Project description:Operational tolerance after solid organ transplantation is defined as stable graft acceptance without immunosuppression while maintaining normal immunocompetence against immunological insults. However, it is not clear yet which cellular and molecular pathways are driving tolerance in these patients and how this state can be induced in order to improve graft survival. Several transcriptomic analysis have shown that transcription signatures in blood can reflect the immunological state associated with operational tolerance in kidney transplant. Nonetheless, epigenetic dynamics orchestrating these transcription signatures have not yet been studied. Here, we performed genome-wide analysis of DNA methylation of kidney transplant recipients with chronic rejection and operational tolerance. Our data showed that chronic rejection and operational tolerance recipients have differential DNA methylation in 2737 genes, indicating that each patient group has a specific epigenetic signature associated with transplant outcome. In addition, we observed that operational tolerance is associated with DNA demethylation in genes involved in immune function, including B cell activation (e.g. ST6GAL1, MS4A1 and MEF2C) and the Th17 differentiation program (e.g. LY9 and BATF), while in CR patients is mostly associated intracellular signaling and ubiquitination pathways. Finally, we used weighted gene co-expression network analysis (WGCNA) to select the more defining epigenetic changes associated with operational tolerance. By this method, we select 12 genomic regions specifically hypomethylated (HIVEP2, HOMER1, UTRN, PTPRO, SP100 and JAZF1) or hypermethylated (EMZ8, EZR-AS, WDR20, NADSYN1, TBCD and MED17) in tolerant patients. Analysis of these genes in stable transplant recipients with immunosuppression showed that these patients have a similar methylation signature to operational tolerance recipients. Overall, these results demonstrate that DNA methylation in blood can mirror the immune status associated with kidney transplant outcome and provides a starting point for understanding the epigenetic mechanisms associated with operational tolerance.

Project description:Long-term allograft survival generally requires lifelong immunosuppression. Rarely, recipients display spontaneous operational tolerance with stable graft function in the absence of immunosuppression. The lack of biological markers of this phenomenon precludes identification of potentially tolerant patients in which immunosuppression could be tapered and hinders the development of new tolerance inducing strategies. The objective of this study was to identify minimally invasive blood biomarkers for operational tolerance and utilize these biomarkers to determine the frequency of this state in immunosupressed patients with stable graft function. Blood gene expression profiles from 75 renal transplant patient cohorts (operational tolerance/acute and chronic rejection/stable graft function on immunosuppression) and 16 healthy individuals were analyzed. A subset of samples was used for microarray analysis where three-class comparison of the different groups of patients identified a tolerant footprint of 49 genes. These biomarkers were applied for prediction of operational tolerance by microarray and real-time PCR in independent test-groups. 33/49 genes correctly segregated tolerance and chronic rejection phenotypes with 99% and 86% specificity. The signature is shared with 1/12 and 5/10 stable patients on triple immunosuppression and low dose steroid monotherapy respectively. The gene signature suggests a pattern of reduced co-stimulatory signaling, immune quiescence, apoptosis and memory T cell responses. This study identifies in the blood of kidney recipients a set of genes associated with operational tolerance that may have utility as a minimally-invasive monitoring tool for guiding immunosuppression titration. Further validation of this tool for safe immunosuppression minimization in prospective clinical trials is warranted. 67 samples were analyzed, no replicates included: 1. TOL (n = 12). patients with long-term stable graft function, without immunosuppression for at least 2 years 2. MIS (n = 10). Patients with stable graft function on steroid monotherapy (<10 mg/day) for 4.6. Calcineurin inhibitors and CellCept were removed in these patients because of previous posttransplant lymphoproliferative disease (n = 6), cancer (n = 2), or uncontrolled infectious disease (n = 2). 3. STA (n = 12). Patients with stable kidney graft function at >5 years posttransplantation while under mycophenolate mofetil or azathioprine and maintenance steroids with (n = 5) or without (n = 7) an associated calcineurin inhibitor. 4. AR (n = 14). Patients experiencing rapid decline (>20% from baseline) in graft function and biopsy-proven AR. 5. CR (n = 11). Patients having a progressive degradation of their renal function (creatinine clearance of <60 ml per min per 1.73 m2 and/or proteinuria of >1.5 g/day) and histological signs CR. 6. N (n = 8). These subjects all had normal blood formulae and no infectious or other concomitant pathology for at least 6 months before the study.

Project description:Survival of solid organ grafts depends on life-long immunosuppression, which results in increased rates of infection and malignancy. Induction of tolerance to allografts would represent the optimal solution for controlling both chronic rejection (CR) and side effects of immunosuppression. Although spontaneous ‘‘operational tolerance’’ can occur in human kidney transplantation, the lack of noninvasive peripheral blood biological markers of this rare phenomenon precludes the identification of potentially tolerant patients in whom immunosuppression could be tapered as well as the development of new tolerance inducing strategies. Here, the potential of high throughput microarray technology to decipher complex pathologies allowed us to study the peripheral blood specific gene expression profile and corresponding EASE molecular pathways associated to operational tolerance in a cohort of human kidney graft recipients. In comparison with patients with CR, tolerant patients displayed a set of 343 differentially expressed genes, mainly immune and defense genes, in their peripheral blood mononuclear cells (PBMC), of which 223 were also different from healthy volunteers. Using the expression pattern of these 343 genes, we were able to classify correctly >80% of the patients in a cross-validation experiment and classified correctly all of the samples over time. Collectively, this study identifies a unique PBMC gene signature associated with human operational tolerance in kidney transplantation. 250 samples were analyzed, replicates included: some patients from the TOL and CR groups were sampled twice with an interval between the two different time points of at least 16 months (mean 25.8 months, range 16–31 months). All the samples were analyzed in duplicate or quadruplicate. 1. TOL (n = 21). patients with stable graft function (blood creatinemia<150 mmol/L, proteinuria<1 g/24 h) for at least 1 year (mean 6.4 years,range 1.6–17.2 years) after complete interruption of immunosuppressive therapy. 2. STA (n = 190). Patients with stable kidney graft function under immunosuppressive therapy. 3. CR (n = 31). Patients having a progressive degradation of their renal function (blood creatinemia ≥150 mmol/L, proteinuria ≥1 g/24 h). CR was diagnosed on a graft biopsy according to the updated Banff criteria. CR was defined by histological signs of chronic allograft arteriopathy (arterial intimal fibrosis with mononuclear cell infiltration) and/or transplant glomerulopathy with glomerular double contours. 4. HV (n = 8). Subjects having normal blood formulae and no infectious or other concomitant pathology for at least 6 months before the study.

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Defining a methylation signature associated with operational tolerance in renal transplant

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