macrophages. It also triggers a cascade of SLE-associated cellular aberrations, including endoplasmic reticulum stress, unfolded protein response, mitochondrial dysfunction, necroptotic cell death, and production of pro-inflammatory cytokines. Parenteral administration of IFN-γ to naïve DRB1*03:01 transgenic mice causes increased serum levels of anti-double stranded DNA antibodies, glomerular immune complex deposition and histopathological renal changes that resemble human lupus nephritis. This study provides evidence for a noncanonical, antigen presentation-independent mechanism of HLA-disease association in SLE and could lay new foundations for our understanding of key molecular mechanisms that trigger and propagate this devastating autoimmune disease.

CD38 Antibody Daratumumab for the Treatment of Chronic Active Antibody-mediated Kidney Allograft Rejection.

BACKGROUND: Late antibody-mediated rejection (AMR) is a major cause of transplant failure. Potential therapeutic targets are plasma cells and natural killer (NK) cells, both expressing high levels of CD38. METHODS: Here, we report the use of CD38 monoclonal antibody daratumumab (9-mo course) in a kidney allograft recipient diagnosed with smoldering myeloma and anti-HLA class II donor-specific antibody-positive chronic active AMR 13 years after transplantation. Patient monitoring included serial HLA single-antigen testing, peripheral blood immune cell phenotyping, as well as follow-up allograft and bone marrow biopsies at 3 and 9 months, including analyses of rejection-related gene expression patterns. RESULTS: Daratumumab led to persistent CD138+ cell depletion in the bone marrow and blood and substantially decreased NK cells counts in blood and graft tissue. At the same time, donor-specific antibody in serum disappeared, and in vitro alloantibody production by CD138+ cells enriched from bone marrow aspirates was abrogated. A 3-month follow-up biopsy revealed a complete resolution of microcirculation inflammation (g+ptc: 3 to 0) and molecular AMR activity (AMR score: 0.79 to <0.2). The same biopsy showed (subclinical) tubulointerstitial inflammation, which prompted steroid treatment. Over an observation period of 12 months, graft function stabilized. CONCLUSIONS: Targeting CD38 for plasma cell and NK cell depletion may be an effective strategy to counteract AMR. Our results may encourage the design of future trials to clarify the role of this innovative treatment concept in organ transplantation.

Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide.

Neutrophil-mediated activation and injury of the endothelium play roles in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap-derived [NET-derived] histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the US Food and Drug Administration-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.

TNFRSF13B genotypes control immune-mediated pathology by regulating the functions of innate B cells.

Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNF receptor superfamily member 13B (TNFRSF13B) variants, which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells' functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of those with antibody-mediated rejection (AMR) but fewer than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses, we investigated alloimmunity and rejection in mice. Cardiac allografts in Tnfrsf13b-mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b-deficient mice were not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased "natural" IgM and compromised complement regulation, leading to complement deposition in allografted hearts and autogenous kidneys. Thus, WT TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhance morbidity associated with immune responses.

Endothelium-protective, histone-neutralizing properties of the polyanionic agent defibrotide.

Neutrophil-mediated activation and injury of the endothelium play a role in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap/NET-derived histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the FDA-approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro , defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo , defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.

Inhibition of EZH2 Ameliorates Lupus-Like Disease in MRL/lpr Mice.

OBJECTIVE: We previously identified a role for EZH2, a transcriptional regulator in inducing proinflammatory epigenetic changes in lupus CD4+ T cells. This study was undertaken to investigate whether inhibiting EZH2 ameliorates lupus-like disease in MRL/lpr mice. METHODS: EZH2 expression levels in multiple cell types in lupus patients were evaluated using flow cytometry and messenger RNA expression data. Inhibition of EZH2 in MRL/lpr mice was achieved by intraperitoneal 3'-deazaneplanocin (DZNep) administration using a preventative and a therapeutic treatment model. Effects of DZNep on animal survival, anti-double-stranded DNA (anti-dsDNA) antibody production, proteinuria, renal histopathology, cytokine production, and T and B cell numbers and percentages were assessed. RESULTS: EZH2 expression levels were increased in whole blood, neutrophils, monocytes, B cells, and CD4+ T cells in lupus patients. In MRL/lpr mice, inhibition of EZH2 by DZNep was confirmed by significant reduction of EZH2 and H3K27me3 in splenocytes. Inhibiting EZH2 with DZNep treatment before or after disease onset improved survival and significantly reduced anti-dsDNA antibody production. DZNep-treated mice displayed a significant reduction in renal involvement, splenomegaly, and lymphadenopathy. Lymphoproliferation and numbers of double-negative T cells were significantly reduced in DZNep-treated mice. Concentrations of circulating cytokines and chemokines, including tumor necrosis factor, interferon-γ, CCL2, RANTES/CCL5, interleukin-10 (IL-10), keratinocyte-derived chemokine/CXCL1, IL-12, IL-12p40, and CCL4/macrophage inflammatory protein 1ß, were decreased in DZNep-treated mice. CONCLUSION: EZH2 is up-regulated in multiple cell types in lupus patients. Therapeutic inhibition of EZH2 abrogates lupus-like disease in MRL/lpr mice, suggesting that EZH2 inhibitors may be repurposed as a novel therapeutic option for lupus patients.