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Background Acute T-cell mediated rejection (TCMR) is usually indicated by alteration in serum-creatinine measurements when considerable transplant damage has already occurred. There is, therefore, a need for non-invasive early detection of immune signals that would precede the onset of rejection, prior to transplant damage. Methods We examined the RT-qPCR expression of 22 literature-based genes in peripheral blood samples from 248 patients in the Kidney Allograft Immune Biomarkers of Rejection Episodes (KALIBRE) study. To account for post-transplantation changes unrelated to rejection, we generated time-adjusted gene-expression residuals from linear mixed-effects models in stable patients. To select genes, we used penalised logistic regression based on 27 stable patients and 27 rejectors with biopsy-proven T-cell-mediated rejection, fulfilling strict inclusion/exclusion criteria. We validated this signature in i) an independent group of stable patients and patients with concomitant T-cell and antibody-mediated-rejection, ii) patients from an independent study, iii) cross-sectional pre-biopsy samples from non-rejectors and iv) longitudinal follow-up samples covering the first post-transplant year from rejectors, non-rejectors and stable patients. Findings A parsimonious TCMR-signature (IFNG, IP-10, ITGA4, MARCH8, RORc, SEMA7A, WDR40A) showed cross-validated area-under-ROC curve 0.84 (0.77–0.88) (median, 2.5th–97.5th centile of fifty cross-validation cycles), sensitivity 0.67 (0.59–0.74) and specificity 0.85 (0.75–0.89). The estimated probability of TCMR increased seven weeks prior to the diagnostic biopsy and decreased after treatment. Gene expression in all patients showed pronounced variability, with up to 24% of the longitudinal samples in stable patients being TCMR-signature positive. In patients with borderline changes, up to 40% of pre-biopsy samples were TCMR-signature positive. Interpretation Molecular marker alterations in blood emerge well ahead of the time of clinically overt TCMR. Monitoring a TCMR-signature in peripheral blood could unravel T-cell-related pro-inflammatory activity and hidden immunological processes. This additional information could support clinical management decisions in cases of patients with stable but poor kidney function or with inconclusive biopsy results.

Background:Use of cell-based medicinal products (CBMPs) represents a state-of-the-art approach for reducing general immunosuppression in organ transplantation. We tested multiple regulatory CBMPs in kidney transplant trials to establish the safety of regulatory CBMPs when combined with reduced immunosuppressive treatment. Methods:The ONE Study consisted of seven investigator-led, single-arm trials done internationally at eight hospitals in France, Germany, Italy, the UK, and the USA (60 week follow-up). Included patients were living-donor kidney transplant recipients aged 18 years and older. The reference group trial (RGT) was a standard-of-care group given basiliximab, tapered steroids, mycophenolate mofetil, and tacrolimus. Six non-randomised phase 1/2A cell therapy group (CTG) trials were pooled and analysed, in which patients received one of six CBMPs containing regulatory T cells, dendritic cells, or macrophages; patient selection and immunosuppression mirrored the RGT, except basiliximab induction was substituted with CBMPs and mycophenolate mofetil tapering was allowed. None of the trials were randomised and none of the individuals involved were masked. The primary endpoint was biopsy-confirmed acute rejection (BCAR) within 60 weeks after transplantation; adverse event coding was centralised. The RTG and CTG trials are registered withClinicalTrials.gov,NCT01656135,NCT02252055,NCT02085629,NCT02244801,NCT02371434,NCT02129881, andNCT02091232. Findings:The seven trials took place between Dec 11, 2012, and Nov 14, 2018. Of 782 patients assessed for eligibility, 130 (17%) patients were enrolled and 104 were treated and included in the analysis. The 66 patients who were treated in the RGT were 73% male and had a median age of 47 years. The 38 patients who were treated across six CTG trials were 71% male and had a median age of 45 years. Standard-of-care immunosuppression in the recipients in the RGT resulted in a 12% BCAR rate (expected range 3·2–18·0). The overall BCAR rate for the six parallel CTG trials was 16%. 15 (40%) patients given CBMPs were successfully weaned from mycophenolate mofetil and maintained on tacrolimus monotherapy. Combined adverse event data and BCAR episodes from all six CTG trials revealed no safety concerns when compared with the RGT. Fewer episodes of infections were registered in CTG trials versus the RGT. Interpretation:Regulatory cell therapy is achievable and safe in living-donor kidney transplant recipients, and is associated with fewer infectious complications, but similar rejection rates in the first year. Therefore, immune cell therapy is a potentially useful therapeutic approach in recipients of kidney transplant to minimise the burden of general immunosuppression.

Background: Kidney transplant recipients (KTRs) with “operational tolerance” (OT) maintain a functioning graft without immunosuppressive (IS) drugs, thus avoiding treatment complications. Nevertheless, IS drugs can influence gene-expression signatures aiming to identify OT among treated KTRs. Methods: We compared five published signatures of OT in peripheral blood samples from 18 tolerant, 183 stable, and 34 chronic rejector KTRs, using gene-expression levels with and without adjustment for IS drugs and regularised logistic regression. Findings: IS drugs explained up to 50% of the variability in gene-expression and 20–30% of the variability in the probability of OT predicted by signatures without drug adjustment. We present a parsimonious consensus gene-set to identify OT, derived from joint analysis of IS-drug-adjusted expression of five published signature gene-sets. This signature, including CD40, CTLA4, HSD11B1, IGKV4–1, MZB1, NR3C2, and RAB40C genes, showed an area under the curve 0⋅92 (95% confidence interval 0⋅88–0⋅94) in cross-validation and 0⋅97 (0⋅93–1⋅00) in six months follow-up samples. Interpretation: We advocate including adjustment for IS drug therapy in the development stage of gene-expression signatures of OT to reduce the risk of capturing features of treatment, which could be lost following IS drug minimisation or withdrawal. Our signature, however, would require further validation in an independent dataset and a biomarker-led trial. Funding: FP7-HEALTH-2012-INNOVATION-1 [305147:BIO-DrIM] (SC,IR-M,PM,DSt); MRC [G0801537/ID:88245] (MPH-F); MRC [MR/J006742/1] (IR-M); Guy's&StThomas’ Charity [R080530]&[R090782]; CONICYT-Bicentennial-Becas-Chile (EN-L); EU:FP7/2007–2013 [HEALTH-F5–2010–260687: The ONE Study] (MPH-F); Czech Ministry of Health [NV19–06–00031] (OV); NIHR-BRC Guy's&StThomas' NHS Foundation Trust and KCL (SC); UK Clinical Research Networks [portfolio:7521].

BACKGROUND: An increased percentage of peripheral transitional B cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B cell receptor (BCR) signalling modulated IL-10 production by B cells, with stimulation via the BCR decreasing CD40-mediated-IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients the increased IL-10 production by B cells was due to an altered CD40 and/or BCR signalling.METHODS: B cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and gender-matched healthy volunteers, were activated via CD40 and BCR, either alone or in combination.RESULTS: In tolerant patients we observed higher percentages of B cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T cells, compared to healthy controls. Furthermore, B cells from tolerant recipients had reduced ERK signalling following BCR-mediated activation compared to healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B cells.CONCLUSION: Altogether our data suggests that the altered response of B cells in tolerant recipients may contribute to long-term stable graft acceptance.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially.

Background: Long-term survival with good allograft function (LTS) after lung transplantation (LT) is mainly limited by the development of chronic lung allograph dysfunction (CLAD). The objective of this study was to identify genes and miRNAs which might contribute to a better understanding of the molecular mechanisms influencing LTS after LT. Methods: The mRNA and miRNAs expression profiles were determined in blood samples from LTS (n=30) and CLAD patients (n=30) by microarray technology. Gene Ontology was used for enrichment analysis and the interactions between miRNAs and genes were studied by network analysis with the mixOmics R package. Results: The analysis of mRNA expression revealed that 458 genes were differentially expressed between LTS versus CLAD patients. Genes related to neutrophil-granulocyte activation and neutrophil degranulation were downregulated in LTS, suggesting a dysregulation of the innate immune system in CLAD patients. Regarding miRNAs expression analysis, 36 mature miRNAs were differentially expressed between both groups. Of these 36 elements, 30 miRNAs had experimentally validated target genes enriched in the set of 458 differentially expressed genes. Biological significance analysis showed that miRNA target genes differentially expressed between LTS and CLAD patients were related to neutrophil degranulation. Conclusion: Differences in both mRNA expression and upstream miRNA regulators have been found between LTS and CLAD patients. These results suggest that innate immune system may play a role in long-term survival after LT. Study financed by ISCIII (PI13/01076), FEDER, FUCAP, SEPAR (138/2016), Astellas, Novartis and Chiesi.

Genetic variation across the human leukocyte antigen loci is known to influence renal-transplant outcome. However, the impact of genetic variation beyond the human leukocyte antigen loci is less clear. We tested the association of common genetic variation and clinical characteristics, from both the donor and recipient, with posttransplant eGFR at different time-points, out to 5 years posttransplantation. We conducted GWAS meta-analyses across 10 844 donors and recipients from five European ancestry cohorts. We also analyzed the impact of polygenic risk scores (PRS), calculated using genetic variants associated with nontransplant eGFR, on posttransplant eGFR. PRS calculated using the recipient genotype alone, as well as combined donor and recipient genotypes were significantly associated with eGFR at 1-year posttransplant. Thirty-two percent of the variability in eGFR at 1-year posttransplant was explained by our model containing clinical covariates (including weights for death/graft-failure), principal components and combined donor-recipient PRS, with 0.3% contributed by the PRS. No individual genetic variant was significantly associated with eGFR posttransplant in the GWAS. This is the first study to examine PRS, composed of variants that impact kidney function in the general population, in a posttransplant context. Despite PRS being a significant predictor of eGFR posttransplant, the effect size of common genetic factors is limited compared to clinical variables.

Purpose Chronic lung allograft dysfunction (CLAD) is the main limiting factor for long-term survival after lung transplantation (LT). However, there is a small number of LT patients which are long-term survivors with good allograft function (LTS). The aim of the present study was to compare the gene expression profile of CLAD and LTS patients and performing an enrichment analysis by using Reactome annotation database. Methods We analyzed whole-blood gene expression profiles of 60 double lung transplant recipients who were included in this multicenter cross-sectional study: 30 patients with CLAD and 30 patients with a stable allograft function 10 years after lung transplantation (LTS). Expression analysis were performed by microarray technology (ClariomTM D Arrays). Results We identified 458 differentially expressed genes (DEGs) between LTS and CLAD patients. Among them, 201 genes were up-regulated and 257 genes were down-regulated. Differentially expressed genes included MMP-8, LTF, TCN1 and OLFM4. Enrichment analysis showed involvement of neutrophil degranulation, class A/1 (Rhodopsin-like receptors), cell surface interaction at the vascular wall, antimicrobial peptides, interleukin-4 and 13 signaling, role of phospholipids in phagocytosis and activation of matrix metalloproteinases pathways. Conclusion It seems that neutrophil degranulation, matrix metalloproteinases and antimicrobial peptides gene expression pathways within others could be involve in differences between CLAD and LTS patients. Study financed by Instituto de Salud Carlos III (PI13/01076); the European Regional Development Fund (FEDER), FUCAP, Astellas, Novartis and Chiesi.

Steroid conversion (HSD11B1, HSD11B2, H6PD) and receptor genes (NR3C1, NR3C2) were examined in kidney-transplant recipients with "operational tolerance" and chronic rejection (CR), independently and within the context of 88 tolerance-associated genes. Associations with cellular types were explored. Peripheral whole-blood gene-expression levels (RT-qPCR-based) and cell counts were adjusted for immunosuppressant drug intake. Tolerant (n = 17), stable (n = 190) and CR patients (n = 37) were compared. Healthy controls (n = 14) were used as reference. The anti-inflammatory glucocorticoid receptor (NR3C1) and the cortisol-activating HSD11B1 and H6PD genes were up-regulated in CR and were lowest in tolerant patients. The pro-inflammatory mineralocorticoid gene (NR3C2) was downregulated in stable and CR patients. NR3C1 was associated with neutrophils and NR3C2 with T-cells. Steroid conversion and receptor genes, alone, enabled classification of tolerant patients and were major contributors to gene-expression signatures of both, tolerance and CR, alongside known tolerance-associated genes, revealing a key role of steroid regulation and response in kidney transplantation.

BACKGROUND: The development of spontaneous kidney transplant tolerance has been associated with numerous B cell-related immune alterations. We have previously shown that tolerant recipients exhibit reduced B cell receptor (BCR) signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether CD4T cells from tolerant recipients also display an antiinflammatory profile that could contribute to graft maintenance.METHODS: CD4T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic-rejection or healthy volunteers. CD4T cells from the 3 groups were compared in terms of their gene expression profile, phenotype and functionally upon activation.RESULTS: Gene expression analysis of transcription factors and signalling proteins, in addition to surface proteins expression and cytokine production, revealed that tolerant recipients possessed fewer Th17 cells and exhibited reduced Th17 responses, relative to patients with chronic rejection or healthy volunteers. Furthermore, impaired TCR signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients.CONCLUSIONS: These data suggest that defective proinflammatory Th17 responses may contribute to the prolonged graft survival and stable graft function, which is observed in tolerant recipients in the absence of immunosuppressive agents.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC), where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal.

Improvements in immunosuppression have modified short-term survival of deceased-donor allografts, but not their rate of long-term failure. Mismatches between donor and recipient HLA play an important role in the acute and chronic allogeneic immune response against the graft. Perfect matching at clinically relevant HLA loci does not obviate the need for immunosuppression, suggesting that additional genetic variation plays a critical role in both short- and long-term graft outcomes. By combining patient data and samples from supranational cohorts across the United Kingdom and European Union, we performed the first large-scale genome-wide association study analyzing both donor and recipient DNA in 2094 complete renal transplant-pairs with replication in 5866 complete pairs. We studied deceased-donor grafts allocated on the basis of preferential HLA matching, which provided some control for HLA genetic effects. No strong donor or recipient genetic effects contributing to long- or short-term allograft survival were found outside the HLA region. We discuss the implications for future research and clinical application.

The complex relationship between Th1 and Th17 cells is incompletely understood. The transcription factor T-bet is best known as the master regulator of Th1 lineage commitment. However, attention is now focused on the repression of alternate T cell subsets mediated by T-bet, particularly the Th17 lineage. It has recently been suggested that pathogenic Th17 cells express T-bet and are dependent on IL-23. However, T-bet has previously been shown to be a negative regulator of Th17 cells. We have taken an unbiased approach to determine the functional impact of T-bet on Th17 lineage commitment. Genome-wide analysis of functional T-bet binding sites provides an improved understanding of the transcriptional regulation mediated by T-bet, and suggests novel mechanisms by which T-bet regulates Th cell differentiation. Specifically, we show that T-bet negatively regulates Th17 lineage commitment via direct repression of the transcription factor IFN regulatory factor-4 (IRF4). An in vivo analysis of the pathogenicity of T-bet–deficient T cells demonstrated that mucosal Th17 responses were augmented in the absence of T-bet, and we have demonstrated that the roles of T-bet in enforcing Th1 responses and suppressing Th17 responses are separable. The interplay of the two key transcription factors T-bet and IRF4 during the determination of T cell fate choice significantly advances our understanding of the mechanisms underlying the development of pathogenic T cells.

As the induction and maintenance of donor-specific tolerance is a central aim in solid organ transplantation, it is essential that clinicians are able to identify and monitor tolerance accurately and reliably. This review highlights recent advances in defining sets of biomarkers in noninvasive samples that may guide minimization and withdrawal of immunosuppression in tolerant recipients.Recent studies in liver and kidney transplant recipients have identified distinct biomarker profiles that are associated with operational tolerance. Although there is some heterogeneity in the findings of these studies, these have suggested novel cellular mechanisms for the development of tolerance.Multiple platforms such as microarray gene expression analysis, flow cytometry, and immune cell functional assays have been used to discover and validate composite sets of biomarkers, which identify recipients with operational tolerance both in liver and kidney transplantation. These studies suggest that distinct cellular and molecular mechanisms lead to the development of tolerance in different transplanted organs. These putative biomarker profiles now need to be validated prospectively in trials of immunosuppression withdrawal and in novel approaches to induce transplant tolerance.

PURPOSE OF REVIEW Balancing the level of anti-rejection therapy is the key challenge facing clinicians managing recipients of a solid organ transplant. Identification of biomarkers in non-invasive samples will allow serial monitoring which can prompt changes in therapy at an earlier stage without the need for invasive tests, and before significant damage to the graft or side effects have occurred. In this review we provide an update on the present status of such biomarker discovery in renal transplantation. RECENT FINDINGS Previous studies focusing on candidate biomarkers have identified a number of genes that have the potential to identify patients undergoing rejection. Advances in technology now allow screening of samples for markers which have led to identification of further genes as well as adding microRNAs and proteins to the list. Similarly, by studying patients in whom anti-rejection therapy has been withdrawn, a gene signature for operational tolerance has been described. SUMMARY It has become clear that to obtain a test suitable for clinical purposes, combinations of markers are required to identify specific clinical phenotypes. Identification and validation of such marker sets in large cohorts is urgently required to allow progression to clinical trials with the ultimate goal of offering recipients personalized anti-rejection therapy regimes.

Assessment of donor-specific alloreactive memory/effector T cell responses using an IFN-γ Elispot assay has been suggested to be a novel immune-monitoring tool for evaluating the cellular immune risk in renal transplantation. Here, we report the cross-validation data of the IFN-γ Elispot assay performed within different European laboratories taking part of the EU RISET consortium. For this purpose, development of a standard operating procedure (SOP), comparisons of lectures of IFN-γ plates assessing intra- and interlaboratory assay variability of allogeneic or peptide stimuli in both healthy and kidney transplant individuals have been the main objectives. We show that the use of a same SOP and count-settings of the Elispot bioreader allow low coefficient variation between laboratories. Frozen and shipped samples display slightly lower detectable IFN-γ frequencies than fresh samples. Importantly, a close correlation between different laboratories is obtained when measuring high frequencies of antigen-specific primed/memory T cell alloresponses. Interestingly, significant high donor-specific alloreactive T cell responses can be similarly detected among different laboratories in kidney transplant patients displaying histological patterns of acute T cell mediated rejection. In conclusion, assessment of circulating alloreactive memory/effector T cells using an INF-γ Elispot assay can be accurately achieved using the same SOP, Elispot bioreader and experienced technicians in kidney transplantation.

Chronic antibody-mediated rejection, a common cause of renal transplant failure, has a variable clinical phenotype. Understanding why some with chronic antibody-mediated rejection progress slowly may help develop more effective therapies. B lymphocytes act as antigen-presenting cells for in vitro indirect antidonor interferon-γ production in chronic antibody-mediated rejection, but many patients retain the ability to regulate these responses. Here we test whether particular patterns of T and B cell antidonor response associate with the variability of graft dysfunction in chronic antibody-mediated rejection. Our results confirm that dynamic changes in indirect antidonor CD4+ T-cell responses correlate with changes in estimated glomerular filtration rates, independent of other factors. Graft dysfunction progressed rapidly in patients who developed unregulated B-cell–driven interferon-γ production. However, conversion to a regulated or nonreactive pattern, which could be achieved by optimization of immunosuppression, associated with stabilization of graft function. Functional regulation by B cells appeared to activate an interleukin-10 autocrine pathway in CD4+ T cells that, in turn, impacted on antigen-specific responses. Thus, our data significantly enhance the understanding of graft dysfunction associated with chronic antibody-mediated rejection and provide the foundation for strategies to prolong renal allograft survival, based on regulation of interferon-γ production.

A novel subset of human regulatory B-cells has recently been described. They arise from within the transitional B-cell subpopulation and are characterised by the production of IL-10. They appear to be of significant importance in regulating T-cell immunity in vivo. Despite this important function, the molecular mechanisms by which they control T-cell activation are incompletely defined. Here we show that transitional B-cells produced more IL-10 and expressed higher levels of IL-10 receptor after CD40 engagement compared to other B-cell subsets. Furthermore, under this stimulatory condition, CD86 expressed by transitional B-cells was down regulated and T-cell proliferation was reduced. We provide evidence to demonstrate that the down-regulation of CD86 expression by transitional B-cells was due to the autocrine effect of IL-10, which in turn leads to decreased T-cell proliferation and TNF-α production. This analysis was further extended to peripheral B-cells in kidney transplant recipients. We observed that B-cells from patients tolerant to the graft maintained higher IL-10 production after CD40 ligation, which correlates with lower CD86 expression compared to patients with chronic rejection. Hence, the results obtained in this study shed light on a new alternative mechanism by which transitional B-cells inhibit T-cell proliferation and cytokine production.

Regulatory T cells (CD4+CD25hiCD127loFOXP3+ T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4+CD25− [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.

In a recent issue Asare et al. (1) assess the utility of a previously identified signature of tolerance, based on the expression of two B-cell receptor genes (IGKV1D-13 and IGKV4-1). The authors set to determine the prevalence of “tolerance” predictions in treated stable kidney transplant recipients (KTRs) and “tolerance” frequency in patients with different immunosuppressant (IS) regimens. They illustrate (Figure 6), very successfully, that changes in the signature are compatible with known alterations of the B-cell compartment elicited by IS drugs. This article is protected by copyright. All rights reserved.

To investigate the role of donor-specific indirect pathway T cells in renal transplant tolerance, we analyzed responses in peripheral blood of 45 patients using the trans-vivo delayed-type hypersensitivity assay. Subjects were enrolled into five groups-identical twin, clinically tolerant (TOL), steroid monotherapy (MONO), standard immunosuppression (SI) and chronic rejection (CR)-based on transplant type, posttransplant immunosuppression and graft function. The indirect pathway was active in all groups except twins but distinct intergroup differences were evident, corresponding to clinical status. The antidonor indirect pathway T effector response increased across patient groups (TOLMONOSICR; p0.0001) whereas antidonor indirect pathway T regulatory response decreased (TOLMONO = SICR; p0.005). This pattern differed from that seen in circulating naïve B-cell numbers and in a cross-platform biomarker analysis, where patients on monotherapy were not ranked closest to TOL patients, but rather were indistinguishable from chronically rejecting patients. Cross-sectional analysis of the indirect pathway revealed a spectrum in T-regulatory:T-effector balance, ranging from TOL patients having predominantly regulatory responses to CR patients having predominantly effector responses. Therefore, the indirect pathway measurements reflect a distinct aspect of tolerance from the recently reported elevation of circulating naïve B cells, which was apparent only in recipients off immunosuppression.

Successful expansion of functional CD4(+) CD25(+) regulatory T cells (T(reg)) ex vivo under good manufacturing practice conditions has made T(reg) -cell therapy in clinical transplant tolerance induction a feasible possibility. In animals, T(reg) cells home to both transplanted tissues and local lymph nodes and are optimally suppressive if active at both sites. Therefore, they have the opportunity to suppress both naïve and memory CD4(+) CD25(-) T cells (Tresp). Clinical transplantation commonly involves depleting therapy at induction (e.g. anti-CD25), which favors homeostatic expansion of memory T cells. Animal models suggest that T(reg) cells are less suppressive on memory, compared with naïve Tresp that mediate allograft rejection. As a result, in the context of human T(reg) -cell therapy, it is important to define the effectiveness of T(reg) cells in regulating naïve and memory Tresp. Therefore, we compared suppression of peripheral blood naïve and memory Tresp by fresh and ex vivo expanded T(reg) cells using proliferation, cytokine production and activation marker expression (CD154) as readouts. With all readouts, naïve human Tresp were more suppressible by approximately 30% than their memory counterparts. This suggests that T(reg) cells may be more efficacious if administered before or at the time of transplantation and that depleting therapy should be avoided in clinical trials of T(reg) cells.

Induction of transplantation tolerance remains the ideal long-term clinical and logistic solution to the current challenges facing the management of renal allograft recipients. In this review, we describe the recent studies and advances made in identifying biomarkers of renal transplant tolerance, from study inceptions, to the lessons learned and their implications for current and future studies with the same goal. With the age of biomarker discovery entering a new dimension of high-throughput technologies, here we also review the current approaches, developments, and pitfalls faced in the subsequent statistical analysis required to identify valid biomarker candidates.

International audience; The long-term stability of renal grafts depends on the absence of chronic rejection. As T cells play a key role in rejection processes, analyzing the T-cell repertoire may be useful for understanding graft function outcomes. We have therefore investigated the power of a new statistical tool, used to analyze the peripheral blood TCR repertoire, for determining immunological differences in a group of 229 stable renal transplant patients undergoing immunosuppression. Despite selecting the patients according to stringent criteria, the patients displayed heterogeneous T-cell repertoire usage, ranging from unbiased to highly selected TCR repertoires; a skewed TCR repertoire correlating with an increase in the CD8(+)/CD4(+) T-cell ratio. T-cell repertoire patterns were compared in patients with clinically opposing outcomes i.e. stable drug-free operationally tolerant recipients and patients with the "suspicious" form of humoral chronic rejection and were found significantly different, from polyclonal to highly selected TCR repertoires, respectively. Moreover, a selected TCR repertoire was found to positively correlate with the Banff score grade. Collectively, these data suggest that TCR repertoire categorization might be included in the calculation of a composite score for the follow-up of patients after kidney transplantation.

In the past decade, an explosion in the number of high-throughput tools for the measurement of different cellular products has occurred. These tools have the potential to further our understanding of human disease and this development has facilitated the identification of new biomarkers in all areas of medicine. In the field of solid organ transplantation, two different areas have developed: the use of biomarkers to predict allograft tolerance for the identification of patients who can be weaned from immunosuppressive therapy, and biomarkers for the prediction of allograft rejection, so that parenchymal damage can be prevented before it becomes irreversible. In this Review, we discuss the development of biomarkers that are indicative of transplant tolerance. Identifying patients in whom donor-specific tolerance has developed would constitute a major advance in the care of organ transplant recipients. This ability would allow the minimization or even the withdrawal of immunosuppressive therapy in selected patients, thus reducing the number of adverse effects and costs, and optimizing long-term graft outcomes. The routine clinical use of these biomarkers, once validated, would bring to the fore the possibility of personalized medicine.

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.

In order to determine the relationships between CD2+ lymphocyte subpopulations and tumour mass, the immunophenotype of natural killer (NK) cells and T lymphocyte subsets was studied in 56 B-chronic lymphocytic leukaemia (B-CLL) patients and 38 healthy subjects. The patients were classified according to their blood lymphocyte count (BLC). Forty patients had BLC30x10(9)/l (low BLC, less tumour mass) and 16 patients had BLC30x10(9)/l (high BLC, larger tumour mass). The percentage of CD3- CD56+ cells, as well as of CD8+, CD8+ CD45RO+ and CD3+ CD57+ T subsets in low BLC patients, were higher than those found in high BLC patients. Conversely, the percentages of CD3+ HLA x DR+, CD4+ and CD4+ CD45RO+ lymphocytes were higher in high BLC patients than in low BLC patients. The CD4/CD8 ratio was decreased in low BLC patients while it was increased in high BLC patients and a significant positive correlation was found between their CD4/CD8 ratio and their BLC. We conclude that in low BLC B-CLL patients there is a decreased percentage of activated helper lymphocytes and an increased percentage of NK cells and activated cytotoxic T lymphocytes. These results suggest a role for NK cells, and helper and cytotoxic T lymphocytes in the control of tumour burden in B-CLL patients.

Background. The trans vivo delayed-type hypersensitivity (DTH) assay has been used for monitoring the immune status of clinical transplant recipients. Here we tested the hypothesis that the assay can reveal control of allograft rejection by CD25 + CD4 + regulatory T cells (Treg). Methods. CBA.Ca (H2 k ) recipients ofheterotopic C57BL/10 (H2 b , B 10) heart transplants were untreated or pretreated with anti-CD4 antibody and donor-specific blood. This protocol has been shown previously to induce operational tolerance to alloantigens that is dependent on CD25 + CD4 + Treg. Four weeks after transplantation leukocytes were harvested and used for the trans vivo DTH assay. Cells were stimulated with irradiated B10 leukocytes or subcellular antigen and injected into ear pinnae of immune deficient CB17.SCID.beige hosts. Results. Stimulation of leukocytes from recipients rejecting B10 cardiac allografts with recall alloantigen caused a "strong" swelling response, whereas similar stimulation of leukocytes from operationally tolerant mice resulted in significantly less swelling (n=17; P=0.003). When CD25 + T cells were depleted from "tolerant" leukocytes, the swelling response triggered was similar to that obtained using cells from rejecting animals (P

International audience; Patients tolerant to a kidney graft display a specific blood cell transcriptional pattern but results from five different studies were inconsistent, raising the question of relevance for future clinical application. To resolve this, we sought to identify a common gene signature, specific functional and cellular components, and discriminating biomarkers for tolerance following kidney transplantation. A meta-analysis of studies identified a robust gene signature involving proliferation of B and CD4 T cells, and inhibition of CD14 monocyte related functions among 96 tolerant samples. This signature was further supported through a cross-validation approach, yielding 92.5% accuracy independent of the study of origin. Experimental validation, performed on new tolerant samples and using a selection of the top-20 biomarkers, returned 91.7% of good classification. Beyond the confirmation of B-cell involvement, our data also indicated participation of other cell subsets in tolerance. Thus, the use of the top 20 biomarkers, mostly centered on B cells, may provide a common and standardized tool towards personalized medicine for the monitoring of tolerant or low-risk patients among kidney allotransplant recipients. These data point to a global preservation of genes favoring the maintenance of a homeostatic and 'healthy' environment in tolerant patients and may contribute to a better understanding of tolerance maintenance mechanisms.

Background: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. Methods: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). Results: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. Conclusions: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis. © 2006 International Society for Analytical Cytology

PUNTOS CLAVE Mecanismos de rechazo inmunologico a los trasplantes. Los linfocitos T inician la respuesta inmune al tejido trasplantado. Los linfocitos B producen aloanticuerpos e intervienen como celulas presentadoras de antigeno. Formas clinicas de rechazo. El rechazo hiperagudo ocurre en las primeras 48 horas por anticuerpos preformados en el suero del receptor que se unen a las celulas endoteliales del tejido con resultado de trombosis intravascular y necrosis. El rechazo agudo ocurre desde varios dias a meses despues y se relaciona con la presentacion directa de los aloantigenos. El rechazo cronico es dificilmente tratable en la actualidad. Linfocitos CD4+ CD25+. Tienen un papel importante en la induccion de tolerancia en el trasplante. Tratamiento del trasplante alogenico. Se basa en la combinacion de diversos inmunosupresores con una fase de induccion y una posterior de mantenimiento entre los que destacan: corticoides, ciclosporina y tacrolimus, rapamicina, micofenolato mofetil y azatioprina.

The analysis of apoptosis in cell populations involves the detection of their specific lineage antigen (LAg) expression. This experimental approach relies on their assumed constant expression, but it is unclear whether such expression is actually maintained during cell death. We examined whether the loss of LAgs is a common feature of apoptotic lymphocytes and whether some might completely lose their LAgs. The changes in the expression of CD3, CD5, CD8, CD4, CD28, CD56, and CD19 were monitored in highly purified lymphocyte populations obtained by negative selection in a fluorescence-activated cell sorter. These were cultured for 24 h with or without phytohemagglutinin or staurosporin. For each LAg-positive subset studied, apoptosis was consistently more common among cells showing partial or total loss of LAg expression compared with cells maintaining their initial LAg levels. The kinetics of expression loss was rapid for CD8, CD56, and CD28, and more than 80% of initial expression was lost in the early stages of apoptosis but was slower for CD3, CD5, and CD4. For CD3 and CD5, expression was dependent on the apoptotic stimulus used. It is interesting that loss of antigen expression was independent of cell size. This phenomenon was also found in nonmanipulated, highly pure CD19 B lymphocytes of peripheral blood mononuclear cells from B chronic lymphocytic leukemia patients. Loss of LAg expression appeared to be a common feature of apoptotic lymphocytes under all the conditions assayed. The different kinetic patterns of LAg loss suggest apoptotic cells might actively regulate this process.

CD4(+)CD25(+) regulatory T cells have been shown to regulate a variety of autoimmune and allogeneic responses in mice and humans. The role of CD4(+)CD25(+) cells in regulating alloresponses in human transplant recipients remains uncertain. Previous research has demonstrated a reduced frequency of direct pathway donor-specific T cells in renal transplant recipients when compared with the frequency of T cells reactive to an HLA-matched third party. A number of mechanisms have been proposed to account for this finding; the purpose of this study was to determine whether CD4(+)CD25(+) cells play a significant role. Twelve stable renal transplant patients were investigated using limiting dilution assay (LDA) and ELISPOT for interferon-gamma to determine the effect of depleting CD4(+)CD25(+) cells on the direct pathway alloresponse. The percentage of CD4(+)CD25(+) cells in the peripheral blood of the study patients was equivalent to that of healthy controls. Furthermore, in no case did depletion of CD4(+)CD25(+) cells significantly increase the frequency of donor-specific T cells detected by LDA. This was also found with ELISPOT in all except one patient, in whom depletion revealed an increased frequency of alloreactive T cell to both donor and third party. Finally, kinetic analysis of the LDA data did not indicate regulation against donor when compared with third party. It is concluded that the action of CD4(+)CD25(+) regulatory cells is not the main mechanism of donor-specific hyporesponsiveness in the direct pathway of allorecognition.

Data from various rodent models have implicated a role for anergic T cells in the maintenance of self and trans- plantation tolerance. The relevance of donor-specific T cell anergy to clinical transplantation, however, has not been dem- onstrated. Previous studies have reported that recipients of solid organ transplant often have reduced frequencies of CD4 T cells with anti-donor direct pathway allospecificity after transplantation. The underlying mechanism(s) of this donor- specific hyporesponsiveness is unclear but likely to contribute to the diminished immunosuppressive requirement of trans- plant patients with time after transplantation. This study shows that ex vivo treatment of CD4 T cells from renal transplant recipients with IL-2 could specifically increase the anti-donor frequency in all the patients with evidence of donor-specific hyporesponsiveness. It also shows that the IL-2-induced re- covery of anti-donor frequency is unlikely to result from non- specific stimulation or selective clonal expansion of activated, allospecific CD4 T cells. Taken together, the data suggest that T cell anergy plays an important role in the direct pathway hyporesponsiveness that evolves in many human renal trans- plant recipients. Heightened interest in clinical transplant tolerance stems from the acquisition of insights into the mechanisms and the advent of novel biologic reagents that may promote such a tolerant state. For these reasons, it is necessary to understand the evolution of anti-donor responses using current approaches to immunosuppression. We and others (1-3) have previously described that a substantial proportion of patients with kidney or other solid organ transplants have markedly reduced num- bers of T cells with direct allospecificity, particularly in long- term live-related transplant recipients. Although markedly de- creased frequencies of direct pathway T cells does not equate to tolerance, it is tempting to speculate that mechanisms that contribute to peripheral T cell tolerance may be operative in diminishing the anti-donor T cell repertoire. It is also important to note that direct pathway hyporesponsiveness can often be observed in patients with chronic transplant rejection. In these patients, it appears that the indirect pathway of anti-donor alloimmunity provides an immunologic drive to this indolent process.

We explored how B-lymphocytes influence in vitro T-cell alloresponses in patients with antibody-mediated rejection (AMR), testing whether B-cells would be preferentially involved in this group of patients. Peripheral blood mononuclear cells were collected from 65 patients having biopsy: 14 patients with AMR and 5 with no pathology on protocol; 38 with AMR and 8 with nonimmunologic damage on 'for cause'. Using enzyme-linked immunosorbent spot assays, we found interferon-γ production by indirect allorecognition in 45 of 119 total samples from the 65 patients. B-cells preferentially processed and presented donor alloantigens in samples from AMR patients. In a further 25 samples, B-cell-dependent allo-specific reactivity was shown by depletion of CD25(+) cells and these individuals had higher percentages of CD4CD25hi cells. In 21 samples, reactivity was shown by depletion of CD19(+) cells, associated with polarized cytokine production toward IL-10 after polyclonal activation by IgG/IgM. Overall, this shows a significant contribution by B-cells to indirect donor-specific T-cell reactivity in vitro in patients with AMR. Active suppression by distinct phenotypes of T- or B-cells in approximately half of the patients indicates that chronic AMR is not characterized by a universal loss of immune regulation. Thus, stratified approaches that accommodate the heterogeneity of cell-mediated immunity might be beneficial to treat graft dysfunction.

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 naive B cells express IL-10 and the proinflammatory cytokine TNF-α simultaneously. TrBs had the highest IL-10/TNF-α 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-α increased the suppressive capacity of both memory and naive B-cell subsets. Thus, the ratio of IL-10/TNF-α 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-α expression (i.e., reduced IL-10/TNF-α 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-α 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.

Background. With adequate immunosuppression the majority of renal allografts are accepted, despite the exceptional vigour of the T cell alloimmune response. Previous work from this laboratory has demonstrated that this is accompanied by significant reductions in the precursor frequencies of anti-donor T cells. We have also shown that parenchymal cells are tolerogenic in vitro. We propose that the reduction in T cell frequencies may be due to the interaction between circulating T cells and potentially tolerogenic graft parenchymal cells. Primed/memory T cells (CD45RO + ) are the only subset capable of reaching the allograft and therefore we would predict that T cell hyporesponsiveness would develop predominantly in the CD45RO + subset due to their trafficking properties. Methods. Frequencies of IL-2 secreting CD45RA + and CD45RO + CD4 + T cells in response to donor and third party stimulator cells were estimated in a series of renal transplant recipients, both before and after transplantation. Results. There were highly significant reductions in the frequencies of donor-specific CD4 + CD45RO + T cells, when adjusted to control for the generalised effects of immunosuppression. There were no significant alterations in the frequencies of donor-specific CD4 + CD45RA + T cells. Conclusions. In renal transplant recipients, donor-specific CD4 + T cell hyporesponsiveness occurs predominantly in CD4 + CD45RO + T cells which is the subset capable of trafficking through the graft.

OBJECTIVE Patients with type 1 diabetes and microalbuminuria are at increased risk of cardiovascular disease (CVD). Abnormalities in vascular progenitor cells, which participate in vascular repair, may be implicated in this susceptibility. RESEARCH DESIGN AND METHODS We studied the number and function of vascular progenitor cells in 22 type 1 diabetic patients with history of microalbuminuria (MA+) and 22 type 1 diabetic patients without history of microalbuminuria (MA−), of similar age, diabetes duration, glycemic control, renal function, and no history of CVD. RESULTS MA+ patients had lower circulating CD34+ and CD34+/CD133+ cell numbers compared with MA− patients (P < 0.006). In in vitro functional assays, MA+ patients had a significantly lower number of colony-forming units and impaired vascular endothelial growth factor (VEGF)-A–mediated tube formation, when compared with MA− patients (P < 0.01). CONCLUSIONS In type 1 diabetic patients with microalbuminuria, a marker of microvascular injury and a risk factor for CVD, circulating vascular progenitor cell number is reduced and function is impaired.

Background —The purpose of this study was to determine whether T cells with indirect allospecificity could be detected in heart transplant recipients with chronic rejection. Methods and Results —Human T-cell clones were used to determine the most effective way to deliver major histocompatibility complex alloantigens for indirect presentation. Seven allograft recipients with evidence of progressive, chronic rejection were selected. Four heart graft recipients with no evidence of chronic rejection were used as controls. Peripheral blood T cells and antigen-presenting cells from the recipients were cultured with frozen/thawed stored donor cells or major histocompatibility complex class I–derived synthetic peptides in limiting dilution cultures and then compared with controls using tetanus toxoid and frozen/thawed third-party cells with no human leukocyte antigens in common with the donor. In 5 of 7 patients analyzed who had chronic rejection, elevated frequencies of T cells with indirect, anti-donor specificity (iHTLf) were detected. No such elevated iHTLf were detected in recipients without chronic rejection. Discussion —iHTLf can be obtained from human transplant recipients, which supports the contention that the indirect pathway is involved in chronic transplant rejection.

Summary Transplantation is a successful treatment for end-stage organ failure. Despite improvements in short-term outcome, long-term survival remains suboptimal because of the morbidity and mortality associated with long-term use of immunosuppression. There is, therefore, a pressing need to devise protocols that induce tolerance in order to minimize or completely withdraw immunosuppression in transplant recipients. In this review we will discuss how regulatory T cells (Tregs) came to be recognized as an attractive way to promote transplantation tolerance. We will summarize the preclinical data, supporting the importance of these cells in the induction and maintenance of immune tolerance and that provide the rationale for the isolation and expansion of these cells for cellular therapy. We will also describe the data from the first clinical trials, using Tregs to inhibit graft-versus-host disease (GVHD) after haematopoietic stem cell transplantation and will address both the challenges and opportunities in human Treg cell therapy.

Discovery of novel immune biomarkers for monitoring of disease prognosis and response to therapy in immune-mediated inflammatory diseases is an important unmet clinical need. Here, we establish a novel framework for immunological biomarker discovery, comparing a conventional (liquid) flow cytometry platform (CFP) and a unique lyoplate-based flow cytometry platform (LFP) in combination with advanced computational data analysis. We demonstrate that LFP had higher sensitivity compared to CFP, with increased detection of cytokines (IFN-γ and IL-10) and activation markers (Foxp3 and CD25). Fluorescent intensity of cells stained with lyophilized antibodies was increased compared to cells stained with liquid antibodies. LFP, using a plate loader, allowed medium-throughput processing of samples with comparable intra- and inter-assay variability between platforms. Automated computational analysis identified novel immunophenotypes that were not detected with manual analysis. Our results establish a new flow cytometry platform for standardized and rapid immunological biomarker discovery with wide application to immune-mediated diseases.

Solid organ transplantation has emerged as the “gold standard” therapy for end-stage organ failure as it improves both quality of life and survival. Despite the progress in short-term graft survival, that is closely associated with the impressive reduction of acute rejections within the first year, long-term graft and patient survival remain almost un-changed and unsatisfactory. Incomplete control of chronic allograft injury but particularly the adverse effects of long-term immunosuppression, such as graft toxicity, diabetes, cardiovascular events, infections, and tumours continue to challenge the long-term success. In general, immunosuppression is applied as one-size-fits-all strategy. This can result in over- and under-immunosuppression of patients with low and high alloresponsiveness, respectively. Trial- and -error strategies to minimize or even completely wean of immunosuppression have a high failure rate. Consequently, there is an unmet medical need to develop biomarkers allowing objective risk stratification of transplant patients. To achieve this goal, we engaged in an academic-industrial partnership. The central focus of the European-wide BIO-DrIM consortium (BIOmarker-Driven IMmmunosuppression) is the implementation of biomarker-guided strategies for personalizing immunosuppression to improve the long-term outcome and to decrease the adverse effects and costs of chronic immunosuppression in solid organ transplant patients. The concept includes four innovative investigator-driven clinical trials designed by the consortium.