Tolerant Kidney Transplant Recipients Research Articles

The N6-methyladenosine writer WTAP contributes to the induction of immune tolerance post kidney transplantation by targeting regulatory T cells

AbstractN6-methyladenosine (m6A) modification is involved in diverse immunoregulation, while the relationship between m6A modification and immune tolerance post kidney transplantation remains unclear. Expression of Wilms tumor 1-associating protein (WTAP), an m6A writer, was firstly detected in tolerant kidney transplant recipients (TOL). Then the role of WTAP on regulatory T (Treg) cell differentiation and function in CD4+ T cells from kidney transplant recipients with immune rejection (IR) was investigated. The potential target of WTAP and effect of WTAP on immune tolerance in vivo were subsequently verified. WTAP was upregulated in CD4+ T cells of TOL and positively correlated with Treg cell proportion. In vitro, WTAP overexpression promoted Treg cell differentiation and enhanced Treg cell-mediated suppression toward naïve T cells. Forkhead box other 1 (Foxo1) was identified as a target of WTAP. WTAP enhanced m6A modification of Foxo1 mRNA in coding sequence (CDS) region, leading to up-regulation of Foxo1. Overexpression of m6A demethylase removed the effect of WTAP overexpression, while Foxo1 overexpression reversed these effects. WTAP overexpression alleviated allograft rejection in model mice, as evidenced by reduced inflammatory response and increased Treg population. Our study suggests that WTAP plays a positive role in induction of immune tolerance post kidney transplant by promoting Treg cell differentiation and function.

Clinical and immunological follow-up of very long-term kidney transplant recipients treated with calcineurin inhibitors indicates dual phenotypes

Operationally tolerant kidney transplant recipients harbor an immunological signature, associated with low rejection risk, and focused on B lymphocytes. Here, we investigated whether patients with long-term transplantation and still on immunosuppressive therapy would present such a signature of low immunological rejection risk, compared to more recently transplanted patients. Of 114 kidney transplant recipients enrolled, 38 with more than 25 years of graft survival and stable graft function under calcineurin inhibitors, were matched with two different groups of transplanted patients (10-15 and 5-7 years after transplantation). Three phenotypes associated with low immunological rejection risk (Tfh, B and regulatory T cells), initially found in operationally tolerant kidney transplant recipients, and the composite score of tolerance (combination of six transcriptomic markers, age at transplantation and age at sampling) were analyzed. We found that very long-term patients were characterized by a significantly lower percentage of total B cells, a significantly higher proportion of CD24HiCD38Lo memory B cells, significantly fewer CD24LoCD38Lo naive B cells, and a significantly lower proportion of PD1HiCCR7Lo Tfh lymphocytes than more recently transplanted patients. This phenotype is associated with a positive composite score of tolerance in patients transplanted for more than 25 years. Thus, our study suggests a dual phenotype in very long-term kidney transplanted patients with an immunological profile associated with low rejection risk.

Regulatory B cells in transplantation: roadmaps to clinic.

Over the last two decades, an additional and important role for B cells has been established in immune regulation. Preclinical studies demonstrate that regulatory B cells (Breg) can prolong allograft survival in animal models and induce regulatory T cells. Operationally tolerant human kidney transplant recipients demonstrate B-cell-associated gene signatures of immune tolerance, and novel therapeutic agents can induce Bregs in phase I clinical trials in transplantation. Our rapidly expanding appreciation of this novel B-cell subtype has made the road to clinical application a reality. Here, we outline several translational pathways by which Bregs could soon be introduced to the transplant clinic.

Research Highlights

Research Highlights

Early expansion of donor-specific Tregs in tolerant kidney transplant recipients

Allograft tolerance, in which a graft is accepted without long-term immunosuppression, could overcome numerous obstacles in transplantation. Human allograft tolerance has been intentionally induced across HLA barriers via combined kidney and bone marrow transplantation (CKBMT) with a regimen that induces only transient chimerism. Tregs are enriched early after CKBMT. While deletional tolerance contributes to long-term tolerance, the role of Tregs remains unclear. We have optimized a method for identifying the donor-specific Treg repertoire and used it to interrogate the fate of donor-specific Tregs after CKBMT. We expanded Tregs with several different protocols. Using functional analyses and T cell receptor sequencing, we found that expanding sorted Tregs with activated donor B cells identified the broadest Treg repertoire with the greatest potency and donor specificity of suppression. This method outperformed both alloantigen stimulation with CTLA4Ig and sequencing of CFSElo cells from the primary mixed lymphocyte reaction. In 3 tolerant and 1 nontolerant CKBMT recipients, we sequenced donor-specific Tregs before transplant and tracked them after transplant. Preexisting donor-specific Tregs were expanded at 6 months after CKBMT in tolerant patients and were reduced in the nontolerant patient. These results suggest that early expansion of donor-specific Tregs is involved in tolerance induction following CKBMT.

Reduced TCR Signaling Contributes to Impaired Th17 Responses in Tolerant Kidney Transplant Recipients.

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 signalling and higher IL-10 production than healthy volunteers. However, it is unclear whether cluster of differentiation (CD)4 T cells from tolerant recipients also display an anti-inflammatory profile that could contribute to graft maintenance. CD4 T cells were isolated from kidney transplant recipients who were identified as being tolerant recipients, patients with chronic rejection or healthy volunteers. CD4 T cells from the 3 groups were compared in terms of their gene expression profile, phenotype, and functionally upon activation. 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 T-cell receptor signalling and altered cytokine cooperation by monocytes contributed to the development of Th17 cells in tolerant recipients. 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.

Current status of tolerance in kidney transplantation.

The attainment of tolerance remains a highly desirable goal in recipients of kidney transplants. Achievement of this goal would extend graft survival and eradicate toxicities related to long-term immunosuppression. Understanding mechanisms of tolerance and strategies to induce tolerance - their risk/benefit profiles - is essential for future success. Mechanistic studies of spontaneously tolerant kidney transplant recipients have uncovered potential roles for B or regulatory T cells, or both, in the maintenance of tolerance. Mixed hematopoietic chimerism has been the most commonly used approach to induce tolerance. Distinct protocols at three major transplant centers have led to successful withdrawal of immunosuppression in a subset of living donor kidney transplant recipients at the expense of complications such as infections and graft versus host disease. The addition of regulatory cell therapies to tolerance induction protocols could enhance success while minimizing complications. This review summarizes the features of spontaneous tolerance in kidney transplant recipients, the results of clinical trials of tolerance induction in the context of living donor kidney transplant, and potential measures to improve the safety and efficacy of tolerance induction strategies.

Longitudinal Studies of a B Cell–Derived Signature of Tolerance in Renal Transplant Recipients

Biomarkers of transplant tolerance would enhance the safety and feasibility of clinical tolerance trials and potentially facilitate management of patients receiving immunosuppression. To this end, we examined blood from spontaneously tolerant renal transplant recipients and patients enrolled in two interventional tolerance trials using flow cytometry and gene expression profiling. Using a previously reported tolerant cohort as well as newly identified tolerant patients, we confirmed our previous finding that tolerance was associated with increased expression of B cell-associated genes relative to immunosuppressed patients. This was not accounted for merely by an increase in total B cell numbers, but was associated with the increased frequencies of transitional and naïve B cells. Moreover, serial measurements of gene expression demonstrated that this pattern persisted over several years, although patients receiving immunosuppression also displayed an increase in the two most dominant tolerance-related B cell genes, IGKV1D-13 and IGLL-1, over time. Importantly, patients rendered tolerant via induction of transient mixed chimerism, and those weaned to minimal immunosuppression, showed similar increases in IGKV1D-13 as did spontaneously tolerant individuals. Collectively, these findings support the notion that alterations in B cells may be a common theme for tolerant kidney transplant recipients, and that it is a useful monitoring tool in prospective trials.

Tolerance signatures in transplant recipients.

The intent of this review was to describe biomarkers that predict or identify individuals who exhibit tolerance to a transplanted organ. The identification of tolerance biomarkers would spare some individuals the toxicity of immunosuppressive agents, enhance the safety of studies to induce tolerance, and provide insights into mechanisms of tolerance that may aid in designing new regimens. Studies of tolerant kidney transplant recipients have revealed an association with B cells. More recent studies have suggested that these B cells may be less mature than from those in nontolerant recipients, and especially suited to suppress alloimmune responses. Biomarkers in tolerant liver transplant patients appear to be distinct from those associated renal tolerance. Most reports have identified an association with natural killer and/or γδ T cells rather than B cells. Recent data indicate biomarkers associated with iron homeostasis within the transplanted liver more accurately predict the tolerant state than do biomarkers expressed in the blood, suggesting that the renal allograft itself, which is infrequently sampled, would be informative. Given the encouraging progress in identifying tolerance biomarkers, it will be important to validate these markers in larger studies of transplant recipients undergoing prospective minimization or withdrawal of immunosuppression.

A different view on immunity: spatial mapping of human T cells over a lifetime.

A different view on immunity: spatial mapping of human T cells over a lifetime.

Monitoring B cell subsets and alloreactivity in kidney transplantation.

B cells are the precursors of antibody producing plasma cells that can give rise to the formation of donor-specific antibodies. However, recent data suggest that besides their role in antibody production, B cells participate in antibody-independent responses, potentially leading to allograft rejection or allograft tolerance. The presence of CD20(+) B cells in kidney graft biopsies has been shown during severe acute rejection episodes and during chronic rejection. Furthermore, operationally tolerant kidney transplant recipients showed a clear B cell dominated fingerprint of tolerance. Several techniques exist to study B cells on different levels. Numerous classification schemes allow for the distinction of many different B cell subsets using flow cytometry. Regardless, data on B cell subsets during stable graft function, rejection or tolerance remain scarce. To obtain a complete picture of the role of B cells during transplantation, antigen specific B cell assays may be required. Therefore, techniques have now been developed that allow for studying the specificity and frequency of HLA specific B cells. Here, we present an overview of the existent assays, panels and techniques intended to characterize peripheral B cells, and the currently available HLA specific B cell functional assays that may allow for monitoring the humoral alloimmune response in transplant recipients.

Tolerant Kidney Transplant Patients Produce B Cells with Regulatory Properties.

Whereas a B cell-transcriptional profile has been recorded for operationally tolerant kidney graft patients, the role that B cells have in this tolerance has not been reported. In this study, we analyzed the role of B cells from operationally tolerant patients, healthy volunteers, and kidney transplant recipients with stable graft function on T cell suppression. Proliferation, apoptosis, and type I proinflammatory cytokine production by effector CD4(+)CD25(-) T cells were measured after anti-CD3/anti-CD28 stimulation with or without autologous B cells. We report that B cells inhibit CD4(+)CD25(-) effector T cell response in a dose-dependent manner. This effect required B cells to interact with T-cell targets and was achieved through a granzyme B (GzmB)-dependent pathway. Tolerant recipients harbored a higher number of B cells expressing GzmB and displaying a plasma cell phenotype. Finally, GzmB(+) B-cell number was dependent on IL-21 production, and B cells from tolerant recipients but not from other patients positively regulated both the number of IL-21(+) T cells and IL-21 production, suggesting a feedback loop in tolerant recipients that increases excessive B cell activation and allows regulation to take place. These data provide insights into the characterization of B cell-mediated immunoregulation in clinical tolerance and show a potential regulatory effect of B cells on effector T cells in blood from patients with operationally tolerant kidney grafts.

Circulating biomarkers of tolerance.

On the basis of reviewed literature here we describe models of tolerance and summarize the evidence of circulating biomarkers suitable for the assessment of immunological risk in organ transplantation. We focused on results of evaluation of specific peripheral immune cell populations and transcripts in peripheral blood of operationally tolerant liver and kidney transplant recipients. Validation of described markers to define potentially tolerant patients before their use in clinical trials is critical.

Frequencies of Circulating B-Cell Subpopulations Before Kidney Transplantation Identify Patients at Risk of Acute Rejection

The response mediated by B lymphocytes has a crucial impact on kidney transplantation due to the role of anti–human leukocyte antigen antibodies in rejection and the contradictory observation of high B-lymphocyte numbers in tolerant kidney transplant recipients. The basis of the contradiction could lay in the different function of B-cell subsets depending on their degree of differentiation. We ought to measure circulating B-lymphocyte percentages in patients with end-stage renal disease before kidney transplantation to identify those with a high risk of acute rejection. Eighty patients on the waiting list for kidney transplantation followed up in our center were recruited from 2010, and samples were taken just before kidney transplantation. Eleven of 80 patients presented an episode of acute rejection (13.75%) and had an increased frequency of switched (SW) B cells compared with the rejection-free group (median [interquartile range] 24.5% [18.6% to 39.6%] vs 15.1 [8.45% to 23.4%]; P = .025). Subsequently, the frequency of SW B cells was assessed as a predicting factor of acute rejection. A value higher than 18.4% predicted patients at risk of suffering an acute rejection episode with a sensitivity of 81.8% and a specificity of 60.9% and an area under the curve of 71.2%. Moreover, a decrease in naïve B-cell subsets was related to patients at risk of acute rejection. The percentage of circulating B-cell subsets before kidney transplantation could be used as biomarker of risk to suffer acute rejection. These unicenter data must be validated in multicenter studies.

B Cells from Tolerant Kidney Transplant Recipients Produce High Levels of CD40L-Mediated IL-10 and Inhibit Th1 Responses after Donor-Antigen Presentation.

Recent studies revealed that tolerance in kidney transplant recipients is associated with the maintenance or expansion of B cells in peripheral circulation. We have thus studied peripheral blood B cells from an independent cohort of operationally tolerant recipients to elucidate whether human B cells are functionally involved in renal transplantation tolerance. We observed that B cells from tolerant patients produced higher levels of IL-10, compared to other groups, exhibited reduced B cell Receptor (BCR) signalling and maintained IL-10 production after BCR/CD40L-double stimulation. Moreover formerly tolerant patients, that subsequently rejected their graft, lost their ability to produce IL-10 in the same conditions. Additionally, B cells from tolerant recipients limited pro-inflammatory Th1 responses by reducing CD86 expression and maintaining CD4+CD25hi cells after donor-antigen presentation. These data suggest that B cells from tolerant recipients are contributing to longer graft survival by increased IL-10 production and by limiting donor-antigen pro-inflammatory responses.Figure: No Caption available.

Prevention of Graft-versus-Host Disease by Adoptive T Regulatory Therapy Is Associated with Active Repression of Peripheral Blood Toll-Like Receptor 5 mRNA Expression

Acute graft-versus-host disease (GVHD) occurs in 40% to 60% of recipients of partially matched umbilical cord blood transplantation (UCBT). In a phase I study, adoptive transfer of expanded CD4+CD25+Foxp3+ natural regulatory T cells (nTregs) resulted in a reduced incidence of grade II-IV acute GVHD. To investigate potential mechanisms responsible for the reduced GVHD risk, we analyzed peripheral blood mononuclear cell mRNA expression of a tolerance gene set previously identified in operation- tolerant kidney transplant recipients, comparing healthy controls and patients who received nTregs and those who did not receive nTregs with and without experiencing GVHD. Samples from patients receiving nTregs regardless of GVHD status showed increased expression of Foxp3 expression, as well as B cell–related tolerance marker. This was correlated with early B cell recovery, predominately of naïve B cells, and nearly normal T cell reconstitution. CD8+ T cells showed reduced signs of activation (HLA-DR+ expression) compared with conventionally treated patients developing GVHD. In contrast, patients with GVHD had significantly increased TLR5 mRNA expression, whereas nTreg-treated patients without GVHD had reduced TLR5 mRNA expression. We identified Lin-HLADR-CD33+CD16+ cells and CD14++CD16− monocytes as the main TLR5 producers, especially in samples of conventionally treated patients developing GVHD. Taken together, these data reveal interesting similarities and differences between tolerant organ and nTreg-treated hematopoietic stem cell transplantation recipients.

Molecular markers of rejection and tolerance: lessons from clinical research

In terms of finding specific molecular markers associated with graft outcome, attempts have been made to study whole genome transcripts using microarray assays or to study the effect of number of genes of interest using quantitative real-time polymerase chain reaction. Using these techniques, molecular phenotypes of rejection have been characterized, and the variability of the clinical outcome besides similar morphology explained in part. Recently, several specific transcripts including naïve B cell regulation have been identified in the peripheral blood of operationally tolerant kidney transplant recipients. The decrease in immature B cell-related transcripts in the peripheral blood in patients with immunosuppression was shown to be associated with acute rejection. Similarly, tolerance-associated antigen 1 transcripts were identified in biopsies and regulatory T cell transcripts in urine and biopsies in patients without rejection. Better understanding of molecular processes associated with allograft rejection or alloantigen hyporesponsiveness/tolerance may help to improve our knowledge about graft pathology and identify novel markers suitable for future monitoring and guided therapy and finally improve the outcome of kidney transplantation.

Higher IL10 Production by Transitional B Cells in Tolerant Kidney Transplant Recipients

Background: Induction of transplantation tolerance would reduce the risk of infection, malignancy and cardiovascular disease caused by immunosupresive drugs in renal transplant recipients. Indices of Tolerance Project, one of the largest studies identifying biomarkers of tolerance, revealed that tolerant kidney transplant recipients, had an expansion of B cells in peripheral blood. In parallel, a B cell subset with regulatory properties has been described in humans as a transitional B cell, characterised by high expression of CD38, CD24 and IL10 production. We set out to test the hypothesis that the expansion of this B cell subset was central in the process of spontaneous tolerance observed in these rare recipients and to test the ability of these cells to present alloantigens. Methods: Peripheral Blood Mononuclear Cells (PBMCs) samples from <tolerant: functionally stable recipients without immunosuppressive drugs, <stable: functionally stable recipients on standard immunosuppression, <chronic rejector: immunosuppressed recipients that presented signs of chronic rejection and <healthy controls were used in the study. B cell subsets from patients were identified by flow cytometry with anti-CD20, anti-CD19, anti-CD27, anti-IgM, anti-IgG, anti-CD24 and anti-CD38. PBMCs were cultured for 3 days alone, with CD40L and CpG in RPMI 10% FCS. After 3 days, activation markers (CD25, CD86 and CD40) were measured by surface staining and IL10, IFN-g and TNF-a were measured by intracellular staining and ELISA. Results: B cells were identified as CD20+CD19+ and memory B cells as CD20+CD19+CD27+. Within the CD27- non-memory B cells we selected the IgD+IgM+ population to study the non-class-switching B cells and then we identified Transitional B cells (regulatory B cells or Bregs) as CD24hiCD38hi. Within the C27- population, transitional B cells were more prevalent in tolerant patients (12,1%), in comparison with stable patients (4,5%), chronic rejectors (2,1%) and healthy controls (7,8%). B cells from all groups of patients increased activation markers and TLR-9 after CD40L and CpG activation, respectively. Tolerant patients displayed a higher percentage of IL10+Bcells in comparison with the rest of the groups in response to CD40L, however the response to CpG was similar in all patient groups. IFN-g-producing Bcells were not identified after any activation and levels of TNF-a were higher after activation with CD40L but not CpG in all of the tested patients. Discussion: CD40L interaction with B cells occurs after encounter with activated T cells. Only in this context B cells from tolerant recipients were producing higher amounts of IL-10. This is a first step towards a firm link between an expansion of transitional B cells and transplantation tolerance. Further studies are under way, to establish alloantigen specificity in this interaction.

B Cell Repopulation after Alemtuzumab Treatment in Kidney Transplant Recipients - Transient Increase in Transitional B Cells and Long Term Dominance of Naïve B Cells

In organ transplantation, the composition of the B cell compartment is increasingly identified as an important determinant for graft outcome. Whereas naïve and transitional B cells have been associated with long-term allograft survival and operational tolerance, memory B cells have been linked to decreased allograft survival. Alemtuzumab induction therapy effectively depletes B cells, followed by rapid repopulation up to levels exceeding base line. The characteristics of the repopulating B cells are currently unknown. We studied the phenotypic and functional characteristics of repopulating B cells longitudinally in 19 kidney transplant recipients, before and at 6, 9 and 12 months after alemtuzumab induction therapy. A transient increase in transitional B cells and cells with phenotypic characteristics of regulatory B cells at 6 months, as well as a long-term dominance in naïve B cells was found in alemtuzumab treated kidney transplant recipients (Figure 1), which was not influenced by conversion from tacrolimus to sirolimus at 6 months. Memory B cells remained virtually absent up to 12 months after transplantation, even though T cells were partially repopulated.[Figure 1]: Figure 1. Repopulating B cells after alemtuzumab induction therapy retain a naïve phenotype long term. (A) Percentage of naïve B cells (IgD+CD27−) within CD19 gate and (B) percentage of memory B cells (both IgD−CD27+ and IgD+CD27+) within CD19 gate (n=7).At all time-points after treatment, B cells showed unaltered proliferative and IgM-producing capacity as compared to pre-transplant samples, whereas the ability to produce IgG was inhibited long-term. In conclusion, induction therapy with alemtuzumab in kidney transplant recipients results in a long-term shift towards naïve B cells with altered phenotypic and functional characteristics. The B cell compartment after alemtuzumab induction therapy resembles that of operationally tolerant kidney transplant recipients, suggesting that lymphocyte depletion with alemtuzumab may, at least partially, create an environment in which tolerance may be achieved.

Absence of Anti-Donor Specific Antibodies in Drug-Free Tolerant Kidney Transplant Recipients

Absence of Anti-Donor Specific Antibodies in Drug-Free Tolerant Kidney Transplant Recipients