Chimerism and tolerance: past, present and future strategies to prolong renal allograft survival.

PURPOSE OF REVIEW: Immunological factors are a major cause of kidney allograft loss. Calcineurin inhibitors (CNIs) have improved short-term kidney allograft survival; however, they in turn contribute to long-term kidney allograft loss from chronic CNI nephrotoxicity. Tolerance induction in transplantation can avoid the long-term adverse effects of immunosuppressive medications. This review aims to critically discuss recent efforts in inducing transplantation tolerance. RECENT FINDINGS: Tolerance induction mediated by chimerism has shown some promise in minimizing or even complete withdrawal of immunosuppressive treatments in kidney allograft recipients. There has been a number of approaches as varied as the number of centres conducting these trials. However, they can be grouped into those mediated by transient microchimerism and those facilitated by more stable macro or full donor chimerism. The success rates in terms of long-term drug-free graft survival has been limited in microchimerism-mediated tolerance induction approaches. Mixed macrochimerism of less than 50% donor may be unstable with mostly the recipient's native immune system overpowering the donor chimeric status.Tolerance induction leading to chimerism has been limited to living donor kidney transplantation and additional long-term outcomes are required. Furthermore, immune monitoring after tolerance induction has faced a limitation in studying due to a lack of sufficient study participants and appropriate study controls. SUMMARY: Tolerance induction is one of several strategies used to prolong kidney allograft survival, but it has not been routinely utilized in clinical practice. However, future applications from the trials to clinical practice remain limited to living donor kidney transplantation. Once further data regarding tolerance inductions exist and practicality becomes widely accepted, tolerance induction may shift the paradigm in the field of kidney transplantation to achieve the best possible outcome of 'One Organ for Life'.

Immunological biomarkers of tolerance in human kidney transplantation: An updated literature review.

The half-life of transplanted kidneys is <10 years. Acute or chronic rejections have a negative impact on transplant outcome. Therefore, achieving to allograft tolerance for improving long-term transplant outcome is a desirable goal of transplantation field. In contrast, there are evidence that distinct immunological characteristics lead to tolerance in some transplant recipients. In contrast, the main reason for allograft loss is immunological responses. Various immune cells including T cells, B cells, dendritic cells, macrophages, natural killer, and myeloid-derived suppressor cells damage graft tissue and, thereby, graft loss happens. Therefore, being armed with the comprehensive knowledge about either preimmunological or postimmunological characteristics of renal transplant patients may help us to achieve an operational tolerance. In the present study, we are going to review and discuss immunological characteristics of renal transplant recipients with rejection and compare them with tolerant subjects.

Extracellular adenosine reversibly inhibits the activation of human regulatory T cells and negatively influences the achievement of the operational tolerance in liver transplantation.

The artificial induction of tolerance in transplantation is gaining strength. In mice, a differential role of extracellular adenosine (eADO) for regulatory and effector T cells (Tregs and Teffs, respectively) has been proposed: inhibiting Teffs and inducing Tregs. The aim of this study was to analyze the action of extracellular nucleotides in human T cells and, moreover, to examine the influence of CD39 and CD73 ectonucleotidases and subsequent adenosine signaling through adenosine 2 receptor (A2 R) in the induction of clinical tolerance after liver transplant. The action of extracellular nucleotides in human T cells was analyzed by in vitro experiments with isolated T cells. Additionally, 17 liver transplant patients were enrolled in an immunosuppression withdrawal trial, and the differences in the CD39-CD73-A2 R axis were compared between tolerant and nontolerant patients. In contrast to the mice, the activation of human Tregs was inhibited similarly to Teffs in the presence of eADO. Moreover, the expression of the enzyme responsible for the degradation of ADO, adenosine deaminase, was higher in tolerant patients with respect to the nontolerant group along the immunosuppression withdrawal. Our data support the idea that eADO signaling and its degradation may play a role in the complex system of regulation of liver transplant tolerance.

Effects of tacrolimus (FK506) and mycophenolate mofetil (MMF) on regulatory T cells and co-inhibitory receptors in the peripheral blood of human liver allograft patients.

Context: Recent studies have shown that a combination treatment of mycophenolate mofetil (MMF) and tacrolimus (FK506) may be an option for organ transplantation patients. Objective: In this study, we detected the effects of FK506 and MMF on the expressions of regulatory T cells (Tregs) and co-inhibitory receptors on Tregs in peripheral blood mononuclear cells (PBMC) of patients with stable phase after liver transplantation. Materials and methods: A total of 35 patients with stable stage after 6 months of liver transplantation were divided into two groups including 20 patients were treated with FK506 monotherapy (FK506 group), and 15 patients with FK506 and MMF combination (FK506 + MMF group). 15 healthy subjects were served as the control. Results: It is found that percentages of CD3+, CD3+CD4+ and CD3+CD8+ T cells in FK506 group are lowered compared to the control group but they are elevated in FK506 + MMF group. Amount of CD4+CD25+CD127low/-Treg cells in CD3+ CD4+T cells in FK506 + MMF group was higher than that in FK506 group and control group. The expressions of co-inhibitory receptors (CTLA-4, PD-1, Tim-3, LAG-3 and TIGIT) on Tregs in FK506 + MMF group were significant higher than those in the FK506 group and control group. The levels of the relative cytokines (TGF-ß and IL-10) in FK506 group are down-regulated compared to the control group. Conclusion: The application of FK506 combined with MMF may be superior to FK506 monotherapy for the patients to further induce the immune tolerance after liver transplantation.

Increased degradation of ATP is driven by memory regulatory T cells in kidney transplantation tolerance.

Regulatory T cells were recently proposed as the central actor in operational tolerance after renal transplantation. Tolerant patients harbor increased FoxP3hi memory Treg frequency and increased demethylation in the Foxp3 Treg-specific demethylated region when compared to stable kidney recipients and exhibit greater memory Treg suppressive capacities and higher expression of the ectonucleotidase CD39. However, in this particular and unique situation the mechanisms of action of Tregs were not identified. Thus, we analyzed the ability of memory Tregs to degrade extracellular ATP in tolerant patients, healthy volunteers, and patients with stable graft function under immunosuppression and determined the role of immunosuppressive drugs on this process. The conserved proportion of memory Tregs leads to the establishment of a pro-tolerogenic balance in operationally tolerant patients. Memory Tregs in tolerant patients display normal capacity to degrade extracellular ATP/ADP. In contrast, memory Tregs from patients with stable graft function do not have this ability. Finally, in vitro, immunosuppressive drugs may favor the lower proportion of memory Tregs in stable patients, but they have no effect on CD39-dependent ATP degradation and do not explain memory Treg lack of extracellular ATP/ADP degradation ability. Thus, intrinsic active regulatory mechanisms may act long after immunosuppressive drug arrest in operationally tolerant patients and may contribute to kidney allograft tolerance via the maintenance of CD39 Treg function.

Glucocorticoids induce corneal allograft tolerance through expansion of monocytic myeloid-derived suppressor cells.

Glucocorticoids (GCs) are the most widely used drugs to prevent transplant rejection; however, it is not yet clear how GCs induce immune tolerance in transplantation. Here, we demonstrate that GCs induce tolerance to corneal allografts in mice through expansion of MHC class II- CD11b+ Ly6C+ monocytes in the bone marrow and mobilization of the cells to spleen, draining lymph nodes, and graft site. The GC-induced CD11b+ Ly6C+ monocytes inhibited T cell proliferation in vitro, and adoptive transfer of the cells improved the survival of corneal allografts. Depletion of CD11b+ Ly6C+ cells in mice during GC treatment abrogated the effects of GCs in prevention of immune rejection. Together, the results identify monocytic myeloid-derived suppressor cells as crucial mediators of the GC-induced tolerance in transplantation.

Longterm Survival and Cost-Effectiveness of Immunosuppression Withdrawal After Liver Transplantation.

Lifelong immunosuppression (IS) after liver transplantation is associated with severe adverse effects and increased recipients' morbidity and mortality. Clinical operational tolerance has been reported in up to 40% in very well-selected recipients. Longterm survival and cost savings within the Italian national health system in operational tolerant recipients is reported. Seventy-five liver recipients were enrolled for IS withdrawal at our institution during the period from April 1998 to December 2015. The study population comprised 32 (42.7%) tolerant patients; 41 (54.7%) nontolerant patients needing uptake of IS after clinical or biopsy-proven rejection; and 2 (2.7%) immediate nontolerant patients who developed early rejection after the first drug reduction. The primary endpoint of the study was to assess the longterm patients and graft outcome; the secondary endpoint was the assessment of cost savings in the context of IS withdrawal. The follow-up was 95.0 months (interquartile range, 22.5-108.5 months). IS withdrawal did not result in patient nor graft loss and resulted in a major cost savings reaching about €630,000. In conclusion, longterm IS withdrawal represents a remarkable cost savings in the health care of liver recipients without exposing them to graft loss.

Anti-LFA-1 induces CD8 T-cell dependent allograft tolerance and augments suppressor phenotype CD8 cells.

The induction of tolerance to transplanted organs is a major objective in transplantation immunology research. Lymphocyte function-associated antigen-1 (LFA-1) interactions have been identified as a key component of the T-cell activation process that may be interrupted to lead to allograft tolerance. In mice, αLFA-1 mAb is a potent monotherapy that leads to the induction of donor-specific transferable tolerance. By interrogating important adaptive and innate immunity pathways, we demonstrate that the induction of tolerance relies on CD8+T-cells. We further demonstrate that αLFA-1 induced tolerance is associated with CD8+CD28-T-cells with a suppressor phenotype, and that while CD8 cells are present, the effector T-cell response is abrogated. A recent publication has shown that CD8+CD28- cells are not diminished by cyclosporine or rapamycin, therefore CD8+CD28- cells represent a clinically relevant population. To our knowledge, this is the first time that a mechanism for αLFA-1 induced tolerance has been described.

Cell therapeutic approaches to immunosuppression after clinical kidney transplantation.

Refinement of immunosuppressive strategies has led to further improvement of kidney graft survival in recent years. Currently, the main limitations to long-term graft survival are life-threatening side effects of immunosuppression and chronic allograft injury, emphasizing the need for innovative immunosuppressive regimens that resolve this therapeutic dilemma. Several cell therapeutic approaches to immunosuppression and donor-specific unresponsiveness have been tested in early phase I and phase II clinical trials in kidney transplantation. The aim of this overview is to summarize current cell therapeutic approaches to immunosuppression in clinical kidney transplantation with a focus on myeloid suppressor cell therapy by mitomycin C-induced cells (MICs). MICs show great promise as a therapeutic agent to achieve the rapid and durable establishment of donor-unresponsiveness in living-donor kidney transplantation. Cell-based therapeutic approaches may eventually revolutionize immunosuppression in kidney transplantation in the near future.

Induction of transplantation tolerance through regulatory cells: from mice to men.

Organ transplantation results in the activation of both innate and adaptive immune responses to the foreign antigens. While these responses can be limited with the use of systemic immunosuppressants, the induction of regulatory cell populations may be a novel strategy for the maintenance of specific immunological unresponsiveness that can reduce the severity of the detrimental side effects of current therapies. Our group has extensively researched different regulatory T-cell induction protocols for use as cellular therapy in transplantation. In this review, we address the cellular and molecular mechanisms behind regulatory T-cell suppression and their stability following induction protocols. We further discuss the use of different hematopoietically derived regulatory cell populations, including regulatory B cells, regulatory macrophages, tolerogenic dendritic cells, and myeloid-derived suppressor cells, for the induction of transplantation tolerance in light of new clinical trials developing therapies with some of these populations.

Tissue-resident T cells, in situ immunity and transplantation.

T cells coordinate rejection of transplanted allografts and are key targets for depletion, immunosuppression, and tolerance induction to promote long-term graft survival. Studies in mouse models and humans generally focus on circulating T cells or those from lymphoid sites; however, vast numbers of T cells reside in multiple peripheral tissue sites including lungs, intestines, liver, and skin as non-circulating, tissue-resident memory T cells (Trm cells). In this review, we define the basic properties of Trm cells, the emerging evidence of their importance for protective immunity, and the potential role of resident versus circulating T cells in transplant rejection and in providing protection to prevalent infections posttransplantation. We also discuss potential susceptibilities and/or resistance of protective Trm to immunosuppression therapies, and how consideration of Trm, their compartmentalization, and specificity can enable improved therapies for targeted inhibition of pathogenic and preservation of protective T-cell subsets.

Biomarkers and possible mechanisms of operational tolerance in kidney transplant patients.

A small number of patients do not reject their graft after weaning from immunosuppressive treatment. Here, we analyze the studies carried out to try to understand the mechanisms involved in this operational transplant tolerance and evaluate the hypotheses proffered on these potential mechanisms.

Recollective homeostasis and the immune consequences of peritransplant depletional induction therapy.

One's cellular immune repertoire is composed of lymphocytes in multiple stages of maturation - the dynamic product of their responses to antigenic challenges and the homeostatic contractions necessary to accommodate immune expansions within physiologic norms. Given that alloreactivity is predominantly a cross-reactive phenomenon that is stochastically distributed throughout the overall T-cell repertoire, one's allospecific repertoire is similarly made up of cells in a variety of differentiation states. As such, the continuous expansion and elimination of activated memory populations, producing a 'recollective homeostasis' of sorts, has the potential over time to alter the maturation state and effector composition of both ones protective and alloreactive T-cell repertoire. Importantly, a T cell's maturation state significantly influences its response to numerous immunomodulatory therapies used in organ transplantation, including depletional antibody induction. In this review, we discuss clinically utilized depletional induction strategies, how their use alters a transplant recipient's cellular immune repertoire, and how a recipient's repertoire influences the clinical effects of induction therapy.

Early mixed chimerism-based preemptive immunotherapy in children undergoing allogeneic hematopoietic stem cell transplantation for acute leukemia.

This retrospective analysis comprises 10-year experience with early posttransplant mixed chimerism-based preemptive intervention. Out of 104 patients, 51 received preemptive immunotherapy. Their outcomes were similar to patients achieving full donor chimerism spontaneously. Among patients receiving intervention, 5-year event-free survival was identical in patients with and without pretransplant residual disease, respectively (68% [95% confidence interval (CI) 38-98%] vs. 69% [95% CI 54-85%] log-rank = 0.4). In patients who received preemptive immunotherapy, chimerism status and residual disease prior to transplant were no longer predictors of poor outcome; however, 41% of the patients with residual disease prior to transplant relapsed early and did not benefit from this strategy.

Pharmacological modulation of cell death in organ transplantation.

New options to pharmacologically modulate fundamental mechanisms of regulated cell death are rapidly evolving and found first clinical applications in cancer therapy. Here, we present an overview on how the recent advances in the understanding of the biology and pharmacology of cell death might influence research and clinical practice in solid organ transplantation. Of particular interest are the novel opportunities related to organ preservation and immunomodulation, which might contribute to promote organ repair and to develop more selective ways to modulate allogeneic immune responses to prevent rejection and induce immunological tolerance.

Targeting Sirtuin-1 prolongs murine renal allograft survival and function.

Current immunosuppressive medications used after transplantation have significant toxicities. Foxp3(+) T-regulatory cells can prevent allograft rejection without compromising protective host immunity. Interestingly, inhibiting the class III histone/protein deacetylase Sirtuin-1 can augment Foxp3(+) T-regulatory suppressive function through increasing Foxp3 acetylation. Here we determined whether Sirtuin-1 targeting can stabilize biological allograft function. BALB/c kidney allografts were transplanted into C57BL/6 recipients with a CD4-conditional deletion of Sirtuin-1 (Sirt1(fl/fl)CD4(cre)) or mice treated with a Sirtuin-1-specific inhibitor (EX-527), and the native kidneys removed. Blood chemistries and hematocrit were followed weekly. Sirt1(fl/fl)CD4(cre) recipients showed markedly longer survival and improved kidney function. Sirt1(fl/fl)CD4(cre) recipients exhibited donor-specific tolerance, accepted BALB/c, but rejected third-party C3H cardiac allografts. C57BL/6 recipients of BALB/c renal allografts that were treated with EX-527 showed improved survival and renal function at 1, but not 10 mg/kg/day. Pharmacologic inhibition of Sirtuin-1 also improved renal allograft survival and function with dosing effects having relevance to outcome. Thus, inhibiting Sirtuin-1 can be a useful asset in controlling T-cell-mediated rejection. However, effects on non-T cells that could adversely affect allograft survival and function merit consideration.

Different Effects of Tacrolimus on Innate and Adaptive Immune Cells in the Allograft Transplantation.

While tacrolimus (FK506) is currently used as immunosuppression therapy in transplant recipient, the immunological mechanism remains unknown. Herein, the immunoregulatory effects of FK506 were investigated in the physiological status and allogeneic skin transplantation. FK506 cannot significantly alter the functions of innate immune cells (macrophages and neutrophils) and adaptive immune cells (T cells) in the physiological status. However, it can effectively delay allogeneic skin-graft rejection through ameliorating the T cell responses, but not myeloid-derived innate immune cell responses. Importantly, it did not affect the allograft recipient macrophage innate immune defence capacity to bacteria. In clinics, FK506 treatment can significantly control the cytokine production in T cells, but not non-T cells. This study shows targeting calcineurin signalling, FK506, to be essential in inducing allograft tolerance, but not to damage the innate defence capacity, validating the immune cell phenotypes as a potential marker in transplantation following FK506 treatment.

Peripheral phenotype and gene expression profiles of combined liver-kidney transplant patients.

BACKGROUND AND AIMS: The beneficial effect of one graft on another has been reported in combined transplantation but the associated mechanisms and biological influence of each graft have not yet been established. METHODS: In multiple analyses, we explored the PBMC phenotype and signature of 45 immune-related messenger RNAs and 754 microRNAs from a total of 235 patients, including combined liver-kidney transplant recipients (CLK), patients with a liver (L-STA) or kidney (K-STA) graft only under classical immunosuppression and patients with tolerated liver (L-TOL) or kidney grafts (K-TOL). RESULTS: CLK show an intermediary phenotype with a higher percentage of peripheral CD19(+) CD24(+) CD38(Low) memory B cells and Helios(+) Treg cells, two features associated with tolerance profiles, compared to L-STA and K-STA (P < 0.05, P < 0.01). Very few miRNA were significantly differentially expressed in CLK vs. K-STA and even fewer when compared to L-STA (35 and 8, P < 0.05). Finally, CLK are predicted to share common miRNA targets with K-TOL and even more with L-TOL (344 and 411, P = 0.005). Altogether CLK display an intermediary phenotype and gene profile, which is closer to that of liver transplant patients, with possible similarities with the profiles of tolerant patients. CONCLUSION: These data suggest that CLK patients show the immunological influence of both allografts with liver having a greater influence.

Combining autologous dendritic cell therapy with CD3 antibodies promotes regulatory T cells and permanent islet allograft acceptance.

Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation.

The regulatory T cell effector molecule fibrinogen-like protein 2 is necessary for the development of rapamycin-induced tolerance to fully MHC-mismatched murine cardiac allografts.

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4(+) and CD8(+) immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-ß and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.