Eva Latulippe

BK Polyomavirus Genomic Integration and Large T Antigen Expression: Evolving Paradigms in Human Oncogenesis

Human polyomaviruses are ubiquitous, with primary infections that typically occur during childhood and subsequent latency that may last a lifetime. Polyomavirus‐mediated disease has been described in immunocompromised patients; its relationship to oncogenesis is poorly understood. We present deep sequencing data from a high‐grade BK virus–associated tumor expressing large T antigen. The carcinoma arose in a kidney allograft 6 years after transplantation. We identified a novel genotype 1a BK polyomavirus, called Chapel Hill BK polyomavirus 2 (CH‐2), that was integrated into the BRE gene in chromosome 2 of tumor cells. At the chromosomal integration site, viral break points were found, disrupting late BK gene sequences encoding capsid proteins VP1 and VP2/3. Immunohistochemistry and in situ hybridization studies demonstrated that the integrated BK virus was replication incompetent. We propose that the BK virus CH‐2 was integrated into the human genome as a concatemer, resulting in alterations of feedback loops and overexpression of large T antigen. Collectively, these findings support the emerging understanding that viral integration is a nearly ubiquitous feature in polyomavirus‐associated malignancy and that unregulated large T antigen expression drives a proliferative state that is conducive to oncogenesis. Based on the current observations, we present an updated model of polyomavirus‐mediated oncogenesis.

2013 Banff Criteria for Chronic Active Antibody-Mediated Rejection: Assessment in a Real-Life Setting

Significant changes in the criteria for chronic active antibody‐mediated rejection (CAABMR) were made in the Banff 2013 classification. These modifications expanded the number of patients diagnosed with CAABMR, with undetermined clinical significance. We compared the 2007 and 2013 criteria for the composite end point of death‐censored graft failure or doubling of serum creatinine in 123 patients meeting the criterion related to the morphologic evidence of chronic tissue injury. In all, 18% and 36% of the patients met the 2007 and 2013 criteria, respectively. For the criterion related to antibody interaction with endothelium, only 25% were positive based on the 2007 definition compared with 82% using the 2013 definition. Cox modeling revealed that a 2013 but not a 2007 diagnosis was associated with the composite end point (adjusted hazard ratio 2.5 [95% confidence interval (CI) 1.2–5.2] vs. 1.6 [95% CI 0.7–3.8], respectively). The 2013 criterion based on both the C4d score and the glomerulitis plus peritubular capillaritis score (g+ptc) was more strongly associated with the end point than the 2007 criterion based only on C4d; however, when dissected by component, only the C4d component was significant. The association with clinical outcomes improved with the 2013 criteria. This is related to the new C4d threshold but not to the g+ptc ≥2 component.

Higher calcineurin inhibitor levels predict better kidney graft survival in patients with de novo donor‐specific anti‐HLA antibodies: a cohort study

The development of de novo anti‐HLA donor‐specific antibodies (dnDSA) is associated with poorer outcomes in kidney transplant recipients. Despite this, antibody screening post‐transplant is not widespread, largely because the optimal management of patients with dnDSA remains undetermined. We hypothesized that in this population, calcineurin inhibitor blood levels would be an independent predictor of graft loss. We analyzed a cohort of unsensitized patients for whom anti‐HLA antibody screening was performed prospectively post‐transplant. During the screening period between January 2005 and April 2016, 42 patients developed dnDSA. There was no difference in the clinical characteristics or the histological scores of patients biopsied for clinical indication versus those biopsied solely due to detection of dnDSA. Cox modeling revealed a strong relationship between mean tacrolimus levels following dnDSA detection and graft loss, with a hazard ratio of 0.49 (95% CI, 0.33–0.75), which persisted following adjustment for established independent predictors (HR, 0.52, 95% CI, 0.30–0.89). Kaplan–Meier analysis by tertiles of tacrolimus levels and receiver operating curve analysis concurred to show that a threshold of 5.3 ng/ml could be predictive of graft loss. These data suggest that anti‐HLA antibody monitoring post‐transplant could guide maintenance immunosuppression and improve graft outcomes.

2,8-Dihydroxyadeninuria-induced progressive renal failure

Adenine phosphoribosyl-transferase (APRT) is the key enzyme that transforms adenine into adenylate monophosphate (AMP). In case of APRT deficiency, xanthine oxidase rapidly oxidizes adenine into 2,8-dihydroxyadenine (2,8-DHA), which is then eliminated by the kidneys through tubular secretion. 2,8-DHA is insoluble at a physiologic range of pH; therefore, it can easily precipitate in the urine. Although subjects with APRT deficiency may have repeated kidney stones, some of these patients may remain asymptomatic throughout life [1,2]. Nevertheless, a handful of reports propose that APRT deficiency can also lead to 2,8-DHA crystal-induced acute or chronic renal failure [3,4]. In this paper, we report a case of a middle-aged man with APRT deficiency that led to 2,8-DHA crystal-induced chronic active tubulointerstitial nephritis.

IL-6 production by monocytes is associated with graft function decline in patients with borderline changes suspicious for acute T-cell-mediated rejection: a pilot study

Although borderline changes (BL) suspicious for acute T‐cell‐mediated rejection represent a diagnostic category, its clinical relevance is questioned leading to heterogeneous therapeutic management. We hypothesized that measuring IL‐6 secretion by peripheral blood mononuclear cells identifies patients with ongoing graft damage. We examined the association between secreted IL‐6 and the change in estimated glomerular filtration rate at 6 months after the biopsy (ΔeGFR). We then conducted phenotypic and functional studies on patient and mouse innate immune cells in the blood and the kidney. In a training set, ΔeGFR was strongly associated with IL‐6 levels, showing a clinically meaningful decline of 4.6 ± 1.5 ml/min per increase in log10 IL‐6 (P = 0.001). These results were consistent after adjustment and were reproduced in a validation cohort. Phenotyping of peripheral blood cells revealed that the main source of IL‐6 was CD14+CD16−CCR2+HLA‐DR+CD86+CD11c+ inflammatory monocytes. There was a significant correlation between IL‐6 secretion and interstitial dendritic cell density in the biopsy. Finally, characterization of mouse kidney dendritic cells revealed that they share features with macrophages and function as effector cells secreting IL‐6. In conclusion, measuring IL‐6 secreted by peripheral blood cells can be useful in the management of patients with BL in the absence of a concurrent inflammatory condition.

Allogeneic dendritic cells stimulated with antibodies against HLA class II polarize naive T cells in a follicular helper phenotype

Follicular helper T cells (Tfh) are crucial for the production of high-affinity antibodies, such as alloantibodies, by providing the signals for B-cell proliferation and differentiation. Here, we demonstrate that human allogeneic dendritic cells (DC) stimulated with antibodies against HLA class II antigens preferentially differentiate human naive CD4+ T cells into Tfh cells. Following coculture with DCs treated with these antibodies, CD4+ T cells expressed CXCR5, ICOS, IL-21, Bcl-6 and phosphorylated STAT3. Blockade of IL-21 abrogated Bcl-6, while addition of the IL-12p40 subunit to the coculture increased CXCR5, Bcl-6, phosphorylated STAT3 and ICOS, indicating that they were both involved in Tfh polarization. We further phenotyped the peripheral T cells in a cohort of 55 kidney transplant recipients. Patients with anti-HLA-II donor-specific antibodies (DSA) presented higher blood counts of circulating Tfh cells than those with anti-HLA-I DSAs. Moreover, there was a predominance of lymphoid aggregates containing Tfh cells in biopsies from patients with antibody-mediated rejection and anti-HLA-II DSAs. Collectively, these data suggest that alloantibodies against HLA class II specifically promote the differentiation of naive T cells to Tfh cells following contact with DCs, a process that might appear in situ in human allografts and constitutes a therapeutic target.

Tacrolimus prevents von Willebrand factor secretion by allostimulated human glomerular endothelium

Little is known about the endothelial injury caused directly by circulating donor‐specific antibodies (DSAs) during antibody‐mediated rejection. von Willebrand factor (vWF) is a highly thrombotic glycoprotein stored in Weibel‐Palade bodies in endothelial cells. It has been shown that its secretion is triggered by allostimulation. Calcineurin‐like phosphatases regulate pathways involved in vWF secretion. Therefore, we hypothesized that tacrolimus would prevent alloantibody‐induced glomerular lesions, in part via inhibition of vWF secretion from endothelial cells. Here, we used a human in vitro model of glomerular endothelium expressing HLA class I and II antigens and demonstrated that anti–HLA class II antibodies elicit a higher endothelial release of vWF than do anti–HLA class I antibodies in cell supernatants. We observed that tacrolimus treatment decreased vWF secretion after stimulation with both classes of anti‐HLA antibodies and decreased platelet adhesion on allostimulated endothelial cells in a microfluidic chamber. In kidney recipients, tacrolimus trough levels were negatively associated with vWF blood levels. These results indicate that direct disruption of hemostasis via vWF secretion is a potential mechanism of antibody‐mediated injury in patients with DSAs. Our results further suggest that the targeting of microcirculation hemostasis may be beneficial to prevent the development of microangiopathic lesions in antibody‐mediated rejection.