Soluble and cell-based markers of immune checkpoint inhibitor-associated nephritis.

BACKGROUND: Non-invasive biomarkers of immune checkpoint inhibitor-associated acute tubulointerstitial nephritis (ICI-nephritis) are urgently needed. Because ICIs block immune checkpoint pathways that include cytotoxic T lymphocyte antigen 4 (CTLA4), we hypothesized that biomarkers of immune dysregulationpreviously defined in patients with congenital CTLA4 deficiency, including elevated soluble interleukin-2 receptor alpha (sIL-2R) and flow cytometric cell-based markers of B and T cell dysregulation in peripheral blood may aid the diagnosis of ICI-nephritis. METHODS: A retrospective cohort of patients diagnosed with ICI-nephritis was compared with three prospectively enrolled control cohorts: ICI-treated controls without immune-related adverse events, patients not on ICIs with hemodynamic acute kidney injury (hemodynamic AKI), and patients not on ICIs with biopsy proven acute interstitial nephritis from other causes (non-ICI-nephritis). sIL-2R level and flow cytometric parameters were compared between groups using Wilcoxon rank sum test or Kruskal-Wallis test. Receiver operating characteristic curves were generated to define the accuracy of sIL-2R and flow cytometric biomarkers in diagnosing ICI-nephritis. The downstream impact of T cell activation in the affected kidney was investigated using archived biopsy samples to evaluate the gene expression of IL2RA, IL-2 signaling, and T cell receptor signaling in patients with ICI-nephritis compared with other causes of drug-induced nephritis, acute tubular injury, and histologically normal controls. RESULTS: sIL-2R level in peripheral blood was significantly higher in patients with ICI-nephritis (N=24) (median 2.5-fold upper limit of normal (ULN), IQR 1.9-3.3), compared with ICI-treated controls (N=10) (median 0.8-fold ULN, IQR 0.5-0.9, p<0.001) and hemodynamic AKI controls (N=6) (median 0.9-fold-ULN, IQR 0.7-1.1, p=0.008). A sIL-2R cut-off point of 1.75-fold ULN was highly diagnostic of ICI-nephritis (area under the curve >96%) when compared with either ICI-treated or hemodynamic AKI controls. By peripheral blood flow cytometry analysis, lower absolute CD8+T cells, CD45RA+CD8+ T cells, memory CD27+B cells, and expansion of plasmablasts were prominent features of ICI-nephritis compared with ICI-treated controls. Gene expressions for IL2RA, IL-2 signaling, and T cell receptor signaling in the kidney tissue with ICI-nephritis were significantly higher compared with controls. CONCLUSION: Elevated sIL-2R level and flow cytometric markers of both B and T cell dysregulation may aid the diagnosis of ICI-nephritis.

Deep learning identified pathological abnormalities predictive of graft loss in kidney transplant biopsies.

Interstitial fibrosis, tubular atrophy, and inflammation are major contributors to kidney allograft failure. Here we sought an objective, quantitative pathological assessment of these lesions to improve predictive utility and constructed a deep-learning-based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte infiltrates. Periodic acid- Schiff stained slides of transplant biopsies (60 training and 33 testing) were used to quantify pathological lesions specific for interstitium, tubules and mononuclear leukocyte infiltration. The pipeline was applied to the whole slide images from 789 transplant biopsies (478 baseline [pre-implantation] and 311 post-transplant 12-month protocol biopsies) in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss. Our model accurately recognized kidney tissue compartments and mononuclear leukocytes. The digital features significantly correlated with revised Banff 2007 scores but were more sensitive to subtle pathological changes below the thresholds in the Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of one-year graft loss, while a Composite Damage Score in 12-month post-transplant protocol biopsies predicted later graft loss. ITASs and Composite Damage Scores outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITASs or Composite Damage Scores also demonstrated significantly higher incidence of estimated glomerular filtration rate decline and subsequent graft damage. Thus, our deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies and demonstrated superior ability for prediction of post-transplant graft loss with potential application as a prevention, risk stratification or monitoring tool.

The pathology and changing epidemiology of dialysis-related cardiac beta-2 microglobulin amyloidosis.

Cardiac amyloidosis in the setting of systemic amyloidosis due to ß2-microglobulin can occur in the setting of long-term dialysis. It has been suggested that newer dialysis techniques may prevent or at least reduce the likelihood of this disorder occurring. Currently, the prevalence and incidence of dialysis-related cardiac ß2-microglobulin amyloidosis are unclear. The published literature regarding dialysis-related cardiac ß2-microglobulin amyloidosis (Aß2M) was reviewed, and a new case of this disorder is reported. The cumulative available data were analyzed for changing patient characteristics over time. Cardiac Aß2M was previously a common condition in patients who had undergone dialysis for 9 or more years with traditional low-flow dialysis membranes. Newer dialysis technologies reduce, but do not normalize, serum ß2-microglobulin levels in chronic dialysis patients. Newer dialysis technologies appear to reduce the risk of developing Aß2M compared with traditional low-flow dialysis membranes. New cases of documented dialysis-related cardiac Aß2M are uncommon. Analysis of the cases of dialysis-related cardiac Aß2M published over the last 3 decades reveals increasing dialysis intervals over time. Thus, new cases of this disorder are typically associated with remote prior dialysis with low-flow membranes. While initially, the majority of cases of dialysis-related cardiac Aß2M were reported from Europe and the United States, more recently, the majority of cases were reported from Japan, where there is a relatively large population of patients on very long-term dialysis. In addition, low-flow dialysis membranes continue to be used in many parts of the world, raising the potential for dialysis-related cardiac Aß2M to be more common in those countries. Dialysis-related osteoarticular Aß2M appears to continue to occur in the setting of chronic dialysis with the use of high-flow membranes. Dialysis-related cardiac Aß2M is currently uncommon and typically associated with the use of low-flow dialysis membranes. However, the condition could potentially occur in the setting of long-term dialysis even with the use of high-flow membranes. SUMMARY: Dialysis-related cardiac ß2-microglobulin amyloidosis frequently occurred in the past in patients who had undergone dialysis for nine or more years. Currently, the condition is uncommon and typically associated with remote prior dialysis with low-flow membranes. There is potential for this condition to continue to afflict patients receiving chronic dialysis with newer dialysis technologies.

Lupus-like Immune Complex-mediated Glomerulonephritis in Patients with Hepatitis C Virus Infection Treated with Oral, Interferon-free, Direct-acting Antiviral Therapy.

Novel, all-oral interferon-free direct-acting antiviral agents have revolutionized the management of hepatitis C virus (HCV) infection by producing exceptional cure rates with minimal adverse events. While provocation or exacerbation of autoimmunity has been reported in HCV-infected patients receiving interferon, this phenomenon has not been reported in patients receiving interferon-free HCV therapy. We report the occurrence of three cases of lupus-like immune complex-mediated glomerulonephritis occurring shortly after exposure to sofosbuvir-based direct-acting antiviral therapies. In all three cases, renal function quickly improved with immunosuppression. However, two of the three patients developed infectious complications of immunosuppression and died. This is the first report of a lupus-like immune complex mediated glomerulonephritis occurring in the context of HCV eradication with all-oral direct-acting antiviral therapies.

Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Immune Complex Tubulointerstitial Nephritis Due to Autoantibodies to the Proximal Tubule Brush Border.

Immune complex tubulointerstitial nephritis due to antibodies to brush border antigens of the proximal tubule has been demonstrated experimentally and rarely in humans. Our patient developed ESRD and early recurrence after transplantation. IgG and C3 deposits were conspicuous in the tubular basement membrane of proximal tubules, corresponding to deposits observed by electron microscopy. Rare subepithelial deposits were found in the glomeruli. The patient had no evidence of SLE and had normal complement levels. Serum samples from the patient reacted with the brush border of normal human kidney, in contrast with the negative results with 20 control serum samples. Preliminary characterization of the brush border target antigen excluded megalin, CD10, and maltase. We postulate that antibodies to brush border antigens cause direct epithelial injury, accumulate in the tubular basement membrane, and elicit an interstitial inflammatory response.

An elevated IgG4 response in chronic infectious aortitis is associated with aortic atherosclerosis.

Recently, it was shown that infectious bacterial aortitis can stimulate an elevated IgG4⁺ plasma cell response in the vessel wall, which could mimic IgG4 aortitis/periaortitis. However, the factors that are associated with an elevated IgG4⁺ plasma cell response in infectious aortitis are unclear. To ascertain these factors, 17 cases of infectious aortitis and 6 cases of non-infectious severe abdominal aortic atherosclerosis were assessed for the magnitude of IgG4⁺ plasma cell response. The degree of IgG4⁺ plasma cell infiltration was determined by immunohistochemistry. Infectious cases were subcharacterized as chronic (>3 weeks duration) or acute (<3 weeks duration) based on the duration of symptoms, and as involving either the ascending aorta or the distal aorta, ie, the descending thoracic and/or abdominal aorta. There was a 5-16-fold greater degree of IgG4⁺ plasma cell infiltration in the chronic distal infectious aortitis group compared with the other three infectious aortitis groups (P ≤ 0.0007), and compared with non-infectious severe abdominal aortic atherosclerosis (P<0.0008). This resulted in a greater IgG4/IgG ratio in the chronic distal infectious aortitis group compared with the acute ascending and acute distal infectious aortitis groups (P<0.03). The degree of IgG4⁺ plasma cell infiltration in chronic distal infectious aortitis overlaps with that seen in the aortitis and periaortitis of IgG4-related disease. In the chronic infectious aortitis cases, the degree of IgG4⁺ plasma cell infiltration was more intense in patients with moderate to severe aortic atherosclerosis compared with those patients with less aortic atherosclerosis (P=0.007). These findings indicate that an elevated IgG4⁺ plasma cell response occurs in the descending thoracic and abdominal aorta in the setting of chronic bacterial infectious aortitis and pre-existing atherosclerosis. This inflammatory response to chronic infection in atherosclerosis-laden aortas may have implications for the development of IgG4-rich inflammatory atherosclerotic aortic aneurysms.

Partial therapeutic response to Rituximab for the treatment of chronic alloantibody mediated rejection of kidney allografts.

BACKGROUND AND OBJECTIVES: Chronic rejection leads to kidney allograft failure and develops in many kidney transplant recipients. One cause of chronic rejection, chronic antibody mediated rejection (CAMR), is attributed to alloantibodies. Maintenance immunosuppression including prednisone, mycophenolate mofetil (MMF) and calcineurin inhibitors may limit alloantibody production in some patients, but many maintain or develop alloantibody production, leading to CAMR. Therefore, no efficacious therapy to treat CAMR is presently available to prevent the progression of CAMR to kidney allograft failure. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: We performed a retrospective review of 31 subjects with CAMR, of which 14 received Rituximab and 17 subjects did not. Response to Rituximab was defined as decline or stabilization of serum creatinine for at least one year. Data reviewed included demographic, clinical, allograft, post-transplant, and pathological variables. Pathological variables in the diagnostic allograft biopsy were scored according to Banff criteria. RESULTS: The median survival time (MST) for allografts in the control group was 439 days, and for the Rituximab treated group was 685 days. The Rituximab group was dichotomous with 8 subjects showing a medial survival time of 1180 days, and 6 subjects having a median survival time of 431 days. The MST for the responders was statistically significant from the non-responders and controls. No pathological parameter distinguished any subset of subjects. CONCLUSIONS: These data show that Rituximab followed by standard maintenance immunosuppression shows a therapeutic effect in the treatment of CAMR, which is confined to a subset of treated subjects, not identifiable a priori.

Up to 9-day survival and control of thrombocytopenia following alpha1,3-galactosyl transferase knockout swine liver xenotransplantation in baboons.

BACKGROUND: With standard miniature swine donors, survivals of only 3 days have been achieved in primate liver-transplant recipients. The recent production of alpha1,3-galactosyl transferase knockout (GalT-KO) miniature swine has made it possible to evaluate xenotransplantation of pig organs in clinically relevant pig-to-non-human primate models in the absence of the effects of natural anti-Gal antibodies. We are reporting our results using GalT-KO liver grafts. METHODS: We performed GalT-KO liver transplants in baboons using an immunosuppressive regimen previously used by our group in xeno heart and kidney transplantation. Post-operative liver function was assessed by laboratory function tests, coagulation parameters and histology. RESULTS: In two hepatectomized recipients of GalT-KO grafts, post-transplant liver function returned rapidly to normal. Over the first few days, the synthetic products of the donor swine graft appeared to replace those of the baboon. The first recipient survived for 6 days and showed no histopathological evidence of rejection at the time of death from uncontrolled bleeding, probably caused by transfusion-refractory thrombocytopenia. Amicar treatment of the second and third recipients led to maintenance of platelet counts of over 40 000 per µl throughout their 9- and 8-day survivals, which represents the longest reported survival of pig-to-primate liver transplants to date. Both of the last two animals nevertheless succumbed to bleeding and enterococcal infection, without evidence of rejection. CONCLUSIONS: These observations suggest that thrombocytopenia after liver xenotransplantation may be overcome by Amicar therapy. The coagulopathy and sepsis that nevertheless occurred suggest that additional causes of coagulation disturbance must be addressed, along with better prevention of infection, to achieve long-term survival.

Pathology after eculizumab in dense deposit disease and C3 GN.

Eculizumab might benefit C3 glomerulopathies mediated by dysregulation of the alternative complement pathway. Here, we report renal biopsy findings before and after eculizumab therapy in three patients with dense deposit disease and two with C3 GN. All pretreatment biopsies had glomerular and tubular basement membrane deposits that stained exclusively for C3 without significant Ig. After 1 year of therapy, there was reduction in active glomerular proliferation and neutrophil infiltration in three of five patients, consistent with effective C5 blockade, which prevents production of chemotactin C5a. One individual with mild mesangial disease had no significant change in activity or chronicity. One patient exhibited persistent activity and worsening chronicity despite therapy. Immunofluorescence showed no significant reduction in C3 or C5b-9, and electron microscopy revealed persistent deposits in all cases, suggesting a long t(1/2) of C5b-9 in extracellular matrix. Normal renal biopsies stained positive for C5b-9 in glomeruli, tubular basement membranes, and vessel walls, albeit at lower intensity than in C3 glomerulopathy. This indication of physiologic levels of C5b-9 activation in normal kidney potentially explains the localization of deposits in patients with dysregulation of the alternative complement pathway. All post-treatment biopsies showed de novo monoclonal staining for IgG-κ in the same distribution as C3 and C5b-9, mimicking monoclonal Ig deposition disease (MIDD). Staining of the γ heavy chain was restricted to the IgG2 and IgG4 subclasses, suggesting the binding of monoclonal eculizumab to C5 in renal tissues. The long-term effects of this apparent drug-tissue interaction are unknown.

Dense IgG4 plasma cell infiltrates associated with chronic infectious aortitis: implications for the diagnosis of IgG4-related disease.

BACKGROUND: IgG4-related aortitis is a newly recognized form of noninfectious aortitis that occurs as part of the spectrum of a systemic disease referred to as IgG4-related disease. IgG4-related aortitis is distinguished from giant cell aortitis and Takayasu aortitis in part by the presence of increased numbers of IgG4-expressing plasma cells. Chronic infectious aortitis can also display lymphoplasmacytic infiltrates, but the degree of IgG4 expression in these cases has not been specifically evaluated. METHODS: Two cases of chronic active infectious abdominal aortitis were prospectively identified. Both were due to gram-positive bacteria, and at least one of the cases was due to chronic active Staphylococcus aureus infection. The degree of IgG4 plasma cell infiltration was assessed by immunohistochemistry. RESULTS: Both cases of chronic infectious aortitis focally displayed high levels of IgG4-expressing plasma cells, greater than 50% of the IgG-expressing plasma cells, and greater than 50 IgG4-expressing plasma cells per high-power field. CONCLUSIONS: Focal dense IgG4 plasma cell infiltrates can be seen in association with chronic infectious aortitis due to gram-positive bacteria, including Staphylococcus aureus. This observation supports the proposal that chronic Staphylococcus aureus infection may stimulate a Th2-mediated elevation in IgG4. The pathologic diagnosis of IgG4-related aortitis should not be based solely on the presence of increased IgG4 plasma cell counts from immunohistochemistry, but requires consideration of the overall pathology, including careful exclusion of infectious aortitis.

Chronic alloantibody mediated rejection.

Alloantibodies clearly cause acute antibody mediated rejection, and all available evidence supports their pathogenic etiology in the development of chronic alloantibody mediated rejection (CAMR). But the slow evolution of this disease, the on-going immunosuppression, the variations in titer of alloantibodies, and variation in antigenic targets all complicate identifying which dynamic factors are most important clinically and pathologically. This review highlights the pathological factors related to the diagnosis of CAMR, the time course and natural history of this disease. What is known about CAMR pathogenesis is discussed including alloantibodies, the role of complement, gene activation, and Fc effector cell function. Therapy, which is problematic for this disease, is also discussed, including on-going and potential therapies and their limitations.

Rapid reduction in donor-specific anti-human leukocyte antigen antibodies and reversal of antibody-mediated rejection with bortezomib in pediatric heart transplant patients.

BACKGROUND: High titer donor-specific antibodies (DSA) and positive crossmatch in cardiac transplant recipients is associated with increased mortality from antibody-mediated rejection (AMR). Although treatment to reduce anti-human leukocyte antigen antibodies using plasmapheresis, intravenous immunoglobulin, and rituximab has been reported to be beneficial, in practice these are often ineffective. Moreover, these interventions do not affect the mature antibody producing plasma cell. Bortezomib, a proteasome inhibitor active against plasma cells, has been shown to reduce DSA in renal transplant patients with AMR. We report here the first use of bortezomib for cardiac transplant recipients in four pediatric heart recipients with biopsy-proven AMR, hemodynamic compromise, positive crossmatch, and high titer class I DSA. METHODS: Patients received four intravenous dose of bortezomib (1.3 mg/m(2)) over 2 weeks with plasmapheresis and rituximab. DSA specificity and strength (mean fluorescence intensity) was determined with Luminex. All had received previous treatment with plasmapheresis, intravenous immunoglobulin, and rituximab that was ineffective. RESULTS: AMR resolved in all patients treated with bortezomib with improvement in systolic function, conversion of biopsy to C4d negative in three patients and IgG negative in one patient, and a prompt, precipitous reduction in DSAs. In three patients who received plasmapheresis before bortezomib, plasmapheresis failed to reduce DSA. In one case, DSA increased after bortezomib but decreased after retreatment. CONCLUSIONS: Bortezomib reduces DSA and may be an important adjunct to treatment of AMR in cardiac transplant recipients. Bortezomib may also be useful in desensitization protocols and in prevention of AMR in sensitized patients with positive crossmatch and elevated DSA.

Contributions of direct and indirect alloresponses to chronic rejection of kidney allografts in nonhuman primates.

The relative contribution of direct and indirect allorecognition pathways to chronic rejection of allogeneic organ transplants in primates remains unclear. In this study, we evaluated T and B cell alloresponses in cynomolgus monkeys that had received combined kidney/bone marrow allografts and myeloablative immunosuppressive treatments. We measured donor-specific direct and indirect T cell responses and alloantibody production in monkeys (n = 5) that did not reject their transplant acutely but developed chronic humoral rejection (CHR) and in tolerant recipients (n = 4) that never displayed signs of CHR. All CHR recipients exhibited high levels of anti-donor Abs and mounted potent direct T cell alloresponses in vitro. Such direct alloreactivity could be detected for more than 1 y after transplantation. In contrast, only two of five monkeys with CHR had a detectable indirect alloresponse. No indirect alloresponse by T cells and no alloantibody responses were found in any of the tolerant monkeys. Only one of four tolerant monkeys displayed a direct T cell alloresponse. These observations indicate that direct T cell alloresponses can be sustained for prolonged periods posttransplantation and result in alloantibody production and chronic rejection of kidney transplants, even in the absence of detectable indirect alloreactivity.