Long-term allograft survival generally requires lifelong immunosuppression (IS). Rarely, recipients display spontaneous ''operational tolerance'' with stable graft function in the absence of IS. The lack of biological markers of this phenomenon precludes identification of potentially tolerant patients in which IS 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 use these biomarkers to determine the frequency of this state in immunosuppressed 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 IS) 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. Thirty-three of 49 genes correctly segregated tolerance and chronic rejection phenotypes with 99% and 86% specificity. The signature is shared with 1 of 12 and 5 of 10 stable patients on triple IS and low-dose steroid monotherapy, respectively. The gene signature suggests a pattern of reduced costimulatory 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 IS titration. Further validation of this tool for safe IS minimization in prospective clinical trials is warranted.kidney transplantation ͉ microarray ͉ tolerant ͉ genomics ͉ immunosuppression titration D espite continuous improvement in renal allograft survival over the last decade, the half-life of renal allografts has increased marginally because of accrual of chronic graft nephropathy from drug-related nephrotoxicity and chronic rejection (1, 2). Patients facing life-long immunosuppression (IS) have increased risk of infection and malignancy (3), whereas insufficient immunosuppressive drug exposure or interruption usually increases rejection risk (4). However, spontaneous and long-term graft acceptance is observed in a small number of patients after solid-organ transplantation (5, 6), years after total withdrawal of immunosuppressive drugs, confirming that a clinical operational state of tolerance to a mismatched graft, described as ''a state of quiescence of the transplanted organ, functioning without a destructive immune response'' (7), can indeed occur and exist in humans. However, the frequency of this observation in the kidney transplant population is unknown and, currently, we cannot identify patients primed to develop this immune adaptation or monitor for the stability of this status of ''operational tolerance.'' The operationally tolerant kidney transpla...

A substantial proportion of long-term kidney graft recipients, including those with a stable renal function in the absence of immunosuppressive therapy, present a skewed T cell receptor (TCR) V␤ chain usage, essentially in the CD8 ؉ subset. This study analyzed in more detail phenotypical and functional alterations of CD8 ؉ lymphocytes in drug-free tolerant patients (DF-Tol) compared with recipients with chronic rejection (CR D rug-free tolerance, defined as long-term maintenance of graft integrity and function without immunosuppression, is a rare event in human kidney transplantation because interruption of immunosuppressive treatment usually leads to acute or chronic graft rejection. Nevertheless, this phenomenon is of unique interest to study the physiologic basis of graft tolerance in humans. On the one hand, long-term drug-free tolerant patients (DF-Tol) represent a unique model to study the extent to which mechanisms of tolerance defined experimentally, such as active suppression by regulatory lymphocytes, ignorance of alloantigens, chimerism, homeostatic regulation or clonal deletion, are relevant to this human situation (1-4). Most studies in rodents analyzed the induction rather than the maintenance phase of tolerance, and discrepancies with the human situation may exist, as exemplified by the role of alloreactive CD8 ϩ central memory cells in rejection and tolerance induction (5,6). On the other hand, the characterization of peculiar immunologic profiles in DF-Tol may be clinically important to identify biologic signatures that are associated with graft tolerance. Considering the major medical and economic burden of chronic immunosuppression and that operational tolerance may be more common than expected but could be masked in long-term immunosuppressed patients, the identification of specific biologic signatures of tolerance could open new perspectives for rational rather than empiric minimizing of immunosuppressive drugs in well-selected patients (6 -10).As a proof of concept of the relevance of the DF-Tol model to study human tolerance, we described recently a number of specific immunologic features in these patients. First, DF-Tol Received February 16, 2005. Accepted October 17, 2005 Published online ahead of print. Publication date available at www.jasn.org.S.B. and J.-P.S. contributed equally to this work.

Kidney transplant is the reference treatment for patients with end-stage renal disease, but patients may develop long-term rejection of the graft. However, some patients do not reject the transplant, but instead are operationally tolerant state despite withdrawal of immunosuppressive treatment. In this second article we outline a microarray-based identification of key leader genes associated respectively to rejection and to operational tolerance of the kidney transplant in humans by utilizing a non/statistical bioinformatic approach based on the identification of "key genes," either as those mostly changing their expression, or having the strongest interconnections. A uniquely informative picture emerges on the genes controlling the human transplant from the detailed comparison of these findings with the traditional statistical SAM (Tusher et al. 2001 Proc Natl Acad Sci USA 98:5116-5121) analysis of the microarrays and with the clinical study carried out in the accompanying part I article.

Animal studies have suggested a potential role for regulatory T cells (Tregs) in allograft tolerance, but these FOXP3ϩ cells seem to be an inherent component of acute rejection (AR) in human recipients of renal transplants. The balance between regulatory cells and effector/cytotoxic cells may determine graft outcome; this balance has not been described for chronic allograft injury. We investigated the expression of key regulatory, effector, and cytotoxic transcripts (i.e., FOXP3, T-bet, and granzyme B, respectively) in the grafts and peripheral blood of long-term-surviving renal transplant patients. We found that, whereas neither intragraft nor peripheral blood FOXP3 or T-bet mRNA could distinguish between rejection and nonrejection status, granzyme B (GrzB) mRNA could: It was significantly increased in the graft and significantly decreased in the peripheral blood of patients with chronic antibody-mediated rejection (CAMR). Quantifying peripheral blood GrzB mRNA demonstrated potential to aid in the noninvasive diagnosis of CAMR. In summary, these data affirm GrzB as a marker not only for AR but also for CAMR. In addition, we identified several previously unreported clinical or demographic factors influencing regulatory/effector/cytotoxic profiles in the peripheral blood, highlighting the necessity to consider confounding variables when considering the use of potential biomarkers, such as FOXP3, for diagnosis or prognosis in kidney transplantation.

Diagnosis of the specific cause of late allograft injury is necessary if more personalized and efficient immunosuppressive regimens are to be introduced. This study sought previously unrecognized biomarkers for specific histologic diagnoses of late graft scarring by comparison of gene sets from published microarray studies. Tribbles-1 (TRIB1), a human homolog of Drosophila tribbles, was identified to be a potentially informative biomarker. For testing this, mRNA expression in 76 graft biopsies, 71 blood samples, and 11 urine samples were profiled from independent cohorts of renal transplant patients with different histologic diagnoses recruited at two European centers. TRIB1 but not TRIB2 or TRIB3 was found to be a potential blood and tissue biomarker of chronic antibody-mediated rejection, an active immune-mediated form of chronic allograft failure associated with a poor prognosis. TRIB1 mRNA levels in peripheral blood mononuclear cells discriminated patients with chronic antibody-mediated rejection from those with other types of late allograft injury with high sensitivity and specificity. TRIB1 was also upregulated in a rodent model of chronic cardiac vasculopathy, suggesting that this biomarker may be useful in other solid-organ transplants and across species. It was determined that TRIB1 is expressed primarily by antigen-presenting cells and activated endothelial cells. Overall, these data support the potential use of TRIB1 as a biomarker of chronic antibody-mediated allograft failure.

Objective. The association of HLA-DRB1 alleles with anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) suggests the potential involvement of T lymphocytes in ACPA-seropositive disease. The purpose of this study was to investigate this hypothesis by systematic histologic and molecular analyses of synovial T cells in ACPA؉ versus ACPA-RA patients.Methods. Synovial biopsy samples were obtained from 158 RA patients. Inflammation was determined histologically and immunohistochemically. RNA was extracted from peripheral blood mononuclear cells and synovial tissues obtained from 11 ACPA؉ RA patients, 7 ACPA-RA patients, and 10 spondylarthritis (SpA) patients (arthritis controls). T lymphocyte clonality was studied by combined quantitative and qualitative T cell receptor CDR3 length distribution (LD) analysis and direct sequencing analysis.Results. ACPA؉ and ACPA-RA patients were similar at both the clinical and histologic levels. At the molecular level, however, patients with ACPA؉ synovitis displayed a marked elevation of qualitative CDR3 LD alterations as compared with those with ACPA-synovitis and with the SpA controls. These differences in CDR3 LD were not observed in the peripheral blood, indicating a selective recruitment and/or local expansion of T cells in the synovial compartment. The CDR3 LD alterations reflected true monoclonal or oligoclonal expansions, as confirmed by direct sequencing of the T cell receptor. The CDR3 LD alterations in RA synovium did not correlate with B cell clonal expansions but were inversely associated with synovial lymphoid neogenesis.Conclusion. The T cell repertoire is specifically restricted in RA patients with ACPA؉ synovitis. Whereas the origin and role of these clonal alterations remain to be determined, our data suggest the preferential involvement of T lymphocytes in ACPAseropositive RA.

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 by a classical statistical microarray analysis and, in the second part, by a nonstatistical analysis.

Due to its low level of nephrotoxicity and capacity to harness tolerogenic pathways, sirolimus (SRL) has been proposed as an alternative to calcineurin inhibitors in transplantation. The exact mechanisms underlying its unique immunosuppressive profile in humans, however, are still not well understood. In the current study, we aimed to depict the in vivo effects of SRL in comparison with cyclosporin A (CSA) by employing gene expression profiling and multiparameter flow cytometry on blood cells collected from stable kidney recipients under immunosuppressant monotherapy. SRL recipients displayed an increased frequency of CD4 + CD25highFoxp3 + T cells. However, this was accompanied by an increased number of effector memory T cells and by enrichment in NFkB-related pro-inflammatory expression pathways and monocyte and NK cell lineage-specific transcripts. Furthermore, measurement of a transcriptional signature characteristic of operationally tolerant kidney recipients failed to detect differences between SRL and CSA-treated recipients. In conclusion, we show here that the blood transcriptional profile induced by SRL monotherapy in vivo does not resemble that of operationally tolerant recipients and is dominated by innate immune cells and NFkB-related pro-inflammatory events. These data provide novel insights on the complex effects of SLR on the immune system in clinical transplantation.