Establishing long-term allograft acceptance without the requirement for continuous immunosuppression, a condition known as allograft tolerance, is a highly desirable therapeutic goal in solid organ transplantation. Determining which recipients would benefit from withdrawal or minimization of immunosuppression would be greatly facilitated by biomarkers predictive of tolerance. In this study, we identified the largest reported cohort to our knowledge of tolerant renal transplant recipients, as defined by stable graft function and receiving no immunosuppression for more than 1 year, and compared their gene expression profiles and peripheral blood lymphocyte subsets with those of subjects with stable graft function who are receiving immunosuppressive drugs as well as healthy controls. In addition to being associated with clinical and phenotypic parameters, renal allograft tolerance was strongly associated with a B cell signature using several assays. Tolerant subjects showed increased expression of multiple B cell differentiation genes, and a set of just 3 of these genes distinguished tolerant from nontolerant recipients in a unique test set of samples. This B cell signature was associated with upregulation of CD20 mRNA in urine sediment cells and elevated numbers of peripheral blood naive and transitional B cells in tolerant participants compared with those receiving immunosuppression. These results point to a critical role for B cells in regulating alloimmunity and provide a candidate set of genes for wider-scale screening of renal transplant recipients.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4 + T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4 + T cells, and a high ratio of forkhead box P3 to α-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

Immunosuppressive drugs can be completely withdrawn in up to 20% of liver transplant recipients, commonly referred to as 'operationally' tolerant. Immune characterization of these patients, however, has not been performed in detail, and we lack tests capable of identifying tolerant patients among recipients receiving maintenance immunosuppression. In the current study we have analyzed a variety of biological traits in peripheral blood of operationally tolerant liver recipients in an attempt to define a multiparameter 'fingerprint' of tolerance. Thus, we have performed peripheral blood gene expression profiling and extensive blood cell immunophenotyping on 16 operationally tolerant liver recipients, 16 recipients requiring on-going immunosuppressive therapy, and 10 healthy individuals. Microarray profiling identified a gene expression signature that could discriminate tolerant recipients from immunosuppression-dependent patients with high accuracy. This signature included genes encoding for c d T-cell and NK receptors, and for proteins involved in cell proliferation arrest. In addition, tolerant recipients exhibited significantly greater numbers of circulating potentially regulatory T-cell subsets (CD4 + CD25 + T-cells and Vd1 + T cells) than either non-tolerant patients or healthy individuals. Our data provide novel mechanistic insight on liver allograft operational tolerance, and † Deceased 12/9/2005 constitute a first step in the search for a non-invasive diagnostic signature capable of predicting tolerance before undergoing drug weaning.

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Chronic kidney disease (CKD), impairment of kidney function, is a serious public health problem, and the assessment of genetic factors influencing kidney function has substantial clinical relevance. Here, we report a meta-analysis of genome-wide association studies for kidney function–related traits, including 71,149 east Asian individuals from 18 studies in 11 population-, hospital- or family-based cohorts, conducted as part of the Asian Genetic Epidemiology Network (AGEN). Our meta-analysis identified 17 loci newly associated with kidney function–related traits, including the concentrations of blood urea nitrogen, uric acid and serum creatinine and estimated glomerular filtration rate based on serum creatinine levels (eGFRcrea) (P < 5.0 × 10−8). We further examined these loci with in silico replication in individuals of European ancestry from the KidneyGen, CKDGen and GUGC consortia, including a combined total of ~110,347 individuals. We identify pleiotropic associations among these loci with kidney function–related traits and risk of CKD. These findings provide new insights into the genetics of kidney function.

Chronic kidney disease (CKD), the result of permanent loss of kidney function, is a major global problem. We identify common genetic variants at chr2p12-p13, chr6q26, chr17q23 and chr19q13 associated with serum creatinine, a marker of kidney function (P=10−10 to 10−15). SNPs rs10206899 (near NAT8, chr2p12-p13) and rs4805834 (near SLC7A9, chr19q13) were also associated with CKD. Our findings provide new insight into metabolic, solute and drug-transport pathways underlying susceptibility to CKD.

Chronic allograft nephropathy (CAN) is the principal cause of late renal allograft failure. This complex process is multifactorial in origin, and there is good evidence for immune-mediated effects. The immune contribution to this process is directed by CD4+ T cells, which can be activated by either direct or indirect pathways of allorecognition. For the first time, these pathways have been simultaneously compared in a cohort of 22 longstanding renal allograft recipients (13 with good function and nine with CAN). CD4+ T cells from all patients reveal donor-specific hyporesponsiveness by the direct pathway according to proliferation or the secretion of the cytokines IL-2, IL-5, and IFN-γ. Donor-specific cytotoxic T cell responses were also attenuated. In contrast, the frequencies of indirectly alloreactive cells were maintained, patients with CAN having significantly higher frequencies of CD4+ T cells indirectly activated by allogeneic peptides when compared with controls with good allograft function. An extensive search for alloantibodies has revealed significant titers in only a minority of patients, both with and without CAN. In summary, this study demonstrates widespread donor-specific hyporesponsiveness in directly activated CD4+ T cells derived from longstanding recipients of renal allografts, whether they have CAN or not. However, patients with CAN have significantly higher frequencies of CD4+ T cells activated by donor Ags in an indirect manner, a phenomenon resembling split tolerance. These findings provide an insight into the pathogenesis of CAN and also have implications for the development of a clinical tolerance assay.

Human B cells with immunoregulatory properties in vitro (Bregs) have been defined by the expression of IL-10 and are enriched in various B-cell subsets. However, proinflammatory cytokine expression in B-cell subsets is largely unexplored. We examined the cytokine profiles of human PBMCs and found that subsets of CD24 hi CD38 hi transitional B cells (TrBs), CD24 hi CD27 + memory B cells, and naïve B cells express IL-10 and the proinflammatory cytokine TNF-a simultaneously. TrBs had the highest IL-10/TNF-a ratio and suppressed proinflammatory helper T cell 1 (Th1) cytokine expression by autologous T cells in vitro more potently than memory B cells did, despite similar IL-10 expression. Whereas neutralization of IL-10 significantly inhibited TrB-mediated suppression of autologous Th1 cytokine expression, blocking TNF-a increased the suppressive capacity of both memory and naïve B-cell subsets. Thus, the ratio of IL-10/TNF-a expression, a measure of cytokine polarization, may be a better indicator of regulatory function than IL-10 expression alone. Indeed, compared with TrB cells from patients with stable kidney graft function, TrBs from patients with graft rejection displayed similar IL-10 expression levels but increased TNF-a expression (i.e., reduced IL-10/TNF-a ratio), did not inhibit in vitro expression of Th1 cytokines by T cells, and abnormally suppressed expression of Th2 cytokines. In patients with graft dysfunction, a low IL-10/TNF-a ratio in TrBs associated with poor graft outcomes after 3 years of follow-up. In summary, these results indicate that B cell-mediated immune regulation is best characterized by the cytokine polarization profile, a finding that was confirmed in renal transplant patients.