Induction Of Donor-specific Tolerance Research Articles

Competitive binding of CD226/TIGIT with poliovirus receptor regulates macrophage polarization and is involved in vascularized skin graft rejection

End-stage organ failure often requires solid organ transplantation. Nevertheless, transplant rejection remains an unresolved issue. The induction of donor-specific tolerance is the ultimate goal in transplantation research. In this study, an allograft vascularized skin rejection model using BALB/c-C57/BL6 mice was established to evaluate the regulation of the poliovirus receptor signaling pathway using CD226 knockout or T cell immunoglobulin and ITIM domain (TIGIT)–crystallizable fragment (Fc) recombinant protein treatment. In the TIGIT-Fc–treated and CD226 knockout groups, graft survival time prolonged significantly, with a regulatory T cell proportion increase and M2-type macrophage polarization. Donor-reactive recipient T cells became hyporesponsive while responding normally after a third-party antigen challenge. In both groups, serum interleukin (IL)-1β, IL-6, IL-12p70, IL-17A, tumor necrosis factor–α, interferon gamma, and monocyte chemoattractant protein-1 levels decreased, and the IL-10 level increased. In vitro, M2 markers, such as Arg1 and IL-10, were markedly increased by TIGIT-Fc, whereas iNOS, IL-1β, IL-6, IL-12p70, tumor necrosis factor–α, and interferon gamma levels decreased. CD226-Fc exerted the opposite effect. TIGIT suppressed TH1 and TH17 differentiation by inhibiting macrophage SHP-1 phosphorylation and enhanced ERK1/2-MSK1 phosphorylation and nuclear translocation of CREB. In conclusion, CD226 and TIGIT competitively bind to poliovirus receptor with activating and inhibitory functions, respectively. Mechanistically, TIGIT promotes IL-10 transcription from macrophages by activating the ERK1/2-MSK1-CREB pathway and enhancing M2-type polarization. CD226/TIGIT–poliovirus receptor are crucial regulatory molecules of allograft rejection.

Gut microbiome and renal transplantation.

Gut microbiome research has been a surge of interest in many branches of medicine in the last decade. Our main aim is to show ability of microbes to infuence the functions of human body, especially in the immune system, and on the other hand to clarify changes in composition of gut microbiome in the post-transplantation period and their function for the long-term survival of the graft and the patient in the context of the occurrence of a wide range of complications. Kidney transplantation with the subsequent use of immunosuppressants and antibiotics affects the composition of gut microbiome. The subsequent development of dysbiosis significantly increases the risk of acute rejection, interstitial fibrosis and tubular atrophy of the graft, post-transplant diarrhoea, organ´s infections and metabolic complications such as post-transplant diabetes mellitus. Also important is the influence of the microorganisms of the gut microbiome on metabolism of immunosuppressants with the production of less effective components and the subsequent necessity of modifying their levels with a higher risk of underdosing and the occurrence of graft rejection. Support of the composition of the gut microbiome in the post-transplantation period in favor of bacteria producing short chain fatty acids (SCFA) is possible by changing of diet with predominance of fiber, the application of probiotics, prebiotics. According to available studies, it can lead to benefits in term of metabolic compensation, to the induction of donor-specific tolerance and many others, with an overall improvement in the quality of patient and graft survival.

Tolerance Induction in Vascularized Composite Allotransplantation-A Brief Review of Preclinical Models.

Pre-clinical studies are an obligatory tool to develop and translate novel therapeutic strategies into clinical practice. Acute and chronic rejection mediated by the recipient's immune system remains an important limiting factor for the (long-term) survival of vascularized composite allografts (VCA). Furthermore, high intensity immunosuppressive (IS) protocols are needed to mitigate the immediate and long-term effects of rejection. These IS regiments can have significant side-effects such as predisposing transplant recipients to infections, organ dysfunction and malignancies. To overcome these problems, tolerance induction has been proposed as one strategy to reduce the intensity of IS protocols and to thereby mitigate long-term effects of allograft rejection. In this review article, we provide an overview about animal models and strategies that have been used to induce tolerance. The induction of donor-specific tolerance was achieved in preclinical animal models and clinical translation may help improve short and long-term outcomes in VCAs in the future.

Treg Therapy for the Induction of Immune Tolerance in Transplantation-Not Lost in Translation?

The clinical success of solid organ transplantation is still limited by the insufficiency of immunosuppressive regimens to control chronic rejection and late graft loss. Moreover, serious side effects caused by chronic immunosuppressive treatment increase morbidity and mortality in transplant patients. Regulatory T cells (Tregs) have proven to be efficient in the induction of allograft tolerance and prolongation of graft survival in numerous preclinical models, and treatment has now moved to the clinics. The results of the first Treg-based clinical trials seem promising, proving the feasibility and safety of Treg therapy in clinical organ transplantation. However, many questions regarding Treg phenotype, optimum dosage, antigen-specificity, adjunct immunosuppressants and efficacy remain open. This review summarizes the results of the first Treg-based clinical trials for tolerance induction in solid organ transplantation and recapitulates what we have learnt so far and which questions need to be resolved before Treg therapy can become part of daily clinical practice. In addition, we discuss new strategies being developed for induction of donor-specific tolerance in solid organ transplantation with the clinical aims of prolonged graft survival and minimization of immunosuppression.

The potential for Treg-enhancing therapies in transplantation.

Since the discovery of regulatory T cells (Tregs) as crucial regulators of immune tolerance against self-antigens, these cells have become a promising tool for the induction of donor-specific tolerance in transplantation medicine. The therapeutic potential of increasing in vivo Treg numbers for a favorable Treg to Teff cell ratio has already been demonstrated in several sophisticated pre-clinical models and clinical pilot trials. In addition to improving cell quantity, enhancing Treg function utilizing engineering techniques led to encouraging results in models of autoimmunity and transplantation. Here we aim to discuss the most promising approaches for Treg-enhancing therapies, starting with adoptive transfer approaches and ex vivo expansion cultures (polyclonal vs. antigen-specific), followed by selective in vivo stimulation methods. Furthermore, we address next generation concepts for Treg function enhancement (CARs, TRUCKs, BARs) as well as the advantages and caveats inherit to each approach. Finally, this review will discuss the clinical experience with Treg therapy in ongoing and already published clinical trials, however, data on long-term results and efficacy is still very limited and many questions that might complicate clinical translation remain open. Here, we discuss the hurdles for clinical translation and elaborate on current Treg based therapeutic options as well as their potencies for improving long-term graft survival in transplantation.

Antibody-Based Preparative Regimens for Cell, Tissue and Organ Transplantation

The ability to successfully transplant cells and organs from a donor into an immunologically disparate recipient is one of the greatest treatment advances in the history of medicine. Nevertheless, acute and chronic rejection, graft versus host disease, and the inability to identify suitable donors continue to be challenges and limit broader application of cell and organ transplantation to the many patients that could benefit. Immunosuppression before and after allogeneic transplant has been found to dramatically improve allograft survival and, despite side effects, has been a mainstay of patient management. Inducing donor-specific tolerance is the holy grail in allotransplantation and is readily established in experimental animals but has been difficult to achieve in patients in settings apart from hematopoietic cell transplantation. Antibody-based conditioning to prepare the recipient is a promising approach towards achieving transplant tolerance in both hematopoietic and solid organ transplant settings, and multiple targets are currently under consideration including those on circulating lymphocytes and hematopoietic stem cells. Here we review progress in the use of antibodies to support cell and tissue transplantation with a particular focus on induction of donor-specific tolerance for solid organ transplantation.

P–418 Different immunoregulatory components at the decidua basalis of oocyte donation pregnancies

Abstract Study question Is the number of regulatory T-cells (Tregs) and immunoregulatory cytokines in the decidua basalis of oocyte donation (OD) pregnancies different compared to naturally conceived pregnancies? Summary answer This study suggests that the immunoregulation at the fetal-maternal interface in OD pregnancies with a higher amount of fetal-maternal HLA mismatches appears to be altered. What is known already Tregs and related immunoregulatory cytokines, such as interleukins, transforming growth factor-β, and galectin–1, play a key role in maintaining tolerance at the decidua basalis in human pregnancy. Previous studies observed decreased numbers of decidual Tregs in miscarriage and preeclamptic pregnancies. These complications occur in higher frequencies in OD pregnancies, which are characterized by more fetal-maternal human leukocyte antigen (HLA) mismatches compared with naturally conceived (NC) and non-donor in vitro fertilization (IVF) pregnancies, since the fetus obtains paternal and donor-derived HLA genes. Consequently, the maternal immune system has to cope with greater immunogenetic dissimilarity. Involved immunoregulatory mechanisms however remain poorly understood. Study design, size, duration: This case-control study included 27 OD, 11 IVF, and 16 NC placentas of uncomplicated pregnancies, which were collected after delivery at 37–42 weeks of gestation between 2005 and 2013. Clinical data, maternal peripheral blood and umbilical cord blood were collected. Participants/materials, setting, methods Decidua basalis was dissected from the placentas, and processed to formalin-fixed, paraffin-embedded slices (4 µm). Immunohistochemical staining for FOXP3, interleukin 10, interleukin 6, galectin–1, transforming growth factor-β, and Flt–1 was performed. Semi-quantitative (FOXP3+ Tregs) and computerized analysis (cytokines), using Image-J software, were executed. Maternal peripheral blood and fetal umbilical cord blood were typed for HLA class I and II, using the Sequence Specific Oligonucleotides PCR technique, to calculate the number of fetal-maternal HLA mismatches. Main results and the role of chance All the deciduae basalis of OD, IVF and NC pregnancies showed FOXP3+ Tregs. No significant differences were found when comparing the three groups for the mean number of FOXP3+ Tregs. However, when the amount of fetal-maternal HLA mismatches was related to the percentage of FOXP3+ Tregs, the Tregs were significantly higher in pregnancies with 4–6 HLA class I mismatches (n = 16), than in those with 0–3 HLA class I mismatches (n = 38; p = 0.029). Furthermore, OD pregnancies express less interleukin 10, interleukin 6, galectin–1 and Flt–1 in the decidua basalis compared to NC pregnancies. Moreover, the amount of interleukin 10 was significantly lower with 3–4 fetal-maternal HLA class II mismatches (p = 0.032). Limitations, reasons for caution This study is limited by a small sample size. Moreover, only term placentas were collected. It would be worthwhile investigating immunological alterations in the decidua throughout the whole gestation, since maternal adaptation of the fetal allograft could be more prominent early in pregnancy. Wider implications of the findings: Unravelling the mechanisms of immunomodulation during OD pregnancy, reflected by a high level of fetal-maternal dissimilarity, could help to reach the ultimate goal in transplantation; the induction of donor-specific tolerance. In addition, it might help to understand the development of complications in OD pregnancy. Trial registration number Not applicable

Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation.Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance.Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p<0.001) and chimerism (7.56% vs. 0.46% at 4 weeks, p=0.003) compared to TCD BM alone. No difference in engraftment (100% vs. 90%, p=0.501) or chimerism (7.56% vs. 7.66% at 4 weeks, p=0.961) was observed compared to fetuses injected with TCD BM at E14 (Figures 1A and 1B). These trends persisted at subsequent time points. Pups injected with TCD BM plus CD4+ chimeric splenocytes displayed normal weight gain and no histologic evidence of GVHD. Survival to adulthood was 100%. Donor cells of all hematopoietic lineages were present in the PB at 6 months of age. On in vivo MLR, pups injected with TCD BM plus CD4+ chimeric splenocytes displayed significantly reduced T cell proliferation compared to B6-sensitized Balb/c mice and P0 pups injected with TCD BM only. T cell proliferation did not differ significantly from fetuses injected with TCD BM at E14 (Figure 1C).Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naïve P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

A Prospective Controlled Trial to Evaluate Safety and Efficacy of in vitro Expanded Recipient Regulatory T Cell Therapy and Tocilizumab Together With Donor Bone Marrow Infusion in HLA-Mismatched Living Donor Kidney Transplant Recipients (Trex001).

Background: The induction of donor-specific immunological tolerance could improve outcome after kidney transplantation. However, no tolerance protocol is available for routine clinical use. Chimerism-based regimens hold promise, but their widespread application is impeded in part by unresolved safety issues. This study tests the hypothesis that therapy with polyclonal recipient regulatory T cells (Tregs) and anti-IL6R (tocilizumab) leads to transient chimerism and achieves pro-tolerogenic immunomodulation in kidney transplant recipients also receiving donor bone marrow (BM) without myelosuppressive conditioning of the recipient.Methods/design: A prospective, open-label, controlled, single-center, phase I/IIa academic study is performed in HLA-mismatched living donor kidney transplant recipients.Study group: Recipients of the study group receive in vitro expanded recipient Tregs and a donor bone marrow cell infusion within 3 days after transplantation and tocilizumab for the first 3 weeks post-transplant. In addition they are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Starting 6 months post-transplant, sirolimus and steroids are withdrawn in a step-wise manner in stable patients.Control group: Recipients of the control group are treated with thymoglobulin, belatacept, sirolimus, and steroids as immunosuppression. Co-primary endpoints of safety (impaired graft function [eGFR <35 mL/min/1.73 m2], graft-vs.-host disease or patient death by 12 months) and efficacy (total leukocyte donor chimerism within 28 days post-transplant) are assessed. Secondary endpoints include frequency of biopsy-proven acute rejection episodes and subclinical rejection episodes on surveillance biopsies, assessment of kidney graft function, and the evaluation whether the study protocol leads to detectable changes in the immune system indicative of pro-tolerogenic immune modulation.Discussion: The results of this trial will provide evidence whether treatment with recipient Tregs and donor BM is feasible, safe and efficacious in leading to transient chimerism. If successful, this combination cell therapy has the potential to become a novel treatment option for immunomodulation in organ transplantation without the toxicities associated with myelosuppressive recipient conditioning.Trial registration: European Clinical Trials Database EudraCT Nr 2018-003142-16 and clinicaltrials.gov NCT03867617.

Myeloid-derived suppressor cells therapy enhance immunoregulatory properties in acute graft versus host disease with combination of regulatory T cells

BackgroundMyeloid-derived suppressor cells (MDSCs) play a critical role in modulating the immune response and promoting immune tolerance in models of autoimmunity and transplantation. Regulatory T cells (Tregs) exert therapeutic potential due to their immunomodulatory properties, which have been demonstrated both in vitro and in clinical trials. Cell-based therapy for acute graft-versus-host disease (aGVHD) may enable induction of donor-specific tolerance in the preclinical setting.MethodsWe investigated whether the immunoregulatory activity of the combination of MDSCs and Tregs on T cell and B cell subset and alloreactive T cell response. We evaluated the therapeutic effects of combined cell therapy for a murine aGVHD model following MHC-mismatched bone marrow transplantation. We compared histologic analysis from the target tissues of each groups were and immune cell population by flow cytometric analysis.ResultsWe report a novel approach to inducing immune tolerance using a combination of donor-derived MDSCs and Tregs. The combined cell-therapy modulated in vitro the proliferation of alloreactive T cells and the Treg/Th17 balance in mice and human system. Systemic infusion of MDSCs and Tregs ameliorated serverity and inflammation of aGVHD mouse model by reducing the populations of proinflammatory Th1/Th17 cells and the expression of proinflammatory cytokines in target tissue. The combined therapy promoted the differentiation of allogeneic T cells toward Foxp3 + Tregs and IL-10-producing regulatory B cells. The combination treatment control also activated human T and B cell subset.ConclusionsTherefore, the combination of MDSCs and Tregs has immunomodulatory activity and induces immune tolerance to prevent of aGVHD severity. This could lead to the development of new clinical approaches to the prevent aGVHD.

A Human-Mouse Hybrid HLA-A2-IG Dimer Inhibits the Proliferation of CD4+T Cells In Vitro

Currently, general immunosuppressive drugs are used to maintain tolerance to allografts. However, these drugs have a major drawback of rendering the patient susceptible to infections and other side effects like malignancy and drug related toxicities with an overall rejection of the organ at some point. Previous studies have shown that MHC-Ig dimers may suppress alloresponsive T cells in a donor specific manner in vitro. This work aimed to answer the question as to whether these dimers will surmount rejection through the direct mechanism of allorecognition by suppressing alloreactive CD8+ T cells. To do this, we first identified two mice models with a single mismatch at the MHC loci. We found and procured white albino NOD mice which happened to be transgenic for HLA-A2 and HLA-A24 molecules. We then constructed a human-mouse hybrid HLA-A2-Ig dimer by overlap-PCR to join parts of two different already cloned plasmids to form the full length HLA-A2β2α1α2murineα3 insert which was then cloned to pcDNA3.1 to form pcDNA3.1HLA-A2β2α1α2murineα3. The IgG2bFc region was added by restriction digestion and ligation to form the plasmid pcDNA3.1HLA-A2β2α1α2murineα3IgG2bFc. Sequencing was done and confirmed that the construction and cloning were successful. The plasmid pcDNA3.1HLA-A2β2α1α2murineα3IgG2bFc was then transfected by electroporation to J558L cells. Screening was done using G418 for 4 weeks in cell culture. We purified the dimer by affinity chromatography and then used ELISA to confirm expression of the dimer. The purified dimer was then used in 1-way MLC experiments where responder cells were mice cells expressing HLA-A24 molecules while stimulator cells were mice cells expressing HLA-A2 molecules. Cell samples were gated on anti-mouse CD3-PE/CY7, anti-mouse CD4-PE, and anti-mouse CD8-APC/CY7. Cell proliferation was analysed using CFSE. Our results showed that the proliferation of CD4+ T cells was inhibited in the presence of the dimer. This work is crucial for subsequent studies aiming to search for induction of donor specific tolerance.

Proteasome Expression in Ovarian Heterotopic Allografts of Wistar and August Rats under Induction of Donor-Specific Tolerance

The aim of this work is to investigate the dynamics for the ovarian tissue engraftment of inbred August rats transplanted to outbred Wistar rats and vice versa on the background of the induction of donor-specific tolerance and to identify the features of proteasome pools in the surviving grafts and tissue replacing the rejected grafts in animals of both groups. By day 14 after transplantation, a slight difference in graft engraftment was revealed in studied recipients: 87% in Wistar rats and 80% in August rats. At the same time, ovarian tissue allografts with well-preserved structure and significant vascularization represented 76% in Wistar rats and only 29% in August rats on day 37. The difference revealed in the remote period is apparently connected with the special condition of the central nervous system of August rats caused by the increased content of monoamines and heat-shock protein 70. The total proteasome level on day 37 was the same both in intact donor tissue and in the surviving grafts, as well as in the tissue replacing the rejected grafts, and did not depend on the donor–recipient differences. However, the increased expression of immune proteasomes was found in the surviving tissue and tissue replacing the rejected grafts compared to intact donor tissue. The surviving grafts were enriched with proteasomes with immune subunit LMP2, and the tissue replacing the rejected grafts was enriched with proteasomes with immune subunit LMP7 equally in August and Wistar rats. In addition, the surviving grafts in both groups of animals were characterized by equally high expression of proteasome activator РА28αβ compared to the replacement and intact donor tissues. Thus, transplant proteasomes containing immune subunits LMP2, and probably associated with the activator RA28αβ, serve as one of the key factors for engraftment regardless of the genetically determined differences of the recipients.

Induction of Donor-Specific Immune Tolerance with Clinical MIC Cell Infusion — a Phase I Study (TOL-1)

▪Background:After transplantation of solid organs like allogeneic kidneys, the administration of immunosuppressive drugs such as cyclosporine A (CSA) and steroids is mandatory. This regimen exerts toxicity to the graft and makes transplant recipients prone to opportunistic infections. Replacement of the immunosuppressive drugs by a transfusion of tolerogenic cells might overcome these noxious side effects. Mitomycin-induced cells (MICs) are donor-derived monocytes that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C and have a myeloid-derived suppressor cell (MDSC) character.Materials and methods:Peripheral blood mononuclear cells (PBMCs) were harvested from living kidney donors by leukapheresis and MIC cells were manufactured under Good Manufacturing Practice (GMP) conditions in the clean room of our University Hospital. Kidney transplant recipients received either 1.5x10E6 MIC cells per kg body weight on day -2 (N=3, group A) or 1.5x10E8 MIC cells per kg body weight on day -2 (N=3, group B) or on day -7 (N=4, group C) before living donor kidney transplantation. Patients received immunosuppressive therapy with cyclosporine a (CSA), enteric coated mycophenolate sodium (EC-MPS) and corticosteroids. The primary outcome was measured by the frequency of adverse events (AEs) on post-transplant day 30 with a follow-up until post-transplant day 360 for all patients.Results:Clinically, all kidney transplant recipients showed a median serum creatinine of 1.4 mg/dL at day 30 and remained stable with a median creatinine of 1.48 mg/dL at day 180 without significant proteinuria (median 10 g/mol creatinine at day 180) and without rejection episode. In total 72 AEs were observed including three severe AEs which were not associated with the MIC cell transfusion. Besides two infectious complications, no positive cross match results, no de novo donor-specific antibodies or rejection episodes were recorded. In group C, a reduction of immunosuppressive therapy was effective in the observational phase with low-dose CSA and low-dose EC-MPS.Immunologically, CD19+ B cells increased up to a median of 300/µL until day 30, followed by a decrease to a median of 35/µL at day 180 in group C. Notably, CD19+CD24highCD38high regulatory B cells were significantly increased from a median of 2% on day 30 to a median of 20% on day 180. The plasma IL-10/TNF-α ratio increased from a median of 0.05 before cell therapy to a median of 0.11 at day 180. Moreover, recipient lymphocytes showed no or only minimal reactivity against irradiated donor PBMCs, while reactivity against 3rd party healthy donor PBMCs in vitro was not impaired.Additionally, the quality assessment demonstrated that MIC cells have the capability to induce tolerogenic dendritic cells (tDCs) by down-regulating the costimulatory molecules CD80 and CD86, and the maturation molecule CD83, while up-regulating the immunosuppressive molecule CD103. MIC-induced tDCs showed the capacity to inhibit donor specific allo-reactive CD4 and CD8 T cell proliferation.Conclusion:A stable function was observed in all transplant recipients receiving the MIC cells product without any allograft injury or rejection episodes even under reduction of conventional therapy with immunosuppressive drugs. MIC cells constitute a novel tool for immunotherapy with a high potential in transplantation medicine. DisclosuresNo relevant conflicts of interest to declare.

Changes in the Content of Mononuclear Liver Cells Expressing Immune Proteasomes after Transplantation of Ovarian Tissues Depending on Donor-Recipient Differences in Rats.

Implantation of ovarian tissue allografts in outbred Wistar rats and inbred August rats against the background of induction of donor-specific tolerance was accompanied by an increase in liver content of mononuclear cells expressing LMP2 immune of proteasome subunit by day 37 after transplantation in comparison with day 0. Graft rejection, on the contrary, was associated with a decrease in the number of LMP2+ cells in the liver of rats of both lines during this period. The difference in the content of these cells and the graft take rate were higher in Wistar rats. The number of mononuclear cells expressing LMP7 immune proteasome subunit in the liver did not change in rats of both lines by day 37 in comparison with day 0. Thus, the level of immune proteasomes with LMP2 subunit in mononuclear cells of the liver is related to fine mechanisms of regulation of immune responses and their shift toward graft take or rejection.

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

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

NK Cells Regulate CD8 T Cell-Mediated Allogeneic Rejection in Immunocompetent Recipients across an MHC Class I Mismatched Barrier

Non-T natural killer (NK) cells are a population of granular lymphoid cells TcR- CD3− CD122+ (NK1.1+ in C57BL/6 mice and DX5+ in Balb/c mice). These innate lymphoid cells mature in the bone marrow and are responsible for killing viral-infected and transformed cells in a non-MHC restricted manner, without the need of prior sensitization due to a recognition mechanism based on the array of germline-encoded inhibitory and activating receptors expressed on the cell surface of NK cells [1]. NK cells modulate CD8 T cell expansion during homeostatic proliferation in response to lymphopenic conditions by killing activated T cells and competing for IL-15 with the proliferating CD8 effector T cells [2]. This NK cell behavior may respond to an adaptive evolutionary mechanism to prevent aggressive anti-viral immune responses during the acute phase of infection and thus attenuate immunopathology [3]. We set up an experimental mouse model of alloreactivity across an MHC class I barrier to evaluate the role of NK cells in CD8 T cell-mediated rejection of bm1 skin allografts in naïve B6 recipients. In this transplant setting, host CD8 T cells are the main effectors T cells involved in the rejection of class I MHC mismatched skin allografts preferentially through the direct pathway of antigen presentation with limited help from CD4 T cells primed by the indirect pathway of antigen presentation. As opposed to previous studies done in lymphopenic immunodeficient recipients [2], depletion of NK cells was performed in immunocompetent C57BL/6 recipients with or without CD8 T depletion, the former displaying homeostatic proliferation in response to lymphopenia due to antibody-mediated elimination of CD8 T cells. We observed that depletion of NK cells in both CD8-repleted and CD8-depleted naïve C57BL/6 immunocompetent mice accelerated significantly bm1 graft rejection compared to non-NK-depleted controls (p<0.0005) (Figure 1). Our data claims for precaution in solid organ transplantation under inducing protocols of extensive depletion of leukocytes, such as ATG or alentuzumab. The immunotherapeutic interventions that include the removal of NK cells may accelerate graft rejection and promote aggressive CD8 T cell cytotoxic responses, which could be refractory to current immunosuppression. It also suggests that enhancing NK-cell functional activity combined with CD8 T cell depletion under tolerogenic regimen that tolerizes CD4 T cells at the time of transplantation may contribute to allogeneic donor-specific tolerance induction by elimination of the direct pathway of antigen presentation and by regulating the recovery of CD8 T cells.Bibliographic

Blockade of adhesion molecule lymphocyte function-associated antigen-1 improves long-term heart allograft survival in mixed chimeras.

The mixed chimerism approach for intentional induction of donor-specific tolerance was shown to be successful in various models from mice to humans. For transplant patients, the approach would obviate the need for long-term immunosuppression and associated side effects; moreover, it would preclude the risk of late graft loss due to chronic rejection. Widespread clinical application is hindered by toxicities related to recipient pre-conditioning. Herein we aimed to investigate a clinically relevant protocol for tolerance induction to cardiac allografts, sparing CD40 blockade or T-cell depletion. B6 mice were conditioned with non-myeloablative total body irradiation, fully mismatched BALB/c bone marrow cells, and short-term therapy, based on either anti- lymphocyte function-associated antigen-1 (anti-LFA-1) or anti-CD40L. Multilineage chimerism was followed by flow-cytometric analysis, tolerance was assessed with skin and heart allografts from fully or major histocompatibility complex-mismatched donors. Mechanisms of tolerance were investigated by analysis of donor-specific antibodies (DSAs), mixed lymphocyte reaction (MLR) assays, and deletion of donor-reactive T cells. We found that the combination of cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4Ig) and rapamycin with LFA-1 blockade enhanced bone marrow engraftment and led to more efficient T-cell engraftment and subsequent tolerization. Although fully mismatched skin grafts were chronically rejected, primarily vascularized heart allografts survived indefinitely and without signs of chronic rejection, independent of minor antigen mismatches. We have demonstarted a robust protocol for the induction of tolerance for cardiac allografts in the absence of CD40 blockade. Our findings demonstrate the potential of a clinically relevant minimal conditioning protocol designed to induce lifelong immunologic tolerance toward cardiac allografts.

Changes in the Expression of Immune Proteasomes in the Liver after the Induction of Portal Tolerance Depending on Donor-Recipient Differences in Rats.

Induction of donor-specific tolerance in outbred Wistar rats (RT1u) and inbred August rats (RT1c) increased the expression of immune proteasome subunits in liver with a peak on day 7 after beginning of the induction. The increase in the level of immune subunits LMP2 and LMP7 was more pronounced in the liver of August rats in comparison with Wistar rats (by 2 and 6 times, respectively), which was associated with higher concentrations of monoamines in the CNS of August rats. After induction of donor-specific tolerance in August and Wistar rats, the immune subunits were in cells of sinusoidal lining and in cells located in sinusoid lumens. It can be suggested that immune proteasomes in these cells producing antigenic peptides for presentation to immunocompetent participate in the suppression of their activity and form the molecular basis for the development of donor-specific tolerance at very early stages of this process.

Vascularized Thymosternal Composite Tissue Allo- and Xenotransplantation in Nonhuman Primates: Initial Experience

Background:Vascularized composite allotransplantation is constrained by complications associated with standard immunosuppressive strategies. Vascularized thymus and bone marrow have been shown to promote prolonged graft survival in composite organ and soft-tissue vascularized composite allotransplantation models. We report development of a nonhuman primate vascularized thymosternal composite tissue transplant model as a platform to address donor-specific immune tolerance induction strategies.Methods:Vascularized thymosternal allograft (skin, muscle, thymus, sternal bone) was transplanted between MHC-mismatched rhesus monkeys (feasibility studies) and baboons (long-term survival studies), with end-to-side anastomoses of the donor aorta and SVC to the recipient common femoral vessels. A male allograft was transplanted to a female’s lower abdominal wall, and clinically applicable immunosuppression was given. Skin biopsies and immunological assays were completed at regular intervals, and chimerism was quantified using polymerase chain reaction specific for baboon Y chromosome.Results:Four allo- and 2 xenotransplants were performed, demonstrating consistent technical feasibility. In 1 baboon thymosternal allograft recipient treated with anti-CD40–based immunosuppression, loss of peripheral blood microchimerism after day 5 was observed and anticipated graft rejection at 13 days. In the second allograft, when cutaneous erythema and ecchymosis with allograft swelling was treated with anti-thymocyte globulin starting on day 6, microchimerism persisted until immunosuppression was reduced after the first month, and the allograft survived to 87 days, 1 month after cessation of immunosuppression treatment.Conclusions:We established both allo- and xeno- composite vascularized thymosternal transplant preclinical models, which will be useful to investigate the role of primarily vascularized donor bone marrow and thymus.