BackgroundThe mouse heterotopic heart transplant model is a widely used system for dissecting mechanisms of graft rejection and identifying new therapeutic targets, owing to its relative technical simplicity and the availability of well-defined inbred strains. One limitation of the model is that monitoring of the non-life sustaining implanted graft by manual palpation does not provide information regarding the magnitude of the anti-donor immune response and is operator-dependent. Building on previous studies suggesting that trans-abdominal ultrasonography can detect graft rejection more precisely than manual palpation, we tested if changes in motion mode (M-mode) ultrasound also correlated with the magnitude of the anti-donor T cell response.MethodsWe performed sets of MHC-mismatched heterotopic heart transplants in mice and quantified the accuracy of palpation vs. ultrasonography in determining graft rejection. In an additional set of mice, we performed mixed lymphocyte reactions to quantify anti-donor T cell immunity at a fixed time point (usual experimental design) or at time points determined by changes in allograft M-mode analysis.ConclusionsThe data demonstrate that ultrasonography is superior to manual palpation for determining graft rejection and newly shows that changes in M-mode associate with the magnitude of anti-donor T cell immunity. These findings are important to increase reproducibility and translational impact of studies using this model.

Introduction IgA nephropathy (IgAN), the most prevalent glomerular disease worldwide, carries a significant risk of kidney failure. Its pathogenesis involves the presence of elevated levels of IgA and galactose-deficient IgA (Gd-IgA), deposition of these antibodies in the kidney mesangium, and complement-mediated glomerular injury, leading to progressive renal function loss. The source and specificity of IgA in this disease remain unclear. We hypothesized that pathogenic IgA results from an immune response to abnormal protein modifications. Methods We used an advanced enzyme-linked immunosorbent assay (ELISA) platform to assess serum IgA and Gd-IgA reactivity to 93 posttranslational modifications and other chemical adducts in 28 patients with biopsy-proven IgAN and 22 healthy controls. Results Carboxymethyl-lysine (CML) was identified as the dominant target of IgA and Gd-IgA, but not IgG in patients with IgAN. This finding was validated in an independent cohort of 15 IgAN cases and 15 controls. In addition, a positive correlation was found between serum CML concentration and IgA reactivity in patients with IgAN, alongside albuminuria. Lastly, immunofluorescence staining observed elevated CML deposition in glomeruli of patients with IgAN than in controls. Conclusion Our studies identify CML as a primary target of circulating IgA in IgAN, suggesting that aberrant responses to this modified self-antigen contribute to disease pathophysiology.

Systemic complement is a major contributor to the onset and progression of kidney graft injury. However, the kidney itself is an important site of complement production. Renal-derived complement plays a key role in graft dysfunction, unlike in some other solid organ transplants. Complement factors are generated by multiple renal cell types under both physiological and pathological conditions. Renal complement production mediates ischemia/reperfusion injury and acute cellular and humoral rejection but protective effects of the complement cascade have been reported as well. More recently, intracellular complement production and activation (complosome) has also been shown to be an important regulator of key metabolic and cellular functions in renal cells and in immune kidney infiltrates, adding complexity to the field. Herein, we review current knowledge on the role of renal-derived complement in the pathophysiology of kidney graft damage and the current landscape of complement targeted therapeutics in kidney transplantation.

Extracellular vesicles (EVs) contain proteins, lipids, and nucleic acids from their cells of origin. By delivering these cargos to distant acceptor cells, EVs modulate many biologic processes, including adaptive immunity. Following transplantation, EV's expressing donor major histocompatibility complexes bind to host dendritic cells (DCs), permitting donor major histocompatibility complexes expression by recipient DCs and priming antidonor T cell responses. The mechanisms through which circulating EVs bind DCs are poorly understood. The complement system opsonizes pathogens and damaged cells and enhances subsequent recognition by antigen-presenting cells through surface-expressed complement receptors. Here, we newly show that complement opsonization of graft-released EVs augments their binding to recipient DCs in a CD11c-dependent manner. Enhanced delivery of donor antigen by EVs induces antidonor T cell responses and graft rejection, which can be mitigated by pharmacologic inhibition of complement activation. Our findings reveal a previously unrecognized mechanism linking complement activation to EV function with important implications for T cell immunity.

Donor-derived exosomes bind recipient dendritic cells (rDCs) and prime allo-T cell responses through semi-direct presentation. The mechanisms that allow efficient exosome binding are unknown. We found that recipient complement C3b opsonizes circulating exosomes after murine cardiac transplant and leads to greater exosome binding and higher donor MHC I expression on rDCs (MFI fold Δ over naive, 9.2 v. 1.9, WT v. C3KO). Moreover, C3-opsonized exosomes from transplanted mice adoptively transferred into naïve untransplanted hosts induce greater T-cell alloresponses than C3KO controls (log10 fold Δ over naive of IFNγ+CD8+ T cells, 1.25 v. 0.12, WT v. C3KO), linking exosome C3b expression and allogeneic T cell priming. DCs highly express CD11c, a component of complement-receptor 4, which can bind C3b among other ligands. Recipient CD11c-deficiency abrogates complement-dependent exosome binding (fold Δ over naive 6.94 v. 3.17, WT v. CD11cKO), supporting our novel hypothesis that exosomes opsonization by C3b enhances their capture by rDCs via CD11c, thereby improving T cell priming. Translationally, we tested sera from human kidney transplant recipients enrolled in a clinical trial testing C1-inhibitor (C1INH) and found that C1INH treatment lowered post-transplant C3b expression on circulating exosomes compared to placebo (C3b MFI fold Δ from baseline 1.2 v. 4.2), and that elevated C3b expression on human exosomes associated with enhanced binding to acceptor DCs ex vivo. NIH R01 AI172899 Antigen and Dendritic Cell Processing, Presentation, and Biology (AGDC)

Normothermic machine perfusion (NMP), the most recent advancement in solid organ preservation, enables ex situ maintenance of grafts in a physiologically active state, offering a significant advantage over traditional cold storage methods. Whereas NMP is now widely adopted for clinical preservation of livers, hearts, and lungs, its application in kidney transplantation remains relatively limited. In this context, NMP holds promise for expanding the use of marginal donor kidneys by enhancing viability assessment and potentially restoring function in grafts that might otherwise be discarded. In addition, NMP provides a valuable platform for studying molecular markers of injury and recovery in human organs, as well as for delivering targeted therapies aimed at modulating immunologic or transcriptomic profiles. Despite its potential, broader clinical implementation is hindered by variability in perfusion devices, protocols, and perfusate compositions across centers, making cross-study comparisons challenging. This review examines the current landscape of kidney NMP and its emerging role in graft reconditioning.

The complement system plays a critical role in modulating adaptive T cell responses. Coordination of the proinflammatory signaling cascade and complement regulators permits efficient T cell priming and survival, while minimizing off-target damage to healthy host cells. In the context of transplantation, anti-donor T cell immunity remains a barrier to long term graft health and complement-targeted therapies have shown the potential to significantly improve patient outcomes. Here we will review our current understanding of complement-mediated T cell function and how these findings may be harnessed in organ transplantation.

Natural killer (NK) cells respond to diseased and allogeneic cells through NKG2A/HLA-E or killer cell immunoglobulin-like receptor (KIR)/HLA-ABC interactions. Correlations between HLA/KIR disparities and kidney transplant pathology suggest an antibody-independent pathogenic role for NK cells in transplantation, but the mechanisms remain unclear. Using CyTOF to characterize recipient peripheral NK cell phenotypes and function, we observed diverse NK cell subsets among participants who responded heterogeneously to allo-stimulators. NKG2A+KIR+ NK cells responded more vigorously than other subsets, and this heightened response persisted after kidney transplantation despite immunosuppression. In test and validation sets from 2 clinical trials, pretransplant donor-induced release of cytotoxicity mediator Ksp37 by NKG2A+ NK cells correlated with reduced long-term allograft function. Separate analyses showed that Ksp37gene expression in allograft biopsies lacking histological rejection correlated with death-censored graft loss. Our findings support an antibody-independent role for NK cells in transplant injury and support further testing of pretransplant, donor-reactive, NK cell-produced Ksp37 as a risk-assessing, transplantation biomarker.

Purpose of reviewAntibody-mediated rejection (AMR) after solid organ transplantation remains an unsolved problem and leads to poor early and late patient outcomes. The complement system is a well recognized pathogenic mediator of AMR. Herein, we review the known molecular mechanisms of disease and results from ongoing clinical testing of complement inhibitors after solid organ transplant.Recent findingsActivation and regulation of the complement cascade is critical not only for the terminal effector function of donor-specific antibodies, but also for the regulation of T and B cell subsets to generate the antidonor humoral response. Donor-specific antibodies (DSA) have heterogenous features, as are their interactions with the complement system. Clinical testing of complement inhibitors in transplant patients have shown good safety profiles but mixed efficacy to date.SummaryThe complement cascade is a critical mediator of AMR and clinical trials have shown early promising results. With the steady emergence of novel complement inhibitors and our greater understanding of the molecular mechanisms linking complement and AMR, there is greater optimism now for new prognostic and therapeutic tools to deploy in transplant patients with AMR.

Purpose of Review Exosomes have garnered increasing interest due to their involvement in a wide array of biological processes, including immunity and regeneration. In this review, we outline our current understanding of the role of exosomes in modulating transplant immune responses and as biomarkers of allograft function or rejection. Recent Findings The exosomal effect on post-transplant immunity is heterogeneous and context dependent. They are critical for priming anti-donor T cell immunity via semi-direct presentation but have also been shown to promote tolerance to graft-expressed non-inherited maternal antigens. Post-transplant, proteomic and gene expression profiling of exosomes collected from blood, urine, or bronchoalveolar lavage can discriminate between cellular and antibody-mediated rejection and as a potential early prognostication tool. Summary Secreted by both the donor and recipient cells after solid organ transplantation, exosomes are mechanistic mediators of the allogeneic immunity and have shown promise as non-invasive biomarkers of graft function.

Human Natural Killer (NK) cells are heterogeneous lymphocytes regulated by variegated arrays of germline-encoded activating and inhibitory receptors. They acquire the ability to detect polymorphic self-antigen via NKG2A/HLA-E or KIR/HLA-I ligand interactions through an education process. Correlations among HLA/KIR genes, kidney transplantation pathology and outcomes suggest that NK cells participate in allograft injury, but mechanisms linking NK HLA/KIR education to antibody-independent pathological functions remain unclear. We used CyTOF to characterize pre- and post-transplant peripheral blood NK cell phenotypes/functions before and after stimulation with allogeneic donor cells. Unsupervised clustering identified unique NK cell subpopulations present in varying proportions across patients, each of which responded heterogeneously to donor cells based on donor ligand expression patterns. Analyses of pre-transplant blood showed that educated, NKG2A/KIR-expressing NK cells responded greater than non-educated subsets to donor stimulators, and this heightened alloreactivity persisted > 6 months post-transplant despite immunosuppression. In distinct test and validation sets of patients participating in two clinical trials, pre-transplant donor-induced release of NK cell Ksp37, a cytotoxicity mediator, correlated with 2-year and 5-year eGFR. The findings explain previously reported associations between NK cell genotypes and transplant outcomes and suggest that pre-transplant NK cell analysis could function as a risk-assessment biomarker for transplant outcomes.

Conditioning regimens used for hematopoietic stem cell transplantation (HCT) can escalate the severity of acute T cell-mediated graft-versus-host disease (GVHD) by disrupting gastrointestinal integrity and initiating lipopolysaccharide (LPS)-dependent innate immune cell activation. Activation of the complement cascade has been associated with murine GVHD, and previous work has shown that alternative pathway complement activation can amplify T cell immunity. Whether and how mannan-binding lectin (MBL), a component of the complement system that binds mannose as well as oligosaccharide components of LPS and lipoteichoic acid, affects GVHD is unknown. In this study, we tested the hypothesis that MBL modulates murine GVHD and examined the mechanisms by which it does so. We adoptively transferred C3.SW bone marrow (BM) cells +/- T cells into irradiated wild type (WT) or MBL-deficient C57Bl/6 (B6) recipients with or without inhibiting MBL-initiated complement activation using C1-esterase inhibitor (C1-INH). We analyzed the clinical severity of disease expression and analyzed intestinal gene and cell infiltration. In vitro studies assessed MBL expression on antigen-presenting cells (APCs) and compared LPS-induced responses of WT and MBL-deficient APCs. MBL-deficient recipients of donor BM +/- T cells exhibited significantly less weight loss over the first 2 weeks post-transplantation weeks compared with B6 controls (P < .05), with similar donor engraftment in the 2 groups. In recipients of C3.SW BM + T cells, the clinical expression of GVHD was less severe (P < .05) and overall survival was better (P < .05) in MBL-deficient mice compared with WT mice. On day-7 post-transplantation, analyses showed that the MBL-deficient recipients exhibited less intestinal IL1b, IL17, and IL12 p40 gene expression (P < .05 for each) and fewer infiltrating intestinal CD11c(+), CD11b(+), and F4/80(+) cells and TCR beta(+), CD4(+), CD4(+)IL17(+), and CD8(+) T cells (P < .05 for each). Ovalbumin or allogeneic cell immunizations induced equivalent T cell responses in MBL-deficient and WT mice, demonstrating that MBL-deficiency does not directly impact T cell immunity in the absence of irradiation conditioning. Administration of C1-INH did not alter the clinical expression of GVHD in preconditioned WT B6 recipients, suggesting that MBL amplifies clinical expression of GVHD via a complement-independent mechanism. WT, but not MBL-deficient, APCs express MBL on their surfaces. LPS-stimulated APCs from MBL-deficient mice produced less proinflammatory cytokines (P < .05) and induced weaker alloreactive T cell responses (P < .05) compared with WT APCs. Together, our data show that MBL modulates murine GVHD, likely by amplifying complement-independent, LPS-initiated gastrointestinal inflammation. The results suggest that devising strategies to block LPS/MBL ligation on APCs has the potential to reduce the clinical expression of GVHD. (C) 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

SHARPIN, together with RNF31/HOIP and RBCK1/HOIL1, form the linear ubiquitin chain assembly complex (LUBAC) E3 ligase that catalyzes M1-linked poly-ubiquitination. Mutations in RNF31/HOIP and RBCK/HOIL1 in humans and Sharpin in mice lead to auto-inflammation and immunodeficiency but the mechanism underlying the immune dysregulation remains unclear. We now show that the phenotype of the Sharpin -/- mice is dependent on CYLD, the deubiquitinase that removes K63-linked poly-ubiquitin chains. The dermatitis, disrupted splenic architecture, and loss of Peyer’s patches in the Sharpin -/- mice were fully reversed in Sharpin -/- Cyld -/- mice. There is enhanced association of RIPK1 with the death-inducing signaling complex (DISC) following TNF stimulation in Sharpin -/- cells, and this is dependent on CYLD since it is reversed in Sharpin -/- Cyld -/- cells. Enhanced RIPK1 recruitment to the DISC in Sharpin -/- cells correlated with impaired phosphorylation of CYLD at serine 418, a modification reported to inhibit its enzymatic activity. The dermatitis in the Sharpin -/- mice was also ameliorated by the conditional deletion of Cyld using LysM-cre or Cx3cr1-cre indicating that CYLD-dependent death of myeloid cells is inflammatory. Our studies reveal that under physiological conditions, TNF- and RIPK1-dependent cell death is suppressed by the linear ubiquitin-dependent inhibition of CYLD. The Sharpin -/- phenotype illustrates the pathological consequences when CYLD inhibition fails. Short Summary In the absence of SHARPIN, cells fail to properly regulate the deubiquitinase CYLD, leading to RIPK1-mediated cell death. Deletion of Cyld reverses the sensitivity of Sharpin -/- cells to TNF-induced cell death, as well as the multi-organ inflammation and immune dysfunction observed in Sharpin -/- mice.

Herein, we report that Shroom3 knockdown, via Fyn inhibition, induced albuminuria with foot process effacement (FPE) without focal segmental glomerulosclerosis (FSGS) or podocytopenia. Interestingly, knockdown mice had reduced podocyte volumes. Human minimal change disease (MCD), where podocyte Fyn inactivation was reported, also showed lower glomerular volumes than FSGS. We hypothesized that lower glomerular volume prevented the progression to podocytopenia. To test this hypothesis, we utilized unilateral and 5/6th nephrectomy models in Shroom3-KD mice. Knockdown mice exhibited less glomerular and podocyte hypertrophy after nephrectomy. FYN-knockdown podocytes had similar reductions in podocyte volume, implying that Fyn was downstream of Shroom3. Using SHROOM3 or FYN knockdown, we confirmed reduced podocyte protein content, along with significantly increased phosphorylated AMPK, a negative regulator of anabolism. AMPK activation resulted from increased cytoplasmic redistribution of LKB1 in podocytes. Inhibition of AMPK abolished the reduction in glomerular volume and induced podocytopenia in mice with FPE, suggesting a protective role for AMPK activation. In agreement with this, treatment of glomerular injury models with AMPK activators restricted glomerular volume, podocytopenia, and progression to FSGS. Glomerular transcriptomes from MCD biopsies also showed significant enrichment of Fyn inactivation and Ampk activation versus FSGS glomeruli. In summary, we demonstrated the important role of AMPK in glomerular volume regulation and podocyte survival. Our data suggest that AMPK activation adaptively regulates glomerular volume to prevent podocytopenia in the context of podocyte injury.

IMPORTANCE:Preliminary reports indicate that acute kidney injury (AKI) is common in coronavirus disease (COVID)-19 patients and is associated with worse outcomes. AKI in hospitalized COVID-19 patients in the United States is not well-described.OBJECTIVE:To provide information about frequency, outcomes and recovery associated with AKI and dialysis in hospitalized COVID-19 patients.DESIGN:Observational, retrospective study.SETTING:Admitted to hospital between February 27 and April 15, 2020.PARTICIPANTS:Patients aged ≥18 years with laboratory confirmed COVID-19 Exposures: AKI (peak serum creatinine increase of 0.3 mg/dL or 50% above baseline). Main Outcomes and Measures: Frequency of AKI and dialysis requirement, AKI recovery, and adjusted odds ratios (aOR) with mortality. We also trained and tested a machine learning model for predicting dialysis requirement with independent validation.RESULTS:A total of 3,235 hospitalized patients were diagnosed with COVID-19. AKI occurred in 1406 (46%) patients overall and 280 (20%) with AKI required renal replacement therapy. The incidence of AKI (admission plus new cases) in patients admitted to the intensive care unit was 68% (553 of 815). In the entire cohort, the proportion with stages 1, 2, and 3 AKI were 35%, 20%, 45%, respectively. In those needing intensive care, the respective proportions were 20%, 17%, 63%, and 34% received acute renal replacement therapy. Independent predictors of severe AKI were chronic kidney disease, systolic blood pressure, and potassium at baseline. In-hospital mortality in patients with AKI was 41% overall and 52% in intensive care. The aOR for mortality associated with AKI was 9.6 (95% CI 7.4-12.3) overall and 20.9 (95% CI 11.7-37.3) in patients receiving intensive care. 56% of patients with AKI who were discharged alive recovered kidney function back to baseline. The area under the curve (AUC) for the machine learned predictive model using baseline features for dialysis requirement was 0.79 in a validation test.CONCLUSIONS AND RELEVANCE:AKI is common in patients hospitalized with COVID-19, associated with worse mortality, and the majority of patients that survive do not recover kidney function. A machine-learned model using admission features had good performance for dialysis prediction and could be used for resource allocation.

The complement system, traditionally considered a component of innate immunity, is now recognized as a crucial mediator of the adaptive immune response in solid organ transplantation. Preclinical and early human trials have demonstrated the importance of complement effector mechanisms in driving allograft injury during specific antigraft immune responses, including ischemia-reperfusion injury, T-cell-mediated rejection, and antibody-mediated rejection, as well as a potential role for complement-derived risk stratification biomarkers. These data support the need for further testing of complement inhibitors in solid organ transplant recipients.