Tolerance In Human Transplantation Research Articles

SAT298 - Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote transplantation tolerance in humans

SAT298 - Low dose interleukin-2 selectively expands circulating regulatory T cells but fails to promote transplantation tolerance in humans

Dominant regulation of long-term allograft survival is mediated by microRNA-142.

Organ transplantation is often lifesaving, but the long-term deleterious effects of combinatorial immunosuppression regimens and allograft failure cause significant morbidity and mortality. Long-term graft survival in the absence of continuing immunosuppression, defined as operational tolerance, has never been described in the context of multiple major histocompatibility complex (MHC) mismatches. Here, we show that miR-142 deficiency leads to indefinite allograft survival in a fully MHC mismatched murine cardiac transplant model in the absence of exogenous immunosuppression. We demonstrate that the cause of indefinite allograft survival in the absence of miR-142 maps specifically to the T cell compartment. Of therapeutic relevance, temporal deletion of miR-142 in adult mice prior to transplantation of a fully MHC mismatched skin allograft resulted in prolonged allograft survival. Mechanistically, miR-142 directly targets Tgfbr1 for repression in regulatory T cells (TREG ). This leads to increased TREG sensitivity to transforming growth factor - beta and promotes transplant tolerance via an augmented peripheral TREG response in the absence of miR-142. These data identify manipulation of miR-142 as a promising approach for the induction of tolerance in human transplantation.

Toward an Integrated View of Operational Tolerance in Human Renal Transplantation: A Systems Biology Perspective.

Operational tolerance (OT) is the phenomenon occurring in human renal and liver transplantation in which the body does not reject the organ after discontinuing immunosuppression for at least a year. We revisited the data generated by The Brazilian Multicenter Study on Operational Tolerance involving different conceptual fields - antigen-specific cytokine response, immune cell numbers and repertoire, signaling pathways, and epigenetics. We integrated our data to pave the way to systems biology thinking and harness debate on potential mechanisms in OT. We present original data on systems biology in OT, connecting potential mechanistic players. Using bioinformatics, we identified three dominant features that discriminate OT from its opposing clinical outcome, chronic rejection (CR). The OT-CR discriminative molecules were FOXP3, GATA3 and STAT6, each corresponding to a differential profile: (1) In FOXP3, OT presents preserved regulatory T cell (Treg) numbers but decreased numbers in CR; (2) in GATA3, increased expression is seen in OT; and (3) in STAT6, decreased monocyte activation is seen in OT. With these variables, we built molecular networks to identify interactions related to OT versus CR. Our first systems biology endeavor gave rise to novel potentially relevant interconnected players in OT mechanisms: FOXP3 connecting to interleukin-9 (IL-9) and IL-35 signaling, suggesting their immunoregulatory involvement in OT. Likewise, GATA3/FOXP3 interactions incrementing/stabilizing FOXP3 transcription suggest participation in keeping healthy FOXP3+ Tregs in OT. We envision that systems biology thinking will greatly contribute to advancing knowledge in human transplantation tolerance in an interactive perspective.

Regulatory/inflammatory cellular response discrimination in operational tolerance

Antigen-specific cellular response is essential in immune tolerance. We tested whether antigen-specific cellular response is differentially modulated in operational tolerance (OT) in renal transplantation with respect to critical antigenic challenges in allotransplantation-donor antigens, pathogenic antigens and self-antigens. We analysed the profile of immunoregulatory (REG) and pro-inflammatory (INFLAMMA) cytokines for the antigen-specific response directed to these three antigen groups, by Luminex. We showed that, in contrast to chronic rejection and healthy individuals, OT gives rise to an immunoregulatory deviation in the cellular response to donor human leucocyte antigen DR isotype peptides, while preserving the pro-inflammatory response to pathogenic peptides. Cellular autoreactivity to the N6 heat shock protein 60 (Hsp60) peptide also showed a REG profile in OT, increasing IL4, IL-5, IL-10 and IL-13. The REG shift of donor indirect alloreactivity in OT, with inhibition of interleukin (IL)-1B, IL-8, IL-12, IL-17, granulocyte colony-stimulating factor, Interferon-γ and monocyte chemoattractant protein-1, indicates that this may be an important mechanism in OT. In addition, the differential REG profile of cellular response to the Hsp60 peptide in OT suggests that REG autoimmunity may also play a role in human transplantation tolerance. Despite cross-reactivity of antigen-specific T cell responses, a systemic functional antigen-specific discrimination takes place in OT.

Differential microRNA Profile in Operational Tolerance: A Potential Role in Favoring Cell Survival.

Background: Operational tolerance (OT) is a state of graft functional stability that occurs after at least 1 year of immunosuppressant withdrawal. MicroRNAs (microRNA) are small non-coding RNAs that downregulate messenger RNA/protein expression of innumerous molecules and are critical for homeostasis. We investigated whether OT in kidney transplantation displays a differential microRNA profile, which would suggest that microRNAs participate in Operational Tolerance mechanisms, and may reveal potential molecular pathways.Methods: We first compared serum microRNA in OT (n = 8) with chronic rejection (CR) (n = 5) and healthy individuals (HI) (n = 5), using a 768-microRNA qPCR-panel. We used the Thermo Fisher Cloud computing platform to compare the levels of microRNAs in the OT group in relation to the other study groups. We performed validation experiments for miR-885-5p, by q-PCR, in a larger number of study subjects (OT = 8, CR = 12, HI = 12), as individual samples.Results: We detected a differential microRNA profile in OT vs. its opposing clinical outcome—CR—suggesting that microRNAs may integrate transplantation tolerance mechanisms. Some miRNAs were detected at higher levels in OT: miR-885-5p, miR-331-3p, miR-27a-5p vs. CR; others, we found at lower levels: miR-1233-3p, miR-572, miR-638, miR-1260a. Considering highly predicted/experimentally demonstrated targets of these miRNAs, bioinformatics analysis revealed that the granzyme B, and death receptor pathways are dominant, suggesting that cell death regulation integrates transplantation tolerance mechanisms. We confirmed higher miR-885-5p levels in OT vs. CR, and vs. HI, in a larger number of subjects.Conclusions: We propose that epigenetics mechanisms involving microRNAs may integrate human transplantation tolerance mechanisms, and regulate key members of the cell death/survival signaling. miR-885-5p could favor cell survival in OT by diminishing the levels of CRADD/RAIDD and CASP3. Nonetheless, given the nature of any complex phenomenon in humans, only cumulative data will help to determine whether this microRNA differential profile may be related to the cause or consequence of operational tolerance.

Broad Impairment of Natural Killer Cells from Operationally Tolerant Kidney Transplanted Patients.

The role of natural killer (NK) cells in organ transplantation is controversial. This study aims to decipher their role in kidney transplant tolerance in humans. Previous studies highlighted several modulated genes involved in NK cell biology in blood from spontaneously operationally tolerant patients (TOLs; drug-free kidney-transplanted recipients with stable graft function). We performed a phenotypic, functional, and genetic characterization of NK cells from these patients compared to kidney-transplanted patients with stable graft function under immunosuppression and healthy volunteers (HVs). Both operationally TOLs and stable patients harbored defective expression of the NKp46 activator receptor and lytic molecules perforin and granzyme compared to HVs. Surprisingly, NK cells from operationally TOLs also displayed decreased expression of the CD16 activating marker (in the CD56Dim NK cell subset). This decrease was associated with impairment of their functional capacities upon stimulation, as shown by lower interferon gamma (IFNγ) production and CD107a membranous expression in a reverse antibody-dependent cellular cytotoxicity (ADCC) assay, spontaneous lysis assays, and lower target cell lysis in the 51Cr release assay compared to HVs. Conversely, despite impaired K562 cell lysis in the 51Cr release assay, patients with stable graft function harbored a normal reverse ADCC and even increased amounts of IFNγ+ NK cells in the spontaneous lysis assay. Altogether, the strong impairment of the phenotype and functional cytotoxic capacities of NK cells in operationally TOLs may accord with the establishment of a pro-tolerogenic environment, despite remaining highly activated after transplantation in patients with stable graft function.

Immunological characteristics of renal transplant tolerance in humans.

Establishing allograft tolerance is a highly desirable therapeutic goal in kidney transplantation, from which recipients would greatly benefit by withdrawing or minimizing immunosuppression. Identifying biomarkers in predicting tolerance or early diagnosing rejection is essential to direct personalized management. Recent findings have revealed that multiple populations of immune cells have involved in promoting long-term graft function or inducing rejection in renal transplant recipients. Thus, roles of immune cells add another level to predict the renal tolerant state; tailoring their functional and/or phenotypic characteristics would provide insights into mechanism involved in transplant tolerance that may aid in designing new therapies. Here, we review these findings and discuss the current understanding immunological characteristics of renal transplant tolerance in humans, and their potential clinical translation to immune tolerance biomarkers.

T helper subsets & regulatory T cells: rethinking the paradigm in the clinical context of solid organ transplantation

Recent years have witnessed remarkable expansion in the knowledge of how various immune/inflammatory cells and T helper (Th) cell subsets, including Th1, Th2, Th9, Th17, Th22, follicular T helper (Tfh) and Treg subpopulations, reciprocally regulate each other. This review highlights current understanding of the Th subsets paradigm, who are the old school players, who are the new kids on the block and how does each come to play in different clinical contexts in solid organ transplantation. The article commences with a brief overview of the development and characteristic cytokine profiles of individual members of the paradigm. However, the main focus of this review is on the current understanding of the Th subset paradigm, and how these unique subpopulations impact host responses towards solid organ allografts. More specifically, it will highlight the recent findings that implicate the paradigm in transplantation. The interplay among different subsets is discussed collectively in the clinical context of pretransplant immunological risk factors such as alloimmunization as well as post-transplant immunological consequences such as rejection. Accumulating evidence suggests that Th17 cells play a role in the development of chronic allograft injury in transplantation of various organs. In vitro, tacrolimus suppressed Th1 and Th2 cells but not Th17 cells. Animal studies suggest that regulatory T cells (Treg)-based therapies could be effective as mechanisms of long-term drug-free transplant tolerance in humans. Indeed, a dual role for TGF-β and Foxp3 in induced tolerance has been proposed, in which TGF-β stimulates Foxp3 expression and is associated with the induction of Treg-facilitating acquisition of tolerance. Exploiting Th subsets' regulatory functions could potentially offer opportunities for immunological interventions in solid organ transplantation.

Preserving the B-Cell Compartment Favors Operational Tolerance in Human Renal Transplantation

Transplanted individuals in operational tolerance (OT) maintain long-term stable graft function after completely stopping immunosuppression. Understanding the mechanisms involved in OT can provide valuable information about pathways to human transplantation tolerance. Here we report that operationally tolerant individuals display quantitative and functional preservation of the B-cell compartment in renal transplantation. OT exhibited normal numbers of circulating total B cells, naive, memory and regulatory B cells (Bregs) as well as preserved B-cell receptor repertoire, similar to healthy individuals. In addition, OT also displayed conserved capacity to activate the cluster of differentiation 40 (CD40)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in Bregs, in contrast, with chronic rejection. Rather than expansion or higher activation, we show that the preservation of the B-cell compartment favors OT.

GATA3 and a dominant regulatory gene expression profile discriminate operational tolerance in human transplantation

Some organ-transplanted patients achieve a state of "operational tolerance" (OT) in which graft function is maintained after the complete withdrawal of immunosuppressive drugs. We used a gene panel of regulatory/inflammatory molecules (FOXP3, GATA3, IL10, TGFB1, TGFBR1/ TBX21, TNF and IFNG) to investigate the gene expression profile in peripheral blood mononuclear cells of renal-transplanted individuals experiencing OT compared to transplanted individuals not displaying OT and healthy individuals (HI). OT subjects showed a predominant regulatory (REG) profile with higher gene expression of GATA3, FOXP3, TGFB1 and TGFB receptor 1 compared to the other groups. This predominant REG gene expression profile displayed stability over time. The significant GATA3 gene and protein expressions in OT individuals suggest that a Th2 deviation may be a relevant pathway to OT. Moreover, the capacity of the REG/INFLAMMA gene panel to discriminate OT by peripheral blood analysis indicates that this state has systemic repercussions.

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans

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 alpha-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.

Identification of a B cell signature associated with renal transplant tolerance in humans

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.

Differential monocyte STAT6 activation and CD4 +CD25 +Foxp3 + T cells in kidney operational tolerance transplanted individuals

In organ transplantation, the immunosuppression withdrawal leads, in most cases, to rejection. Nonetheless, a special group of patients maintain stable graft function after complete withdrawal of immunosuppression, achieving a state called “operational tolerance.” The study of such patients may be important to understand the mechanisms involved in human transplantation tolerance. We compared the profile of CD4 +CD25 +Foxp3 + T cells and the signaling pathways IL-6/STAT3 (signal transducers and activators of transcription) and IL-4/STAT6 in peripheral blood mononuclear cells of four kidney transplant groups: (i) operational tolerance (OT), (ii) chronic allograft nephropathy (CR), (iii) stable graft function under standard immunosuppression (Sta), (iv) stable graft function under low immunosuppression, and (v) healthy individuals. Both CR and Sta displayed lower numbers and percentages of CD4 +CD25 +Foxp3 + T cells compared with all other groups ( p < 0.05). The OT patients displayed a reduced activation of the IL-4/STAT6 pathway in monocytes, compared with all other groups ( p < 0.05). The lower numbers of CD4 +CD25 +Foxp3 + T cells observed in CR individuals may be a feature of chronic allograft nephropathy. The differential OT signaling profile, with reduced phosphorylation of STAT6, in monocytes' region, suggests that some altered function of STAT6 signaling may be important for the operational tolerance state.

Tolerance is the achievable ‘Holy Grail’ in transplantation

Purpose of review Despite great progress in organ transplantation, long-term outcomes are far from satisfactory and induction of tolerance remains the ‘Holy Grail’ for transplant clinicians. Although challenges remain, recent developments in our understanding of the mechanisms of tolerance in animal models and humans have moved us closer to the ultimate goal of achieving clinical transplantation tolerance in humans. This review highlights the newest developments in the quest to achieve such a state. Recent findings Both deletional and nondeletional mechanisms, achieved by a variety of strategies, are involved in tolerance induction. Regulation carried out by Foxp3 expressing CD25+CD4+ regulatory T (Treg) cells is considered one of the most important nondeletional mechanisms of tolerance. Tolerance can be achieved and maintained by manipulating costimulatory signals, which closely influence the function of Treg cells. Summary A variety of depletional and nondepletional strategies targeting the T cells, B cells, dendritic cells, and natural killer cells have all been shown to be important in the development and function of Treg cells. Treg cell therapy holds great promise for revolutionizing the field of transplantation. A vast body of experimental data exists indicating that tolerance can be now achieved; the challenge, however, rests in translating this success to the clinic.

Donor-Specific Allograft Tolerance by Administration of Recipient-Derived Immature Dendritic Cells and Suboptimal Immunosuppression

We recently showed that injection of recipient-type immature bone marrow-derived dendritic cells (iBMDCs) the day before transplantation induced a significant prolongation of allograft survival. This study aimed at improving the administration protocol to induce allograft tolerance. Various amounts of iBMDCs were administered to syngeneic LEW.1A rats before and after transplantation of an allogeneic LEW.1W heart, with or without additional suboptimal immunosuppression. Allograft survival was not improved by repeated injections of syngeneic iBMDCs or by additional treatment with low-dose rapamycin. Combining injections of iBMDCs and LF 15-0195 showed a striking synergistic effect and induced definitive allograft acceptance in 92% of recipients. Tolerant recipients accepted donor-type, but not third-party type skin grafts, suggesting the development of regulatory mechanisms capable of maintaining donor-specific tolerance. The reported findings may contribute to the development of new therapeutic strategies to induce transplantation tolerance in humans.

Regulatory T cells under scrutiny

Purpose of review There is increasing evidence that regulatory T cells play a central role in the control of both reactivity to self-antigens and alloimmune response. This review focuses on the importance of regulatory T cells in clinical transplantation as a marker of hyporesponsiveness against donor antigens and as a potential therapeutic tool for tolerance induction. Recent findings Several subsets of regulatory T cells with distinct phenotypes and mechanisms of action have now been identified. They constitute a network of heterogeneous CD4+ or CD8+ T cell subsets and other minor T-cell populations with mechanisms of action that can be cell-contact dependent or independent. Considerable progress has been made in characterization of regulatory T cells, such as identification of FoxP3 as a specific marker. Regulatory T cells not only play a main role in maintaining self tolerance and preventing autoimmune disease, but can also be induced by tolerance protocols and seem to play a key role in preventing allograft rejection, as demonstrated in many animal models. Of particular interest, in stable transplant patients CD4+CD25+ and CD8+CD28− regulatory T cells have been recently shown to modulate immune response toward donor antigens in the indirect and direct pathway, respectively. This finding raises the possibility that such regulatory T cells also have a role in induction or maintenance of transplant tolerance in humans. Summary Regulatory T cells have been shown to be an essential tool by which the immune system can actively control immune responses. Future developments of transplantation research would probably have regulatory T cells as protagonists for immunologic monitoring as well as for induction of transplant tolerance.

Approaches to transplantation tolerance in humans.

Although transplantation tolerance to organ allografts has been achieved using a wide variety of immunologic interventions in laboratory animals, few tolerance induction protocols with complete immunosuppressive drug withdrawal have been tested in humans. Preclinical and clinical studies of the use of total lymphoid irradiation for the induction of chimeric and nonchimeric tolerance are summarized here.

Induction of transplantation tolerance in humans using stem cell transplants prenatally or postnatally

Induction of transplantation tolerance in humans using stem cell transplants prenatally or postnatally