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Immunosuppressive Drug-Free Operational Immune Tolerance in Human Kidney Transplant Recipients: Blood Gene Expression Analysis Tolerance in Human Kidney Transplant Recipients.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE47755 | GEO | 2017/02/09

SECONDARY ACCESSION(S): PRJNA212316

REPOSITORIES: GEO

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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: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:Background Immune tolerance and persistent mixed chimerism can be achieved reproducibly after combined organ and hematopoietic cell transplantation in mice conditioned with total lymphoid irradiation plus anti-thymocyte globulin. We studied the safety and reproducibility of this approach in a cohort of kidney transplant patients, and tried to identify immune monitoring procedures that can predict tolerance and guide complete immunosuppressive drug withdrawal. Methods Ten patients conditioned with 10 doses of total lymphoid irradiation and 5 doses of anti-thymocyte globulin were given kidney transplants and an injection of CD34+ hematopoietic progenitor cells and T cells from HLA matched donors. Blood cell monitoring included changes in chimerism, balance of T cell subsets, gene expression, and responses to donor alloantigens. Results Nine of 10 patients developed multi-lineage chimerism without graft versus host disease (GVHD), and all had excellent graft function at the last observation point. Five of these with chimerism persisting for at least 12 months were completely withdrawn from immunosuppressive drugs for 6 to 35 months. Blood cells from patients off drugs showed development of specific unresponsiveness to donor alloantigens, M-bM-^@M-^\toleranceM-bM-^@M-^] profiles on gene microarrays, early high ratios of regulatory versus conventional naM-CM-/ve T cells, and early high levels of chimerism among NK cells. Conclusions Total lymphoid irradiation, and anti-thymocyte globulin promoted the development of persistent chimerism and tolerance in a cohort of patients given kidney transplants and donor cell injections. Assays were identified that can assist in the safe withdrawal of immunosuppressive drugs. 45 Agilent Microarray samples were conducted, including 16 tolerance patients, 10 chronic rejection patients, 5 healthy normal controls, and 7 paired pre and post-transplant induced tolerance patients. The aim is to see whether induced tolerance patients show operational tolerance gene expression signature and can withdraw or minimize the immunosuppression regimens.

Project description:This study corresponds to a re-analysis and meta-analysis of the five transcriptomic studies related to spontaneous tolerance to renal allograft in human. It examines the transcriptome of peripheral blood samples from six distinct clinical groups: healthy volunteers (HV); tolerant patients (TOL); stable patients on minimal immunosuppression (MIS) or maintained on classical immunosuppressive therapy (STA); patients showing either signs of chronic rejection (CR) or acute rejection (AR). The initial studies are the following: -Study 1 from Braud C. et al. (J Cell Biochem., 2008) published under GEO accession number GSE47755 and comprising 250 unique samples (8 HV; 21 TOL; 190 STA; 31 CR). -Study 2 from Brouard S. et al. (Proc Natl Acad Sci U S A., 2007) published under GEO accession number GSE47683 and comprising 67 unique samples (8 HV; 12 TOL; 10 MIS; 12 STA; 11 CR; 14 AR). - Study 3 from Lozano JJ. et al. (Am J Transplant., 2011) published under GEO accession number GSE22707 and comprising 42 unique samples (6 HV; 12 TOL; 12 STA; 12 CR). - Study 4 from Newell KA. et al. (J Clin Invest., 2010) published under GEO accession number GSE22229 and comprising 58 unique samples (12 HV; 19 TOL; 27 STA). - Study 5 from Sagoo P. et al. (J Clin Invest. 2010) published under GEO accession number GSE14655 and comprising two distinct cohorts of patients. The IOT (“Indices of Tolerance”) cohort (5a) corresponds to a European group of patients and totalizes 74 unique samples (8 HV; 10 TOL; 11 MIS; 36 STA; 9 CR). The ITN (“Immune Tolerance in Transplantation”) cohort (5b) corresponds to an American group of patients and totalizes 105 unique samples (20 HV; 22 TOL; 11 MIS; 34 STA; 18 CR). The final data matrix corresponds to the combination of the data from the five independent studies and examines the expression of 1846 meta-genes (MG) across 596 unique blood samples. The 1846 meta-genes correspond to the genes being measured across the five datasets and form a virtual microarray platform (TOL-HUMAN-G1846). The 596 examined samples can be divided into 62 HV, 96 TOL, 32 MIS, 311 STA, 81 CR, and 14 AR.

Project description:Being able to identify patients in whom immunological tolerance has been established or is developing would allow an individually tailored approach to post-transplant management of kidney allograft recipients. Ex vivo immunological monitoring was performed on samples from five groups of European renal transplant recipients (“IOT samples”): ten drug-free tolerant recipients who were functionally stable despite remaining immunosuppression-free for more than one year (Tol-DF); also functionally stable patients on minimal immunosuppression (<10 mg/day prednisone, s-LP); stable patients maintained with calcineurin inhibitors (s-CNI); stable patients maintained on CNI-free immunosuppression regimen (s-nCNI); patients showing signs of chronic rejection (CR) and healthy controls (HC). Among the investigation of other biomarkers and bioassays, gene expression profiles were generated on custom Agilent 8x15K 60mer oligonucleotide microarrays (“RISET 2.0”) on the IOT cohort (training set) and on an independent cohort of patients from the ITN (USA) that contained similar groups of patients and included 23 tolerant recipients (“ITN samples”, test set). Set of genes were identified, whose expression on whole blood allowed the identification of 100% of the tolerant recipients in the training set and 84% in the test set. Keywords: classification of clinical samples, tolerance prediction IOT samples: 13 samples from 10 Tol-DF patients, 16 samples from 11 s-LP patients, 8 samples from 8 s-nCNI patients, 40 samples from 28 s-CNI patients, 10 samples from 9 CR patients, and 8 samples from 8 HC donors were analysed on a custom Agilent 8x15K 60mer oligonucleotide microarray encomprising 5069 probes in triplicates. The microarray is dedicated to transplantation research and was designed based on current literature and published and unpublished data provided by the RISET consortium. For the probe selection procedure we specially focused on the detection of multiple transcript variants of a gene, on optimized hybridization properties of the probes, and on the avoidance of crosshybridization. The sampling dates of replicate samples from the same patient range from one day to 1.5 years. ITN samples: 31 samples from 23 Tol-DF patients, 14 samples from 11 s-LP patients, 52 samples from 34 s-CNI patients, 25 samples from 18 CR patients, and 20 samples from 20 HC donors were analysed on the same microarray platform and served as test set. The samples of the two cohorts differ in the protocol of RNA preparation.

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Immunosuppressive Drug-Free Operational Immune Tolerance in Human Kidney Transplant Recipients: Blood Gene Expression Analysis Tolerance in Human Kidney Transplant Recipients.

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