Dysfunction of infiltrating cytotoxic CD8+ T cells within the graft promotes murine kidney allotransplant tolerance.

Tolerance of mouse kidney allografts arises in grafts that develop regulatory Tertiary Lymphoid Organs (rTLOs). scRNAseq data and adoptive transfer of alloreactive T cells post-transplant showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required since adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8 KO recipients resulted in acceptance and not rejection. Analysis of scRNAseq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call "defensive tolerance." This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.

A humanized IL-2 mutein expands Tregs and prolongs transplant survival in preclinical models.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

Trained immunity - basic concepts and contributions to immunopathology.

Trained immunity is a functional state of the innate immune response and is characterized by long-term epigenetic reprogramming of innate immune cells. This concept originated in the field of infectious diseases - training of innate immune cells, such as monocytes, macrophages and/or natural killer cells, by infection or vaccination enhances immune responses against microbial pathogens after restimulation. Although initially reported in circulating monocytes and tissue macrophages (termed peripheral trained immunity), subsequent findings indicate that immune progenitor cells in the bone marrow can also be trained (that is, central trained immunity), which explains the long-term innate immunity-mediated protective effects of vaccination against heterologous infections. Although trained immunity is beneficial against infections, its inappropriate induction by endogenous stimuli can also lead to aberrant inflammation. For example, in systemic lupus erythematosus and systemic sclerosis, trained immunity might contribute to inflammatory activity, which promotes disease progression. In organ transplantation, trained immunity has been associated with acute rejection and suppression of trained immunity prolonged allograft survival. This novel concept provides a better understanding of the involvement of the innate immune response in different pathological conditions, and provides a new framework for the development of therapies and treatment strategies that target epigenetic and metabolic pathways of the innate immune system.

Delivery of costimulatory blockade to lymph nodes promotes transplant acceptance in mice.

The lymph node (LN) is the primary site of alloimmunity activation and regulation during transplantation. Here, we investigated how fibroblastic reticular cells (FRCs) facilitate the tolerance induced by anti-CD40L in a murine model of heart transplantation. We found that both the absence of LNs and FRC depletion abrogated the effect of anti-CD40L in prolonging murine heart allograft survival. Depletion of FRCs impaired homing of T cells across the high endothelial venules (HEVs) and promoted formation of alloreactive T cells in the LNs in heart-transplanted mice treated with anti-CD40L. Single-cell RNA sequencing of the LNs showed that anti-CD40L promotes a Madcam1+ FRC subset. FRCs also promoted the formation of regulatory T cells (Tregs) in vitro. Nanoparticles (NPs) containing anti-CD40L were selectively delivered to the LNs by coating them with MECA-79, which binds to peripheral node addressin (PNAd) glycoproteins expressed exclusively by HEVs. Treatment with these MECA-79-anti-CD40L-NPs markedly delayed the onset of heart allograft rejection and increased the presence of Tregs. Finally, combined MECA-79-anti-CD40L-NPs and rapamycin treatment resulted in markedly longer allograft survival than soluble anti-CD40L and rapamycin. These data demonstrate that FRCs are critical to facilitating costimulatory blockade. LN-targeted nanodelivery of anti-CD40L could effectively promote heart allograft acceptance.

Targeting IL-6 to prevent cardiac allograft rejection.

Outcomes following heart transplantation remain suboptimal with acute and chronic rejection being major contributors to poor long-term survival. IL-6 is increasingly recognized as a critical pro-inflammatory cytokine involved in allograft injury and has been shown to play a key role in regulating the inflammatory and alloimmune responses following heart transplantation. Therapies that inhibit IL-6 signaling have emerged as promising strategies to prevent allograft rejection. Here, we review experimental and pre-clinical evidence that supports the potential use of IL-6 signaling blockade to improve outcomes in heart transplant recipients.

Intra-Organ Delivery of Nanotherapeutics for Organ Transplantation.

Targeted delivery of therapeutics through the use of nanoparticles (NPs) has emerged as a promising method that increases their efficacy and reduces their side effects. NPs can be tailored to localize to selective tissues through conjugation to ligands that bind cell-specific receptors. Although the vast majority of nanodelivery platforms have focused on cancer therapy, efforts have begun to introduce nanotherapeutics to the fields of immunology as well as transplantation. In this review, we provide an overview from a clinician's perspective of current nanotherapeutic strategies to treat solid organ transplants with NPs during the time interval between organ harvest from the donor and placement into the recipient, an innovative technology that can provide major benefits to transplant patients. The use of ex vivo normothermic machine perfusion (NMP), which is associated with preserving the function of the organ following transplantation, also provides an ideal opportunity for a localized, sustained, and controlled delivery of nanotherapeutics to the organ during this critical time period. Here, we summarize previous endeavors to improve transplantation outcomes by treating the organ with NPs prior to placement in the recipient. Investigations in this burgeoning field of research are promising, but more extensive studies are needed to overcome the physiological challenges to achieving effective nanotherapeutic delivery to transplanted organs discussed in this review.

A Mixed-chimerism Protocol Utilizing Thymoglobulin and Belatacept Did Not Induce Lung Allograft Tolerance, Despite Previous Success in Renal Allotransplantation.

BACKGROUND: In kidney transplantation, long-term allograft acceptance in cynomolgus macaques was achieved using a mixed-chimerism protocol based on the clinically available reagents, rabbit anti-thymocyte globulin (ATG), and belatacept. Here, we have tested the same protocol in cynomolgus macaques transplanted with fully allogeneic lung grafts. METHODS: Five cynomolgus macaques underwent left orthotopic lung transplantation. Initial immunosuppression included equine ATG and anti-IL6RmAb induction, followed by triple-drug immunosuppression for 4 mo. Post-transplant, a nonmyeloablative conditioning regimen was applied, including total body and thymic irradiation. Rabbit ATG, belatacept, anti-IL6RmAb, and donor bone marrow transplantation (DBMT) were given, in addition to a 28-d course of cyclosporine. All immunosuppressant drugs were stopped on day 29 after DBMT. RESULTS: One monkey rejected its lung before DBMT due to AMR, after developing donor-specific antibodies. Two monkeys developed fatal post-transplant lymphoproliferative disorder, and both monkeys had signs of cellular rejection in their allografts upon autopsy. The remaining 2 monkeys showed severe cellular rejection on days 42 and 70 post-DBMT. Cytokine analysis suggested higher levels of pro-inflammatory markers in the lung transplant cohort, as compared to kidney recipients. CONCLUSION: Although the clinically applicable protocol showed success in kidney transplantation, the study did not show long-term survival in a lung transplant model, highlighting the organ-specific differences in tolerance induction.

The Fourth International Workshop on Clinical Transplant Tolerance.

The International Workshop on Clinical Transplant Tolerance is a biennial meeting that aims to provide an update on the progress of studies of immunosuppression minimization or withdrawal in solid organ transplantation. The Fourth International Workshop on Clinical Tolerance was held in Pittsburgh, Pennsylvania, September 5-6, 2019. This report is a summary of presentations on the status of clinical trials designed to minimize or withdraw immunosuppressive drugs in kidney, liver, and lung transplantation without subsequent evidence of rejection. All protocols had in common the use of donor or recipient cell therapy combined with organ transplantation. The workshop also included presentations of mechanistic studies designed to improve understanding of the cellular and molecular basis of tolerance and to identify potential predictors/biomarkers of tolerance. Strategies to enhance the safety of hematopoietic cell transplantation and to improve patient selection/risk stratification for clinical trials were also discussed.

Trained immunity in organ transplantation.

Consistent induction of donor-specific unresponsiveness in the absence of continuous immunosuppressive therapy and toxic effects remains a difficult task in clinical organ transplantation. Transplant immunologists have developed numerous experimental treatments that target antigen-presentation (signal 1), costimulation (signal 2), and cytokine production (signal 3) to establish transplantation tolerance. While promising results have been obtained using therapeutic approaches that predominantly target the adaptive immune response, the long-term graft survival rates remain suboptimal. This suggests the existence of unrecognized allograft rejection mechanisms that contribute to organ failure. We postulate that trained immunity stimulatory pathways are critical to the immune response that mediates graft loss. Trained immunity is a recently discovered functional program of the innate immune system, which is characterized by nonpermanent epigenetic and metabolic reprogramming of macrophages. Since trained macrophages upregulate costimulatory molecules (signal 2) and produce pro-inflammatory cytokines (signal 3), they contribute to potent graft reactive immune responses and organ transplant rejection. In this review, we summarize the detrimental effects of trained immunity in the context of organ transplantation and describe pathways that induce macrophage training associated with graft rejection.

Translational impact of NIH-funded nonhuman primate research in transplantation.

The National Institutes of Health (NIH) has long supported using nonhuman primate (NHP) models for research on kidney, pancreatic islet, heart, and lung transplantation. The primary purpose of this research has been to develop new treatments for down-modulating or preventing deleterious immune responses after transplantation in human patients. Here, we discuss NIH-funded NHP studies of immune cell depletion, costimulation blockade, regulatory cell therapy, desensitization, and mixed hematopoietic chimerism that either preceded clinical trials or prevented the human application of therapies that were toxic or ineffective.

Early immune biomarkers and intermediate-term outcomes after heart transplantation: Results of Clinical Trials in Organ Transplantation-18.

Clinical Trials in Organ Transplantation-18 (CTOT-18) is a follow-up analysis of the 200-subject multicenter heart transplant CTOT-05 cohort. CTOT-18 aimed to identify clinical, epidemiologic, and biologic markers associated with adverse clinical events past 1 year posttransplantation. We examined various candidate biomarkers including serum antibodies, angiogenic proteins, blood gene expression profiles, and T cell alloreactivity. The composite endpoint (CE) included death, retransplantation, coronary stent, myocardial infarction, and cardiac allograft vasculopathy. The mean follow-up was 4.5 ± SD 1.1 years. Subjects with serum anti-cardiac myosin (CM) antibody detected at transplantation and at 12 months had a higher risk of meeting the CE compared to those without anti-CM antibody (hazard ratio [HR] = 2.9, P = .046). Plasma VEGF-A and VEGF-C levels pretransplant were associated with CE (odds ratio [OR] = 13.24, P = .029; and OR = 0.13, P = .037, respectively). Early intravascular ultrasound findings or other candidate biomarkers were not associated with the study outcomes. In conclusion, anti-CM antibody and plasma levels of VEGF-A and VEGF-C were associated with an increased risk of adverse events. Although this multicenter report supports further evaluation of the mechanisms through which anti-CM antibody and plasma angiogenesis proteins lead to allograft injury, we could not identify additional markers of adverse events or potential novel therapeutic targets.

Maintaining T cell tolerance of alloantigens: Lessons from animal studies.

Achieving host immune tolerance of allogeneic transplants represents the ultimate challenge in clinical transplantation. It has become clear that different cells and mechanisms participate in acquisition versus maintenance of allograft tolerance. Indeed, manipulations which prevent tolerance induction often fail to abrogate tolerance once it has been established. Hence, elucidation of the immunological mechanisms underlying maintenance of T cell tolerance to alloantigens is essential for the development of novel interventions that preserve a robust and long lasting state of allograft tolerance that relies on T cell deletion in addition to intra-graft suppression of inflammatory immune responses. In this review, we discuss some essential elements of the mechanisms involved in the maintenance of naturally occurring or experimentally induced allograft tolerance, including the newly described role of antigen cross-dressing mediated by extracellular vesicles.

Dosing optimization of CCR4 immunotoxin for improved depletion of CCR4+ Treg in nonhuman primates.

Recently, we have developed a diphtheria toxin-based recombinant anti-human CCR4 immunotoxin for targeting CCR4+ tumors and Tregs. In this study, we further optimized the dosing schedule for improved CCR4+ Treg depletion. We have demonstrated that up to a 90% depletion was achieved and the depletion extended to approximately 2 weeks in the peripheral blood and more than 48 days in the lymph node at 25 µg·kg-1 , BID for 8 consecutive days in cynomolgus monkeys. Expansion was observed including monocytes and NK cells. Antibody against the CCR4 immunotoxin was detected after approximately 2 weeks, affecting further depletion efficacy for multiple course treatment.

Prevalence of polyreactive innate clones among graft--infiltrating B cells in human cardiac allograft vasculopathy.

BACKGROUND: Cardiac allograft vasculopathy (CAV) has been associated with graft-infiltrating B cells, although their characteristics are still unclear. In this study we examined the frequency, localization and reactivity profile of graft-infiltrating B cells to determine their contribution to the pathophysiology of CAV. METHODS: B cells, plasma cells and macrophages were examined by immunohistochemistry in 56 allografts with CAV, 49 native failed hearts and 25 autopsy specimens. A total of 102 B-cell clones were immortalized directly from the infiltrates of 3 fresh cardiac samples with CAV. Their secreted antibodies were assessed using enzyme-linked immunoassay and flow cytometry. RESULTS: B-cell infiltration was observed around coronary arteries in 93% of allograft explants with CAV. Comparatively, intragraft B cells were less frequent and less dense in the intraventricular myocardium from where routine biopsies are obtained. Plasma cells and macrophages were also detected in 85% and 95% of explants, respectively. Remarkably, B-cell infiltrates were not associated with circulating donor-specific antibodies (DSA) or prior episodes of antibody-mediated rejection (AMR). Among all B-cell clones generated from 3 explants with CAV, a majority secreted natural antibodies reactive to multiple autoantigens and apoptotic cells, a characteristic of innate B cells. CONCLUSIONS: Our study reveals a high frequency of infiltrating B cells around the coronary arteries of allografts with CAV, independent of DSA or AMR. These cells are enriched for innate B cells with a polyreactive profile. The findings shift the focus from conventional DSA-producing B cells to the potentially pathogenic polyreactive B cells in the development of clinical CAV.

Preclinical and clinical studies for transplant tolerance via the mixed chimerism approach.

Based upon observations in murine models, we have developed protocols to induce renal allograft tolerance by combined kidney and bone marrow transplantation (CKBMT) in non-human primates (NHP) and in humans. Induction of persistent mixed chimerism has proved to be extremely difficult in major histocompatibility complex (MHC)-mismatched primates, with detectable chimerism typically disappearing within 30-60 days. Nevertheless, in MHC mismatched NHP, long-term immunosuppression-free renal allograft survival has been achieved reproducibly, using a non-myeloablative conditioning approach that has also been successfully extended to human kidney transplant recipients. CKBMT has also been applied to the patients with end stage renal disease with hematologic malignancies. Renal allograft tolerance and long-term remission of myeloma have been achieved by transient mixed or persistent full chimerism. This review summarizes the current status of preclinical and clinical studies for renal and non-renal allograft tolerance induction by CKBMT. Improving the consistency of tolerance induction with less morbidity, extending this approach to deceased donor transplantation and inducing tolerance of non-renal transplants, are critical next steps for bringing this strategy to a wider range of clinical applications.

Ontak-like human IL-2 fusion toxin.

Ontak® is a FDA-approved diphtheria toxin-based recombinant fusion toxin for treatment of human CD25+ cutaneous T cell lymphoma (CTCL). However, it has been discontinued clinically due to the production issue related to the bacterial expression system with difficult purification. Recently we have developed monovalent and bivalent human IL-2 fusion toxins targeting human CD25+ cells using advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system. In vitro efficacy characterization using human CD25+ HUT102/6TG cells demonstrated that both monovalent and bivalent isoforms are potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform. In this study, we further assessed the in vivo efficacy of the human IL-2 fusion toxins using human CD25+ HUT102/6TG tumor-bearing NSG mouse model. The data demonstrated that both monovalent and bivalent human IL-2 fusion toxins significantly prolonged the survival of the human CD25+ tumor-bearing NSG mice in a dose-dependent manner. Then we further assessed the residual tumor cells from the HUT102/6TG tumor-bearing NSG mice using the residual tumor cell bearing NSG mouse model. The results demonstrated that the residual tumor cells were still sensitive to the continual treatment with the human IL-2 fusion toxin. This yeast-expressed human IL-2 fusion toxin will be a promising candidate to replace the clinically discontinued Ontak®.

Diphtheria toxin-based anti-human CD19 immunotoxin for targeting human CD19+ tumors.

CD19 is expressed on normal and neoplastic B cells and is a promising target for immunotherapy. However, there is still an unmet need to further develop novel therapeutic drugs for the treatment of the refractory/relapsing human CD19+ tumors. We have developed a diphtheria toxin-based anti-human CD19 immunotoxin for targeting human CD19+ tumors. We have constructed three isoforms of the CD19 immunotoxin: monovalent, bivalent, and foldback diabody. In vitro binding affinity and efficacy analysis demonstrated that the bivalent isoform had the highest binding affinity and in vitro efficacy. The in vivo efficacy of the CD19 immunotoxins was assessed using human CD19+ JeKo-1 tumor-bearing NOD/SCID IL-2 receptor γ-/- (NSG) mouse model. In these animals, CD19 immunotoxins significantly prolonged the median survival from 31 days in controls to 34, 36, and 40 days in animals receiving the monovalent isoform, foldback diabody isoform, and bivalent isoform, respectively. The bivalent CD19 immunotoxin is a promising therapeutic drug candidate for targeting relapsing/refractory human CD19+ tumors.

Induced regulatory T cells in allograft tolerance via transient mixed chimerism.

Successful induction of allograft tolerance has been achieved in nonhuman primates (NHPs) and humans via induction of transient hematopoietic chimerism. Since allograft tolerance was achieved in these recipients without durable chimerism, peripheral mechanisms are postulated to play a major role. Here, we report our studies of T cell immunity in NHP recipients that achieved long-term tolerance versus those that rejected the allograft (AR). All kidney, heart, and lung transplant recipients underwent simultaneous or delayed donor bone marrow transplantation (DBMT) following conditioning with a nonmyeloablative regimen. After DBMT, mixed lymphocyte culture with CFSE consistently revealed donor-specific loss of CD8+ T cell responses in tolerant (TOL) recipients, while marked CD4+ T cell proliferation in response to donor antigens was found to persist. Interestingly, a significant proportion of the proliferated CD4+ cells were FOXP3+ in TOL recipients, but not in AR or naive NHPs. In TOL recipients, CD4+FOXP3+ cell proliferation against donor antigens was greater than that observed against third-party antigens. Finally, the expanded Tregs appeared to be induced Tregs (iTregs) that were converted from non-Tregs. These data provide support for the hypothesis that specific induction of iTregs by donor antigens is key to long-term allograft tolerance induced by transient mixed chimerism.

Treg depletion in non-human primates using a novel diphtheria toxin-based anti-human CCR4 immunotoxin.

Regulatory T cells (Treg) play an important role in modulating the immune response and has attracted increasing attention in diverse fields such as cancer treatment, transplantation and autoimmune diseases. CC chemokine receptor 4 (CCR4) is expressed on the majority of Tregs, especially on effector Tregs. Recently we have developed a diphtheria-toxin based anti-human CCR4 immunotoxin for depleting CCR4(+) cells in vivo. In this study, we demonstrated that the anti-human CCR4 immunotoxin bound and depleted monkey CCR4(+) cells in vitro. We also demonstrated that the immunotoxin bound to the CCR4(+)Foxp3(+) monkey Tregs in vitro. In vivo studies performed in two naive cynomolgus monkeys revealed 78-89% CCR4(+)Foxp3(+) Treg depletion in peripheral blood lasting approximately 10 days. In lymph nodes, 89-96% CCR4(+)Foxp3(+) Tregs were depleted. No effect was observed in other cell populations including CD8(+) T cells, other CD4(+) T cells, B cells and NK cells. To our knowledge, this is the first agent that effectively depleted non-human primate (NHP) Tregs. This immunotoxin has potential to deplete effector Tregs for combined cancer treatment.

Twelve-Hour Hypothermic Machine Perfusion for Donor Heart Preservation Leads to Improved Ultrastructural Characteristics Compared to Conventional Cold Storage.

BACKGROUND Hypothermic machine perfusion of donor hearts has the theoretical advantage of continuous aerobic metabolism and washes out toxic metabolic byproducts. Here, we studied the effect of hypothermic machine perfusion on cardiac myocyte integrity when hearts are preserved for longer ischemic times (12 hours). MATERIAL AND METHODS Pig hearts were harvested and stored in Celsior® solution for 12 hours using either conventional cold storage on ice (12 h CS, n=3) or pulsatile perfusion with the Paragonix Sherpa Perfusion™ Cardiac Transport System at different flow rates (12 h PP, n=3 or 12 h PP low flow, n=2). After cold preservation, hearts were reperfused using an LV isovolumic Langendorff system. Controls (n=3) were reperfused immediately after organ harvest. Biopsies were taken from the apex of the left ventricle before storage, after storage and after reperfusion to measure ATP and endothelin-1 content in the tissue. TUNEL staining for signs of apoptosis and electron microscopy of the donor hearts were performed. RESULTS 12 h PP hearts showed significantly more weight gain than 12 h CS and controls after preservation. Pulsatile perfused hearts showed less ATP depletion, lower endothelin-1 levels and less apoptosis after preservation compared to CS. Electron microscopy showed damaged muscle fibers, endothelial cell rupture, and injury of mitochondria in the 12 h CS group, while machine perfusion could preserve the cell structures. CONCLUSIONS Hypothermic machine perfusion of donor hearts can preserve the cell structures better than conventional cold storage in prolonged ischemic times. Hypothermic pulsatile perfusion may therefore enable longer preservation times of donor hearts. Whether this method is able to avoid primary graft failure after orthotopic heart transplantation remains to be evaluated in further studies.