Induction Of Transplantation Tolerance Research Articles

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

T-cell Immunometabolism: Therapeutic Implications in Organ Transplantation.

Although solid-organ transplantation has evolved steadily with many breakthroughs in the past 110 y, many problems remain to be addressed, and advanced therapeutic strategies need to be considered. T-cell immunometabolism is a rapidly advancing field that has gathered much attention recently, providing ample mechanistic insight from which many novel therapeutic approaches have been developed. Applications from the field include antitumor and antimicrobial therapies, as well as for reversing graft-versus-host disease and autoimmune diseases. However, the immunometabolism of T cells remains underexplored in solid-organ transplantation. In this review, we will highlight key findings from hallmark studies centered around various metabolic modes preferred by different T-cell subtypes (categorized into naive, effector, regulatory, and memory T cells), including glycolysis, glutaminolysis, oxidative phosphorylation, fatty acid synthesis, and oxidation. This review will discuss the underlying cellular signaling components that affect these processes, including the transcription factors myelocytomatosis oncogene, hypoxia-inducible factor 1-alpha, estrogen-related receptor alpha, and sterol regulatory element-binding proteins, along with the mechanistic target of rapamycin and adenosine monophosphate-activated protein kinase signaling. We will also explore potential therapeutic strategies targeting these pathways, as applied to the potential for tolerance induction in solid-organ transplantation.

Gut microbes enlarged the protective effect of transplanted regulatory B cells on rejection of cardiac allografts

Regulatory B cells (Bregs) play an important role in maintaining immune homeostasis and have the potential to induce tolerance. Previous work has found that Breg cells are involved in heart transplantation tolerance. However, the effect of Breg on the transplantation tolerance and the underlying mechanisms remain to be clarified. Using a within-species heart transplantation model, we aimed to investigate the role of CD19+CD5+CD1dhigh Bregs isolated from transplanted mice in preventing transplant rejection in vivo. We also explored the effects of CD40 and tumor necrosis factor receptor-associated factor 6 (TRAF6) ubiquitin ligase on Breg-mediated prolongation of survival in heart transplant (HT) mice, and the regulatory effects of downstream Cdk4 and Cdk6 proteins on dendritic cells (DCs), which clarified the function and molecular mechanism of Breg cells in HT mice. Our data suggest that adoptive transfer of the transplanted Bregs served as an effective tolerance-inducing mechanism in HT mice and was involved in the CD40-TRAF6 signaling pathway in DCs. Moreover, DCs collected from the Breg treated HT mice also prolonged the survival of HT mice. Furthermore, DC-specific knockout of TRAF6 diminished Breg-mediated prolongation of survival in HT mice. Interestingly, gut microbes from donors increased the survival of cardiac allografts both in both the absence and presence of Bregs but were not implicated in CD40-TRAF6 signaling. These findings reveal a role of Breg cells in the induction of transplantation tolerance through the blockade of the CD40-TRAF6 signaling pathway, which might be used in the treatment of HT in the clinic.

Imaging Tolerance Induction in Neonatal Mice: Hierarchical Interplay Between Allogeneic Adult and Neonatal Immune Cells.

In Medawar's murine neonatal tolerance model, injection of adult semiallogeneic lymphohematopoietic cells (spleen cells [SC] and bone marrow cells [BMC]) tolerizes the neonatal immune system. An eventual clinical application would require fully allogeneic (allo) cells, yet little is known about the complex in vivo/in situ interplay between those cells and the nonconditioned neonatal immune system. To this end, labeled adult SC and BMC were injected into allogeneic neonates; interactions between donor and host cells were analyzed and modulated by systematic depletion/inactivation of specific donor and host immune effector cell types. Consistent with effector cell compositions, allo-SC and allo-SC/BMC each induced lethal acute graft-versus-host disease, whereas allo-BMC alone did so infrequently. CD8 T cells from SC inoculum appeared naïve, while those of BMC were more memory-like. Age-dependent, cell-type dominance defined the interplay between adult donor cells and the neonatal host immune system such that if the dominant adult effector type was removed, then the equivalent neonatal one became dominant. Depletion of donor/host peripheral T cells protected against acute graft-versus-host disease and prolonged heart allograft survival; peripheral CD8 T-cell depletion together with CD4 T cell-costimulation blockade induced more robust tolerance. This comprehensive study provides direct observation of the cellular interplay between allogeneic donor and host immune systems, adds to our previous work with semiallogeneic donor cells, and provides important insights for robust tolerance induction. Induction of transplant tolerance in neonates will likely require "crowd sourcing" of multiple tolerizing cell types and involve depletion of immune effector cells with costimulation blockade.

Cytokine and epigenetic regulation of programmed death-ligand 1 in stem cell differentiation and cancer cell plasticity.

Programmed death-ligand 1 (PD-L1), an immune checkpoint ligand, is recognized as a potential target for cancer immunotherapy as well as for the induction of transplantation tolerance. However, how the crosstalk between stem cell programming and cytokine signaling regulates PD-L1 expression during stem cell differentiation and cancer cell plasticity remains unclear. Herein, we reported that PD-L1 expression was regulated by SOX2 during embryonic stem cell (ESC) differentiation and lung cancer cell plasticity. PD-L1 was induced during ESC differentiation to fibroblasts and was downregulated during SOX2-mediated reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs). Furthermore, SOX2 activation affected cancer cell plasticity and inhibited PD-L1 expression in lung cancer cells. We discovered that the H3K27ac signal at the PD-L1 locus was enhanced during ESC differentiation to fibroblasts as well as during cancer plasticity of SOX2-positive lung cancer cells to SOX2-negative counterparts. Romidepsin, an epigenetic modifier, induced PD-L1 expression in lung cancer cells, whereas TGF-β stimulation downregulated SOX2 but upregulated PD-L1 expression in lung cancer cells. Furthermore, in addition to PD-L1, the expressions of EGFR and its ligand HBEGF were downregulated by activation of endogenous SOX2 expression during lung cancer cell plasticity and iPSC reprogramming, and the activation of EGFR signaling by HBEGF upregulated PD-L1 expression in lung cancer cells. Together, our results reveal the crosstalk between SOX2 programming and cytokine stimulation influences PD-L1 expression, and these findings may provide insights into PD-L1-mediated therapeutics.

CTLA4Ig Primed Donor Lymphocyte Infusions (DLI): A Novel Approach to Natural Killer Cell Immunotherapy Following Haploidentical PBSC Transplantation for Advanced Hematological Malignancies

We have earlier shown that early use of prophylactic G-CSF mobilized DLI resulted in improved disease-free survival following post-transplantation cyclophosphamide (PTCy) based haploidentical HSCT in patients with relapsed/refractory myeloid malignancies. CD56 enriched donor cell infusion following PTCy for similar cohort resulted in rapid proliferation of mature NK cells with attenuation of GVHD. However, the relapse rate was higher than that with early DLI (50% vs 21.4%). CTLA4Ig has been shown to be effective in attenuating T cell activation and induce transplantation tolerance in preclinical models. NK cells on the other hand are resistant to CTLA4Ig and in fact might demonstrate better anti-tumour effect in presence of CTLA4Ig as CD86 is a putative activation receptor. To explore this phenomenon, we employed sequential CTLA4Ig primed DLI following PTCy based haploidentical HSCT.The study group (CTLA4Ig-DLI) had 24 patients (4-65 years; myeloid malignancies-10, Lymphoid malignancies-14) received Abatacept (CTLA4Ig) at 10 mg/kg on day -1 followed by PBSC and sequentially on days +6, +20 and +35 followed 12 hours later by DLI of 5 x 106 CD3 cells/kg containing 0.03-1 x106/kg CD56+ cells. PTCy was administered on days +3 and +4 with cyclosporine from day +5 to day +60 and subsequent rapid tapering. The outcome and immune reconstitution of the CTLA4Ig-DLI group was compared with the cohort of patients who received DLI without abatacept (n=21; DLI group)All patients engrafted at a median of 15 days (11-20 days). The incidence of acute GVHD was 9.7% compared to 31% in the DLI group (p=0.08) and that of chronic GVHD was 28% vs 41.2% (p=0.4). Three patients required treatment for CMV infection with no non-relapse mortality (0% vs 19.9% in DLI group; p=0.03). The relapse incidence was 25.4% vs 21.4% in the DLI group (p=0.7) with an overall survival of 82% vs 70.5% at 18 months (p=0.4). In the CTLA4Ig-DLI group only 1/10 patients with myeloid malignancies relapsed compared to 5/14 in the lymphoid group. Absolute CD56+CD3- and CD56+CD16+ NK cells were significantly higher in the CTLA4Ig-DLI group at day +30 (175±162 vs 73±34; p=0.008 and 95±121 vs 32±19.1; p=0.02 )and +60 (134±107 vs 98±55; p=0.1 and 84 ±87 vs 30±10; p=0.01). The CD56+16+ NK cell population in the CTLA4Ig-DLI group had a higher expression of KIR receptors at day +30. Within the CTLA4Ig-DLI group, patients not experiencing relapse had a significantly higher CD56+16+ NK cell populations at day +30 (125±130 cells/microl vs 15 ± 8±; p=0.004) and day +60 (104±93 cells/microl vs 25± 16; p=0.006).CTLA4Ig primed DLI resulted in greater proliferation of NK cells with mature phenotype than the DLI group with comparatively lower incidences of acute GVHD and NRM and improved relapse-free survival, particularly in those with myeloid malignancies. Higher proliferation of mature NK cells at 30 and 60 days post-transplant was associated with a lower risk of relapse following CTLA4Ig primed DLI. Thus, this novel approach enabled early institution of adoptive immunotherapy to mitigate the risk of relapse in advanced leukemia without increase in GVHD or NRM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Post-Transplantation Cyclophosphamide and Short-Term Methotrexate for Tolerance Induction in HLA-Haploidentical Transplantation for Severe Aplastic Anemia

Background. Haploidentical related donors (HIDs) are attractive donors for patients with severe aplastic anemia (SAA) because they are readily available for nearly all the patients. Bone marrow transplant (BMT) using post-transplant cyclophosphamide (PTCy) has been shown effective to cross the immune barriers between the recipients and their HIDs. Short-term low dose of methotrexate (ST-MTX) has long been adopted either alone or combined with calcineurin inhibitors to get across the immune barrier in hematopoietic stem cell transplant (HSCT).Aim of the study. We presented the outcome of 15 patients with SAA who underwent haploidentical transplant (HIT) using T-cell-replete peripheral graft and combination of PTCy with ST-MTX.Patients and grafts. We analyzed the medical records of 15 patients with SAA between Jan 2015 and July 2017. The transplants were either upfront (8/15) or rescue treatment (7/15). The median age of the patients was 16 (range 5-39) years and 66.7% (10/15) were male. All the patients received granulocyte colony-stimulating factor (G-CSF) mobilized peripheral graft from haploidentical related donors. The median mononuclear cells (MNCs) was 12.4 × 108 cells/kg recipient ideal body weight (IBW) with the range of (7.0-22.0) × 108 cells/kg, the CD34+ cell was 7.5 × 106 cells/kg with the range of (5-23) × 106 cells/kg and the CD3+ cells was 2.7 × 108 cells/kg with the range of (1.8-5.0) × 108 cells/kg.Conditioning regimen and GvHD prophylaxis. We used a TBI-free transplantation platform named FBC+PTCy/ST-MTX. Fludarabine was administered at 40 mg/m2 IV daily for 5 days. Busulfan was delivered in a total dose of 6.4mg/kg. Cyclophosphamide was given 14.5 mg/kg daily for 2 days. PTCy was administered at 60mg/kg/day IV on days +3 and +4 post-transplant. Low doses of MTX were delivered on days +1, +2 and +5 post-transplant at 15mg/m2, 10mg/m2 and 10mg/m2. Cyclosporine was given from day +6 through day +180 with the target through level 150~180ng/ml and then was tapered until 12 months post transplant. Mycophenolate mofetil (MMF) was given from day +6 to +35 days for the first 3 patients and then was eliminated for the following patients.Results.Neither primary nor secondary graft failure (GF) was seen in this study. Neutrophil engrafted at 15 days (range, 13-18 days). Platelets engrafted at 19 days (range, 14-36 days) and recovered over 100 × 109/L at 38 days (range, 14-77 days). The time to total lymphocytes recovery over 0.5 × 109/L for 3 consecutive days was 34 days (range, 25-72 days). The accumulated engraftment of neutrophil and platelets were both 100% at +40 days. (Figure 1-A, B.)The cumulative incidence of aGvHD at 100 days was 20.0%(3/15). Two patients had grade I skin aGvHD and the other one had grade II skin aGvHD. No grade III-IV aGvHD was seen. 12 patients were evaluable for the incidence of cGvHD and none of them developed moderate or sever cGvHD with a median of 150 (102-806) days post transplant. (Figure 1-C, D.) During the follow-up, one patients had skin cGvHD and received systemic therapy with corticoid. The treatment was discontinued after 8 months post transplant. All 3 patients, who survived beyond 12 months post transplant at the end of our follow-up, were off immunosuppress.With a median follow-up of 230 days (range 41-806 days), all the patients are alive. They are transfusion independent with karnofsky performance status scale (KPS) scores 100.Conclusion.This study shows that haploidentical related transplant can be successfully applied using T-cell-repleted peripheral grafts. ST-MTX may facilitate PTCy to achieve a stable engraftment with a minimal risk of GvHD in HIT for patients with SAA. Our approach should expand the donor pool for patients with SAA. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Harnessing Mechanisms of Immune Tolerance to Improve Outcomes in Solid Organ Transplantation: A Review.

Survival after solid organ transplantation (SOT) is limited by chronic rejection as well as the need for lifelong immunosuppression and its associated toxicities. Several preclinical and clinical studies have tested methods designed to induce transplantation tolerance without lifelong immune suppression. The limited success of these strategies has led to the development of clinical protocols that combine SOT with other approaches, such as allogeneic hematopoietic stem cell transplantation (HSCT). HSCT prior to SOT facilitates engraftment of donor cells that can drive immune tolerance. Recent innovations in graft manipulation strategies and post-HSCT immune therapy provide further advances in promoting tolerance and improving clinical outcomes. In this review, we discuss conventional and unconventional immunological mechanisms underlying the development of immune tolerance in SOT recipients and how they can inform clinical advances. Specifically, we review the most recent mechanistic studies elucidating which immune regulatory cells dampen cytotoxic immune reactivity while fostering a tolerogenic environment. We further discuss how this understanding of regulatory cells can shape graft engineering and other therapeutic strategies to improve long-term outcomes for patients receiving HSCT and SOT.

Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance.

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

Development of Donor Recipient Chimeric Cells of bone marrow origin as a novel approach for tolerance induction in transplantation.

Cell therapies and chimerism-based strategies are currently the most successful approach for tolerance induction in transplantation. This study aimed to establish and characterize novel Donor Recipient Chimeric Ccell (DRCC) therapy of bone marrow (BM) origin presenting donor-recipient phenotype to support tolerance induction. Ex vivo fusions of fully MHC-mismatched BM cells from ACI (RT1a) and Lewis (RT1l) rats were performed using polyethylene-glycol (PEG). The creation of rat DRCC was tested by flow cytometry (FC), confocal microscopy and PCR. FC characterized DRCC's phenotype (CD3, CD4, CD8, CD45, CD90, CD11b/c, CD45RA, OX-82, or CD4/CD25) and apoptosis, while mixed lymphocyte reaction assessed DRCC's immunogenicity and colony forming unit assay tested DRCC's differentiation and proliferation. DRCC's polyploidy was evaluated using Hoechst33342 staining and COMET assay tested genotoxicity of fusion procedure. ELISA analyzed the secretion of IL-2, IL-4, IL-10, TGFß1, IFNγ and TNFα by DRCC at day 1, 5 and 14 post-fusion. The DRCC's phenotype after long-term culturing was assessed by reverse-transcription PCR. The chimeric state of DRCC was confirmed. Fusion did not change the expression of hematopoietic markers compared to BM controls. Although an increased number of early and late apoptotic (Annexin V+/Sytox blue- and Annexin V+/Sytox blue+, respectively) DRCC was detected at 24h post-fusion, the number significantly decreased at day 5 (38.4%±3.1% and 22.6%±2.5%, vs. 28.3%±2.5% and 13.9%±2.6%, respectively, P<0.05). DRCC presented decreased immunogenicity, increased expression of IL-10 and TGFβ1 and proliferative potential comparable to BM controls. The average percentage of tetraploid DRCC was 3.1%±0.2% compared to 0.96%±0.1% in BM controls. The lack of damage to the DRCC's DNA content supported the DRCC's safety. In culture, DRCC maintained proliferation for up to 28 days while preserving hematopoietic profile. This study confirmed feasibility of DRCC creation via ex vivo PEG mediated fusion. The created DRCC revealed pro-tolerogenic properties indicating potential immunomodulatory effect of DRCC therapy when applied in vivo to support tolerance induction in solid organ and vascularized composite allograft transplantation.

A Large-Scale Bank of Organ Donor Bone Marrow and Matched Mesenchymal Stem Cells for Promoting Immunomodulation and Transplant Tolerance.

Induction of immune tolerance for solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. This would obviate the need for life-long immunosuppression which is associated with serious adverse outcomes, such as infections, cancers, and renal failure. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of living donor renal transplantation, the mixed chimerism approach has not achieved durable immune tolerance on a large scale in preclinical or clinical trials with other solid organs or vascular composite allotransplants (VCA). Ossium Health has established a bank of cryopreserved bone marrow (BM), termed “hematopoietic progenitor cell (HPC), Marrow,” recovered from deceased organ donor vertebral bodies. This new source for hematopoietic cell transplant will be a valuable resource for treating hematological malignancies as well as for inducing transplant tolerance. In addition, we have discovered and developed a large source of mesenchymal stem (stromal) cells (MSC) tightly associated with the vertebral body bone fragment byproduct of the HPC, Marrow recovery process. Thus, these vertebral bone adherent MSC (vBA-MSC) are matched to the banked BM obtained from each donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than traditional BM-MSC; however, their abundance is >1,000-fold higher than obtainable from living donor BM aspirates. Based on our own unpublished data as well as reports published by others, MSC facilitate chimerism, especially at limiting hematopoietic stem and progenitor cell (HSPC) numbers and increase safety by controlling and/or preventing graft-vs.-host-disease (GvHD). Thus, vBA-MSC have the potential to facilitate mixed chimerism, promote complementary peripheral immunomodulatory functions and increase safety of BM infusions. Both HPC, Marrow and vBA-MSC have potential use in current VCA and solid organ transplant (SOT) tolerance clinical protocols that are amenable to “delayed tolerance.” Current trials with HPC, Marrow are planned with subsequent phases to include vBA-MSC for tolerance of both VCA and SOT.

Apoptotic Donor Cells in Transplantation.

Despite significant advances in prevention and treatment of transplant rejection with immunosuppressive medications, we continue to face challenges of long-term graft survival, detrimental medication side effects to both the recipient and transplanted organ together with risks for opportunistic infections. Transplantation tolerance has so far only been achieved through hematopoietic chimerism, which carries with it a serious and life-threatening risk of graft versus host disease, along with variability in persistence of chimerism and uncertainty of sustained tolerance. More recently, numerous in vitro and in vivo studies have explored the therapeutic potential of silent clearance of apoptotic cells which have been well known to aid in maintaining peripheral tolerance to self. Apoptotic cells from a donor not only have the ability of down regulating the immune response, but also are a way of providing donor antigens to recipient antigen-presenting-cells that can then promote donor-specific peripheral tolerance. Herein, we review both laboratory and clinical evidence that support the utility of apoptotic cell-based therapies in prevention and treatment of graft versus host disease and transplant rejection along with induction of donor-specific tolerance in solid organ transplantation. We have highlighted the potential limitations and challenges of this apoptotic donor cell-based therapy together with ongoing advancements and attempts made to overcome them.

Recent Advances in Costimulatory Blockade to Induce Immune Tolerance in Liver Transplantation

Liver transplantation is an effective therapy for end-stage liver disease. However, most postoperative patients must take immunosuppressive drugs to prevent organ rejection. Interestingly, some transplant recipients have normal liver function and do not experience organ rejection after the withdrawal of immunosuppressive agents. This phenomenon, called immune tolerance, is the ultimate goal in clinical transplantation. Costimulatory molecules play important roles in T cell-mediated immune responses and the maintenance of T cell tolerance. Blocking costimulatory pathways can alter T cell responses and prolong graft survival. Better understanding of the roles of costimulatory molecules has facilitated the use of costimulatory blockade to effectively induce immune tolerance in animal transplantation models. In this article, we review the state of the art in costimulatory pathway blockade for the induction of immune tolerance in transplantation and its potential application prospects for liver transplantation.

Strategies for Liver Transplantation Tolerance.

Liver transplant (LT) recipients require life-long immunosuppression (IS) therapy to preserve allograft function. The risks of chronic IS include an increased frequency of malignancy, infection, renal impairment, and other systemic toxicities. Despite advances in IS, long-term LT outcomes have not been improved over the past three decades. Standard-of-care (SoC) therapy can, in rare cases, lead to development of operational tolerance that permits safe withdrawal of maintenance IS. However, successful IS withdrawal cannot be reliably predicted and, in current prospective studies, is attempted several years after the transplant procedure, after considerable exposure to the cumulative burden of maintenance therapy. A recent pilot clinical trial in liver tolerance induction demonstrated that peri-transplant immunomodulation, using a regulatory T-cell (Treg) approach, can reduce donor-specific alloreactivity and allow early IS withdrawal. Herein we review protocols for active tolerance induction in liver transplantation, with a focus on identifying tolerogenic cell populations, as well as barriers to tolerance. In addition, we propose the use of novel IS agents to promote immunomodulatory mechanisms favoring tolerance. With numerous IS withdrawal trials underway, improved monitoring and use of novel immunomodulatory strategies will help provide the necessary knowledge to establish an active liver tolerance induction protocol for widespread use.

Tregs and Mixed Chimerism as Approaches for Tolerance Induction in Islet Transplantation.

Pancreatic islet transplantation is a promising method for the treatment of type 1 and type 3 diabetes whereby replacement of islets may be curative. However, long-term treatment with immunosuppressive drugs (ISDs) remains essential for islet graft survival. Current ISD regimens carry significant side-effects for transplant recipients, and are also toxic to the transplanted islets. Pre-clinical efforts to induce immune tolerance to islet allografts identify ways in which the recipient immune system may be reeducated to induce a sustained transplant tolerance and even overcome autoimmune islet destruction. The goal of these efforts is to induce tolerance to transplanted islets with minimal to no long-term immunosuppression. Two most promising cell-based therapeutic strategies for inducing immune tolerance include T regulatory cells (Tregs) and donor and recipient hematopoietic mixed chimerism. Here, we review preclinical studies which utilize Tregs for tolerance induction in islet transplantation. We also review myeloablative and non-myeloablative hematopoietic stem cell transplantation (HSCT) strategies in preclinical and clinical studies to induce sustained mixed chimerism and allograft tolerance, in particular in islet transplantation. Since Tregs play a critical role in the establishment of mixed chimerism, it follows that the combination of Treg and HSCT may be synergistic. Since the success of the Edmonton protocol, the feasibility of clinical islet transplantation has been established and nascent clinical trials testing immune tolerance strategies using Tregs and/or hematopoietic mixed chimerism are underway or being formulated.

The Pursuit of Regulatory T Cells in the Induction of Transplant Tolerance.

Organ transplantation is a preferred treatment option for patients with end-stage organ failure. However, transplant induces a robust rejection response that necessitates life-long immunosuppression, which often leads to a plethora of comorbidities. Thus, the goal of transplantation is to achieve a state of tolerance wherein the host permanently accepts the transplanted organ while maintaining normal immune responses to other antigens. Regulatory T cells (Tregs) play an important role in realizing this goal and are being explored in both animal models and human clinical trials. In this chapter, we discuss the key principles of transplant rejection and Treg biology, as well as the status of human clinical trials utilizing Tregs as cellular therapy. We discuss how the current immunosuppressive drugs are utilized in transplantation in favoring an increased Treg to T effector cell ratio, different approaches in generation of therapeutic Tregs, and various facets in Treg trial designs in the clinic. Such clinical trials provided many opportunities to leverage our understanding of Tregs in transplantation. They also demonstrated Tregs as a safe cellular therapy for human use, but the efficacy of this treatment has yet to be fully realized.

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

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. 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. 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'.

Benefit of Belatacept in Cord Blood-Derived Regulatory T Cell-Mediated Suppression of Alloimmune Response

The role of Regulatory T cells (Tregs) in tolerance induction post-transplantation is well-established, but Tregs adoptive transfer alone without combined immunosuppressants have failed so far in achieving clinical outcomes. Here we applied a set of well-designed criteria to test the influence of commonly used immunosuppressants (belatacept, tacrolimus, and mycophenolate) on cord blood-derived Tregs (CB-Tregs). Our study shows that while none of these immunosuppressants modulated the stability and expression of homing molecules by CB-Tregs, belatacept met all other selective criteria, shown by its ability to enhance CB-Tregs-mediated in vitro suppression of the allogeneic response without affecting their viability, proliferation, mitochondrial metabolism and expression of functional markers. In contrast, treatment with tacrolimus or mycophenolate led to reduced expression of functional molecule GITR in CB-Tregs, impaired their viability, proliferation and mitochondrial metabolism. These findings indicate that belatacept could be considered as a candidate in Tregs-based clinical immunomodulation regimens to induce transplant tolerance.

Soluble CD83 inhibits acute rejection by up regulating TGF-β and IDO secretion in rat liver transplantation

BackgroundAllogeneic transplantation immune tolerance is currently a hot research issue and soluble CD83(sCD83) is a novel immunomodulator with great potential in inducing transplantation tolerance. ObjectiveTo investigate the mechanism of the immune tolerance effect of sCD83 on rat liver transplantation. MethodA rat liver transplantation model was established to study the effects of sCD83 on the expression levels of IL-2, IL-10, and TGF-β in peripheral blood and the mRNA expressions of foxp3, TGF-β, and Indoleamine 2,3-dioxygenase (IDO) in liver. The expression changes of costimulatory molecules CD80, CD86, and MHC-II on the surface of DC cells and the expressions of IDO + DC cell, TGF-β + CD4 + T cell, and CD4 + CD25 + Foxp3 + T cell were analyzed and compared. ResultssCD83 alleviated the rejection activity index (RAI) of rat liver transplantation in the early stage, increased the expressions of TGF-β, IL-10 in peripheral blood and the mRNAs of IDO, TGF-β and foxp3 in the transplanted liver, and down-regulated the expressions of MHC-II, CD86, and CD80 in DC cells, resulting in significant increased numbers of tolerogenic TGF-β + CD4 + T cells, Treg cells, and IDO + DC cells with low expression. ConclusionsCD83 inhibited acute rejection after liver transplantation in an allogeneic rat, and the mechanism was associated with the effect that sCD83 increased the expression of TGF-β, activated IDO immunosuppressive pathway, and increased tolerogenic DC cells and Treg cells.