Hematopoietic cell-based and non-hematopoietic cell-based strategies for immune tolerance induction in living-donor renal transplantation: A systematic review.

INTRODUCTION: Despite its use to prevent acute rejection, lifelong immunosuppression can adversely impact long-term patient and graft outcomes. In theory, immunosuppression withdrawal is the ultimate goal of kidney transplantation, and is made possible by the induction of immunological tolerance. The purpose of this paper is to review the safety and efficacy of immune tolerance induction strategies in living-donor kidney transplantation, both chimerism-based and non-chimerism-based. The impact of these strategies on transplant outcomes, including acute rejection, allograft function and survival, cost, and immune monitoring, will also be discussed. MATERIALS AND METHODS: Databases such as PubMed, Scopus, and Web of Science, as well as additional online resources such as EBSCO, were exhaustively searched. Adult living-donor kidney transplant recipients who developed chimerism-based tolerance after concurrent bone marrow or hematopoietic stem cell transplantation or those who received non-chimerism-based, non-hematopoietic cell therapy using mesenchymal stromal cells, dendritic cells, or regulatory T cells were studied between 2000 and 2021. Individual sources of evidence were evaluated critically, and the strength of evidence and risk of bias for each outcome of the transplant tolerance study were assessed. RESULTS: From 28,173 citations, 245 studies were retrieved after suitable exclusion and duplicate removal. Of these, 22 studies (2 RCTs, 11 cohort studies, 6 case-control studies, and 3 case reports) explicitly related to both interventions (chimerism- and non-chimerism-based immune tolerance) were used in the final review process and were critically appraised. According to the findings, chimerism-based strategies fostered immunotolerance, allowing for the safe withdrawal of immunosuppressive medications. Cell-based therapy, on the other hand, frequently did not induce tolerance except for minimising immunosuppression. As a result, the rejection rates, renal allograft function, and survival rates could not be directly compared between these two groups. While chimerism-based tolerance protocols posed safety concerns due to myelosuppression, including infections and graft-versus-host disease, cell-based strategies lacked these adverse effects and were largely safe. There was a lack of direct comparisons between HLA-identical and HLA-disparate recipients, and the cost implications were not examined in several of the retrieved studies. Most studies reported successful immunosuppressive weaning lasting at least 3 years (ranging up to 11.4 years in some studies), particularly with chimerism-based therapy, while only a few investigators used immune surveillance techniques. The studies reviewed were often limited by selection, classification, ascertainment, performance, and attrition bias. CONCLUSIONS: This review demonstrates that chimerism-based hematopoietic strategies induce immune tolerance, and a substantial number of patients are successfully weaned off immunosuppression. Despite the risk of complications associated with myelosuppression. Non-chimerism-based, non-hematopoietic cell protocols, on the other hand, have been proven to facilitate immunosuppression minimization but seldom elicit immunological tolerance. However, the results of this review must be interpreted with caution because of the non-randomised study design, potential confounding, and small sample size of the included studies. Further validation and refinement of tolerogenic protocols in accordance with local practice preferences is also warranted, with an emphasis on patient selection, cost ramifications, and immunological surveillance based on reliable tolerance assays.

A Machine Learning Analysis of Prognostic Genes Associated With Allograft Tolerance After Renal Transplantation.

In this study, we aimed to identify transplantation tolerance (TOL)-related gene signature and use it to predict the different types of renal allograft rejection performances in kidney transplantation. Gene expression data were obtained from the Gene Expression Omnibus (GEO) database, differently expressed genes (DEGs) were performed, and the gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were also conducted. The machine learning methods were combined to analyze the feature TOL-related genes and verify their predictive performance. Afterward, the gene expression levels and predictive performances of TOL-related genes were conducted in the context of acute rejection (AR), chronic rejection (CR), and graft loss through heatmap plots and the receiver operating characteristic (ROC) curves, and their respective immune infiltration results were also performed. Furthermore, the TOL-related gene signature for graft survival was conducted to discover gene immune cell enrichment. A total of 25 TOL-related DEGs were founded, and the GO and KEGG results indicated that DEGs mainly enriched in B cell-related functions and pathways. 7 TOL-related gene signature was constructed and performed delightedly in TOL groups and different types of allograft rejection. The immune infiltration analysis suggested that gene signature was correlated with different types of immune cells. The Kaplan-Meier (KM) survival analysis demonstrated that BLNK and MZB1 were the prognostic TOL-related genes. Our study proposed a novel gene signature that may influence TOL in kidney transplantation, providing possible guidance for immunosuppressive therapy in kidney transplant patients.

Donor antigen-specific regulatory T cell administration to recipients of live donor kidneys: A ONE Study consortium pilot trial.

Regulatory T cells (Tregs) can inhibit cellular immunity in diverse experimental models and have entered early phase clinical trials in autoimmunity and transplantation to assess safety and efficacy. As part of the ONE Study consortium, we conducted a phase I-II clinical trial in which purified donor antigen reactive (dar)-Tregs (CD4+CD25+CD127lo) were administered to 3 patients, 7 to 11 days after live donor renal transplant. Recipients received a modified immunosuppression regimen, without induction therapy, consisting of maintenance tacrolimus, mycophenolate mofetil, and steroids. Steroids were weaned off over 14 weeks. No rejection was seen on any protocol biopsy. Therefore, all patients discontinued mycophenolate mofetil 11 to 13 months posttransplant, per protocol. An early for-cause biopsy in 1 patient, 5 days after dar-Treg infusion, revealed absence of rejection and accumulation of Tregs in the kidney allograft. All patients had Treg-containing lymphoid aggregates evident on protocol biopsies performed 8 months posttransplant. The patients are now all >6 years posttransplant on tacrolimus monotherapy with excellent graft function. None experienced rejection episodes. No serious adverse events were attributable to Treg administration. These results support a favorable safety profile of dar-Tregs administered early after renal transplant, suggest early biopsy might be an instructive research endpoint and provide preliminary evidence of potential immunomodulatory activity.

Responses of regulatory and effector T-cells to low-dose interleukin-2 differ depending on the immune environment after allogeneic stem cell transplantation.

CD4+Foxp3+ regulatory T cells (Tregs) play a central role in the maintenance of immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). Tregs promptly respond to low concentrations of IL-2 through the constitutive expression of high-affinity IL-2 receptors. It has been reported that low-dose IL-2 therapy increased circulating Tregs and improved clinical symptoms of chronic GVHD. Clinical studies of IL-2 therapy so far have mainly targeted patients in the chronic phase of transplantation when acute immune responses has subsided. However, the biological and clinical effects of exogenous IL-2 in an acute immune environment have not been well investigated. In the current study, we investigated the impact of exogenous IL-2 therapy on the post-transplant homeostasis of T cell subsets which influence the balance between GVHD and GVL in the acute phase, by setting the various immune environments early after HSCT in murine model. We initially found that 5,000 IU of IL-2 was enough to induce the active proliferation of Treg without influencing other conventional T cells (Tcons) when administered to normal mice. However, activated Tcons showed the response to the same dose of IL-2 in recipients after allogeneic HSCT. In a mild inflammatory environment within a threshold, exogenous IL-2 could effectively modulate Treg homeostasis with just limited influence to activated T cells, which resulted in an efficient GVHD suppression. In contrast, in a severely inflammatory environment, exogenous IL-2 enhanced activated T cells rather than Tregs, which resulted in the exacerbation of GVHD. Of interest, in an immune-tolerant state after transplant, exogenous IL-2 triggered effector T-cells to exert an anti-tumor effect with maintaining GVHD suppression. These data suggested that the responses of Tregs and effector T cells to exogenous IL-2 differ depending on the immune environment in the host, and the mutual balance of the response to IL-2 between T-cell subsets modulates GVHD and GVL after HSCT. Our findings may provide useful information in the optimization of IL-2 therapy, which may be personalized for each patient having different immune status.

The Inability of Ex Vivo Expanded Mesenchymal Stem/Stromal Cells to Survive in Newborn Mice and to Induce Transplantation Tolerance.

An encounter of the developing immune system with an antigen results in the induction of immunological areactivity to this antigen. In the case of transplantation antigens, the application of allogeneic hematopoietic cells induces a state of neonatal transplantation tolerance. This tolerance depends on the establishment of cellular chimerism, when allogeneic cells survive in the neonatally treated recipient. Since mesenchymal stem/stromal cells (MSCs) have been shown to have low immunogenicity and often survive in allogeneic recipients, we attempted to use these cells for induction of transplantation tolerance. Newborn (less than 24 h old) C57BL/6 mice were injected intraperitoneally with 5 × 106 adipose tissue-derived MSCs isolated from allogeneic donors and the fate and survival of these cells were monitored. The impact of MSC application on the proportion of cell populations of the immune system and immunological reactivity was assessed. In addition, the survival of skin allografts in neonatally treated recipients was tested. We found that in vitro expanded MSCs did not survive in neonatal recipients, and the living MSCs were not detected few days after their application. Furthermore, there were no significant changes in the proportion of individual immune cell populations including CD4+ cell lineages, but we detected an apparent shift to the production of Th1 cytokines IL-2 and IFN-γ in neonatally treated mice. However, skin allografts in the MSC-treated recipients were promptly rejected. These results therefore show that in vitro expanded MSCs do not survive in neonatal recipients, but induce a cytokine imbalance without induction of transplantation tolerance.

Th17/Treg cell balance in stable liver transplant recipients.

BACKGROUND: Immune monitoring of transplanted patients may provide a reliable basis for the individualization of immunosuppressive therapy. In addition, it might be applied for realizing the early and non-invasive diagnosis of acute allograft rejection. METHODS: Percentages of TCD4 + IL-17+ (Th17) and TCD4 + CD25 + CD127dim/- (Treg) cells, as well as serum levels of interleukin (IL)-17 and transforming growth factor (TGF)-ß1, were evaluated in 30 stable patients using flow cytometry and ELISA techniques before and six months after liver transplantation. Besides, the same cells and cytokines were quantified in 10 recipients with acute allograft rejection. RESULTS: Six months post-transplant, the percentage of Th17 and Treg cells in the peripheral blood of stable liver transplant recipients reduced significantly, but the Th17/Treg ratios were comparable to the pre-transplant period (1.24 vs. 1.56); however, Th17/Treg ratios in the rejection group was significantly higher than in the stable recipients (4.06 vs. 1.56, P-value = 0.001). Stable patients showed decreased amounts of serum IL-17 which was remarkably lower than in the rejection group (P-value = 0.01). Moreover, there was a significant correlation between the serum level of IL-17 and the percentage of Th17 cells (P-value <0.001). Th17 frequency was negatively associated with the liver allograft function. Notably, TGF-ß1 levels differed neither between pre-and post-transplant samplings nor between stable and rejection groups. CONCLUSION: Six months after liver transplantation, the mean Th17/Treg ratio in stable recipients remained comparable to the pre-transplant values; however, it was significantly elevated in patients with acute allograft rejection, suggesting the Th17/Treg ratio as a probable predictor of acute rejection.

Biomarkers of immune tolerance in kidney transplantation: an overview.

Kidney failure, one of the most prevalent diseases in the world and with increasing incidence, is associated with substantial morbidity and mortality. Currently available modes of kidney replacement therapy include dialysis and kidney transplantation. Though kidney transplantation is the preferred and ideal mode of kidney replacement therapy, this modality, however, is not without its risks. Kidney transplant recipients are constantly at risk of complications associated with immunosuppression, namely, opportunistic infections (e.g., Epstein-Barr virus and cytomegalovirus infections), post-transplant lymphoproliferative disorder, and complications associated with immunosuppressants (e.g., calcineurin inhibitor- and corticosteroid-associated new onset diabetes after transplantation and calcineurin inhibitor-associated nephrotoxicity). Transplantation tolerance, an acquired state in which immunocompetent recipients have developed donor-specific unresponsiveness, may be the Holy Grail in enabling optimal allograft survival and obviating the risks associated with immunosuppression in kidney transplant recipients. This review aims to discuss the biomarkers available to predict, identify, and define the transplant immune tolerant state and various tolerance induction strategies. Regrettably, pediatric patients have not been included in any tolerance studies and this should be the focus of future studies.

Progress in islet xenotransplantation: Immunologic barriers, advances in gene editing, and tolerance induction strategies for xenogeneic islets in pig-to-primate transplantation.

Islet transplantation has emerged as a curative therapy for diabetes in select patients but remains rare due to shortage of suitable donor pancreases. Islet transplantation using porcine islets has long been proposed as a solution to this organ shortage. There have already been several small clinical trials using porcine islets in humans, but results have been mixed and further trials limited by calls for more rigorous pre-clinical data. Recent progress in heart and kidney xenograft transplant, including three studies of pig-to-human xenograft transplant, have recaptured popular imagination and renewed interest in clinical islet xenotransplantation. This review outlines immunologic barriers to islet transplantation, summarizes current strategies to overcome these barriers with a particular focus on approaches to induce tolerance, and describes an innovative strategy for treatment of diabetic nephropathy with composite islet-kidney transplantation.

Cytokines in Liver Transplantation.

Cytokines, soluble mediators of the immune system, play a critical role in the pathogenesis of autoimmune, allergic and infectious diseases. They are also implicated in the initiation and development of allograft rejection. During recent years, there have been considerable advances in generating novel anti-cytokine agents with promoted efficacy and safety, which could be administrated for managing dysregulated cytokine secretion; besides, gene therapy for overexpression of immunomodulatory cytokines has shown substantial improvements. Liver transplantation has been established as a life-saving treatment for end-stage hepatic diseases but the growing number of recipients urge for improved post-transplant care including tolerance induction, infection control and resolving immunosuppressant drugs adverse effects. Cytokines with a wide range of proinflammatory and regulatory properties might be considered as potential therapeutic targets for selective suppression or enhancement of the immune responses in recipients. In the present review, we aimed to summarize the positive and negative effects of cytokines on liver allograft in addition to their prognostic and therapeutic values.

Large Animal Models of Vascularized Composite Allotransplantation: A Review of Immune Strategies to Improve Allograft Outcomes.

Over the past twenty years, significant technical strides have been made in the area of vascularized composite tissue allotransplantation (VCA). As in solid organ transplantation, the allogeneic immune response remains a significant barrier to long-term VCA survival and function. Strategies to overcome acute and chronic rejection, minimize immunosuppression and prolong VCA survival have important clinical implications. Historically, large animals have provided a valuable model for testing the clinical translatability of immune modulating approaches in transplantation, including tolerance induction, co-stimulation blockade, cellular therapies, and ex vivo perfusion. Recently, significant advancements have been made in these arenas utilizing large animal VCA models. In this comprehensive review, we highlight recent immune strategies undertaken to improve VCA outcomes with a focus on relevant preclinical large animal models.

Immunomodulating functions of human leukocyte antigen-G and its role in graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapeutic strategy to treat several hematological malignancies and non-hematological malignancies. However, graft-versus-host disease (GVHD) is a frequent and serious transplant-related complication which dramatically restrains the curative effect of allo-HSCT and a significant cause of morbidity and mortality in allogeneic HCT recipients. Effective prevention of GVHD mainly depends on the induction of peripheral immune tolerance. Human leukocyte antigen-G (HLA-G) is a non-classical MHC class I molecule with a strong immunosuppressive function, which plays a prominent role in immune tolerance. HLA-G triggers different reactions depending on the activation state of the immune cells and system. It also exerts a long-term immune tolerance mechanism by inducing regulatory cells. In this present review, we demonstrate the immunomodulatory properties of human leukocyte antigen-G and highlight the role of HLA-G as an immune regulator of GVHD. Furthermore, HLA-G could also serve as a good predictor of GVHD and represent a new therapeutic target for GVHD.

Delayed non-myeloablative irradiation to induce long-term allograft acceptance in a large animal lung transplantation model.

We previously induced long-term allograft acceptance in an allogeneic lung transplantation (LTx) model in miniature swine using perioperative non-myeloablative irradiation (IRR) combined with infusion of donor specific alloantigen. In order to improve clinical applicability, we delayed induction with irradiation in this study. Left sided single LTx was performed in minipigs. Group 1 received non-myeloablative irradiation (7Gy thymus and 1.5Gy whole body IRR) before LTx and a perioperative donor specific splenocyte infusion (SpTx). Group 2 received perioperative SpTx but delayed IRR three days after LTx. Group 3 was exposed to delayed IRR without SpTx. Whereas 4 out of 7 animals from the non-delayed group never rejected their grafts and were electively sacrificed on postoperative day (POD) +500, all animals from group 2 rejected their grafts before POD 108. In group 3, 3 out of 8 animals developed long-term allograft acceptance. In all groups, donor leukocyte chimerism peaked up to 20% in peripheral blood one hour after reperfusion of the lung. Group 1 maintained prolonged chimerism beyond POD 7, whereas chimerism levels in groups 2 and 3 decreased continuously thereafter. Delayed irradiation has the potential to improve long-term graft survival, yet not as efficient as a perioperative conditioning protocol.

Immunosuppression in Liver Transplantation: State of the Art and Future Perspectives.

BACKGROUND: Novel drugs and combinations for immunosuppression (IS) after liver transplantation is one main reason for improved graft and patient survival seen in the last decades. The backbone of IS is still steroids and calcineurin inhibitors, although novel drugs are being introduced, such as the mammalian target of rapamycin inhibitors (mTOR inhibitor). The challenge today, along with increased patient survival, is the adverse effects of long-term use of immunosuppressive drugs, mainly nephrotoxicity and other serious adverse effects. Concepts: The ultimate outcome after liver transplantation would be achieving tolerance, a state where all IS can be withdrawn. In the meantime, different approaches to reduce and withdraw IS have been tested out in different clinical trials with the aim to reduce the adverse effects of steroids and calcineurin inhibitors. This has formed the basis of today's clinical practice. The different combinations of immunosuppressive drugs have included mTOR inhibitor such as everolimus and different induction drugs such as anti-interleukin 2 receptor antibodies. Regarding induction drugs, lymphocyte depleting (alemtuzumab and ATG) and non-depleting agents, such as basiliximab, have shown advantageous effects. SUMMARY: Alongside steroid and calcineurin inhibitors reduction or elimination, current strategies for post-liver transplantation immunosuppression explore combinations of novel agents. The gauge (or yardstick) here is the fine balance between the adverse effects of IS drugs and the risk of rejection. Long-term maintenance IS regimens, development of tolerance and antibody-mediated rejection are also discussed in this review.

Enhancement of Antigen Presentation by Deletion of Viral Immune Evasion Genes Prevents Lethal Cytomegalovirus Disease in Minor Histocompatibility Antigen-Mismatched Hematopoietic Cell Transplantation.

Hematoablative treatment followed by hematopoietic cell transplantation (HCT) for reconstituting the co-ablated immune system is a therapeutic option to cure aggressive forms of hematopoietic malignancies. In cases of family donors or unrelated donors, immunogenetic mismatches in major histocompatibility complex (MHC) and/or minor histocompatibility (minor-H) loci are unavoidable and bear a risk of graft-vs.-host reaction and disease (GvHR/D). Transient immunodeficiency inherent to the HCT protocol favors a productive reactivation of latent cytomegalovirus (CMV) that can result in multiple-organ CMV disease. In addition, there exists evidence from a mouse model of MHC class-I-mismatched GvH-HCT to propose that mismatches interfere with an efficient reconstitution of antiviral immunity. Here we used a mouse model of MHC-matched HCT with C57BL/6 donors and MHC-congenic BALB.B recipients that only differ in polymorphic autosomal background genes, including minor-H loci coding for minor-H antigens (minor-HAg). Minor-HAg mismatch is found to promote lethal CMV disease in absence of a detectable GvH response to an immunodominant minor-HAg, the H60 locus-encoded antigenic peptide LYL8. Lethality of infection correlates with inefficient reconstitution of viral epitope-specific CD8+ T cells. Notably, lethality is prevented and control of cytopathogenic infection is restored when viral antigen presentation is enhanced by deletion of immune evasion genes from the infecting virus. We hypothesize that any kind of mismatch in GvH-HCT can induce "non-cognate transplantation tolerance" that dampens not only a mismatch-specific GvH response, which is beneficial, but adversely affects also responses to mismatch-unrelated antigens, such as CMV antigens in the specific case, with the consequence of lethal CMV disease.

First-in-human high-cumulative-dose stem cell therapy in idiopathic pulmonary fibrosis with rapid lung function decline.

Previous phase I studies demonstrated safety and some beneficial effects of mesenchymal stem cells (MSCs) in patients with mild to moderate idiopathic pulmonary fibrosis (IPF). The aim of our study was to evaluate the safety, tolerability, and efficacy of a high cumulative dose of bone marrow MSCs in patients with rapid progressive course of severe to moderate IPF. Twenty patients with forced ventilation capacity (FVC) ≥40% and diffusing capacity of the lung for carbon monoxide (DLCO) ≥20% with a decline of both >10% over the previous 12 months were randomized into two groups: one group received two intravenous doses of allogeneic MSCs (2 × 108 cells) every 3 months, and the second group received a placebo. A total amount of 1.6 × 109 MSCs had been administered to each patient after the study completion. There were no significant adverse effects after administration of MSCs in any patients. In the group of MSC therapy, we observed significantly better improvement for the 6-minute walk distance in 13 weeks, for DLCO in 26 weeks, and for FVC in 39 weeks compared with placebo. FVC for 12 months in the MSCs therapy group increased by 7.8% from baseline, whereas it declined by 5.9% in the placebo group. We did not find differences between the groups in mortality (two patients died in each group) or any changes in the high-resolution computed tomography fibrosis score. In patients with IPF and a rapid pulmonary function decline, therapy with high doses of allogeneic MSCs is a safe and promising method to reduce disease progression.

Impact of infection on transplantation tolerance.

Allograft tolerance is the ultimate goal of organ transplantation. Current strategies for tolerance induction mainly focus on inhibiting alloreactive T cells while promoting regulatory immune cells. Pathogenic infections may have direct impact on both effector and regulatory cell populations, therefore can alter host susceptibility to transplantation tolerance induction as well as impair the quality and stability of tolerance once induced. In this review, we will discuss existing data demonstrating the effect of infections on transplantation tolerance, with particular emphasis on the role of the stage of infection (acute, chronic, or latent) and the stage of tolerance (induction or maintenance) in this infection-tolerance interaction. While the deleterious effect of acute infection on tolerance is mainly driven by proinflammatory cytokines induced shortly after the infection, chronic infection may generate exhausted T cells that could in fact facilitate transplantation tolerance. In addition to pathogenic infections, commensal intestinal microbiota also has numerous significant immunomodulatory effects that can shape the host alloimmunity following transplantation. A comprehensive understanding of these mechanisms is crucial for the development of therapeutic strategies for robustly inducing and stably maintaining transplantation tolerance while preserving host anti-pathogen immunity in clinically relevant scenarios.

Notch-1 Inhibition Promotes Immune Regulation in Transplantation Via Regulatory T Cell-Dependent Mechanisms.

BACKGROUND: Transplantation is the treatment of choice for many patients with end-stage organ disease. Despite advances in immunosuppression, long-term outcomes remain suboptimal, hampered by drug toxicity and immune-mediated injury, the leading cause of late graft loss. The development of therapies that promote regulation while suppressing effector immunity is imperative to improve graft survival and minimize conventional immunosuppression. Notch signaling is a highly conserved pathway pivotal to T-cell differentiation and function, rendering it a target of interest in efforts to manipulate T cell-mediated immunity. METHODS: We investigated the pattern of Notch-1 expression in effector and regulatory T cells (Tregs) in both murine and human recipients of a solid-organ transplant. Using a selective human anti-Notch-1 antibody (aNotch-1), we examined the effect of Notch-1 receptor inhibition in full major histocompatibility complex-mismatch murine cardiac and lung transplant models, and in a humanized skin transplant model. On the basis of our findings, we further used a genetic approach to investigate the effect of selective Notch-1 inhibition in Tregs. RESULTS: We observed an increased proportion of Tregs expressing surface and intracellular (activated) Notch-1 in comparison with conventional T cells, both in mice with transplants and in the peripheral blood of patients with transplants. In the murine cardiac transplant model, peritransplant administration of aNotch-1 (days 0, 2, 4, 6, 8, and 10) significantly prolonged allograft survival in comparison with immunoglobulin G-treated controls. Similarly, aNotch-1 treatment improved both histological and functional outcomes in the murine lung transplant model. The use of aNotch-1 resulted in a reduced proportion of both splenic and intragraft conventional T cells, while increasing the proportion of Tregs. Furthermore, Tregs isolated from aNotch-1-treated mice showed enhanced suppressive function on a per-cell basis, confirmed with selective Notch-1 deletion in Tregs (Foxp3EGFPCreNotch1fl/fl). Notch-1 blockade inhibited the mammalian target of rapamycin pathway and increased the phosphorylation of STAT5 (signal transducer and activator of transcription 5) in murine Tregs. Notch-1low Tregs isolated from human peripheral blood exhibited more potent suppressive capacity than Notch-1high Tregs. Last, the combination of aNotch-1 with costimulation blockade induced long-term tolerance in a cardiac transplant model, and this tolerance was dependent on CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) signaling. CONCLUSIONS: Our data reveal a promising, clinically relevant approach for immune modulation in transplantation by selectively targeting Notch-1.

Optimizing PLG nanoparticle-peptide delivery platforms for transplantation tolerance using an allogeneic skin transplant model.

A robust regimen for inducing allogeneic transplantation tolerance involves pre-emptive recipient treatment with donor splenocytes (SP) rendered apoptotic by 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide(ECDI) treatment. However, such a regimen is limited by availability of donor cells, cost of cell procurement, and regulatory hurdles associated with cell-based therapies. Nanoparticles (NP) delivering donor antigens are a promising alternative for promoting transplantation tolerance. Here, we used a B6.C-H-2bm12(bm12) to C57BL/6(B6) skin transplant model involving a defined major histocompatibility antigen mismatch to investigate design parameters of poly(lactide-co-glycolide) (PLG) NPs delivering peptides containing the donor antigen for optimizing skin allograft survival. We showed that an epitope-containing short peptide (P1) was more effective than a longer peptide (P2) at providing graft protection. Importantly, the NP and P1 complex (NP-ECDI-P1) resulted in a significant expansion of graft-infiltrating Tregs. Interestingly, in comparison to donor ECDI-SP that provided indefinite graft protection, NP-ECDI-P1 targeted different splenic phagocytes and skin allografts in these recipients harbored significantly more graft-infiltrating CD8+IFN-γ+ cells. Collectively, the current study provides initial engineering parameters for a cell-free and biocompatible NP-peptide platform for transplant immunoregulation. Moreover, it also provides guidance to future NP engineering endeavors to recapitulate the effects of donor ECDI-SP as a goal for maximizing tolerance efficacy of NP formulations.

In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment.

In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)-derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM-HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene-engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. Stem Cells 2019;37:1176-1188.

Immune tolerance of tissue-engineered skin produced with allogeneic or xenogeneic fibroblasts and syngeneic keratinocytes grafted on mice.

Organs are needed for the long-term replacement of diseased or wounded tissues. Various technologies based on cells seeded in synthetic or biomaterial scaffolds, or scaffold-free methods have been developed in order to produce substitutes that mimic native organs and tissues. For cell-based approaches, the use of living allogeneic fibroblasts could potentially lead to the production of "off-the-shelf" bioengineered organs/tissues. However, questions remain regarding the outcome of allogeneic grafts in terms of persistence of allogeneic cells, tolerance and the host immune reaction against the tissue after implantation. To evaluate graft tolerance of engineered-tissues containing non-autologous fibroblasts, tissue-engineered skin substitutes (TESs) produced with syngeneic, allogeneic or xenogeneic fibroblasts associated with syngeneic, allogeneic or xenogeneic epithelial cells were grafted in mice as primary and secondary grafts. The immune response was evaluated by histological analysis and immunodetection of M2 macrophages, CD4- and CD8-positive T cells, 15, 19, 35 and 56 days after grafting. Tissue-engineered skin composed of non-autologous epithelial cells were rejected. In contrast, TESs composed of non-autologous fibroblasts underlying syngeneic epithelial cells were still present 56 days after grafting. This work shows that TES composed of non-autologous fibroblasts and autologous epithelial cells are not rejected after grafting. STATEMENT OF SIGNIFICANCE: We found that tissue-engineered skin substitutes produced by a scaffold-free cell-based approach from allogeneic fibroblasts and autologous epithelial cells are not rejected after grafting and allow for the permanent coverage of a full-thickness skin wounds. In the field of tissue engineering, these findings open the possibility of selecting a human fibroblastic or stromal cell population based on its biological properties and adequate biosafety, banking it, in order to produce "ready-to-use" bioengineered organs/tissues that could be grafted to any patient without eliciting immune reaction after grafting. Our results can be generalized to any organs produced from fibroblasts. Thus, it is a great step with multiple applications in tissue engineering and transplantation.