Donor-specific Immune Tolerance Research Articles

Modified immune cells induce donor-specific immune tolerance

Modified immune cells induce donor-specific immune tolerance

Myeloid-derived suppressor cells promote allograft survival by suppressing regulatory T cell dysfunction in high-risk corneal transplantation

Highly inflamed and neovascularized corneal graft beds are known as high-risk (HR) environments for transplant survival. One of the primary factors leading to this rejection is reduction in the suppressive function of regulatory T cells (Treg). Our results show that myeloid-derived suppressor cells (MDSC) counteract interleukin-6–mediated Treg dysfunction by expressing interleukin-10. Additionally, MDSC maintain forkhead box P3 stability and their ability to suppress IFN-γ+ Th1 cells. Administering MDSC to HR corneal transplant recipients demonstrates prolonged graft survival via promotion of Treg while concurrently suppressing IFN-γ+ Th1 cells. Moreover, MDSC-mediated donor-specific immune tolerance leads to long-term corneal graft survival as evidenced by the higher survival rate or delayed survival of a second-party C57BL/7 (B6) graft compared to those of third-party C3H grafts observed in contralateral low-risk or HR corneal transplantation of BALB/c recipient mice, respectively. Our study provides compelling preliminary evidence demonstrating the effectiveness of MDSC in preventing Treg dysfunction, significantly improving graft survival in HR corneal transplantation, and showing promising potential for immune tolerance induction.

INNATE IMMUNE CELLSIN IMMUNE TOLERANCE AFTER LIVER TRANSPLANTATION

Currently, liver transplantation is the most effective treatment for end-stage liver disease. Immuno-suppressive agents are required to be taken after the operations, which have significantly reduced rejection rates and improved the short-term (<1 year) survival rates. However, post-transplant complications related to the immuno-suppressive therapy have led to the development of new protocols aimed at protecting renal function and preventing de novo cancer and dysmetabolic syndrome. Donor specific immune tolerance, which means the mature immune systems of recipients will not attack the grafts under the conditions without any immunosuppression therapies, is considered the optimal state after liver transplantation. There have been studies that have shown that some patients can reach this immune tolerance state after liver transplantation. The intrahepatic immune system is quite different from that in other solid organs, especially the innate immune system. It contains a variety of liver specific cells, such as liver-derived dendritic cells, Kupffer cells, liver sinusoidal endothelial cells, liver-derived natural killer (NK) cells, natural killer T (NKT) cells, and so on. Depending on their specific structures and functions, these intrahepatic innate immune cells play important roles in the development of intrahepatic immune tolerance. In this article, in order to have a deeper understanding of the tolerogenic functions of liver, we summarized the molecular mechanisms of immune tolerance induced by intrahepatic innate immune cells after liver transplantation.

Induction of Immune Tolerance in Islet Transplantation Using Apoptotic Donor Leukocytes.

Allogeneic islet transplantation has become an effective treatment option for severe Type 1 diabetes with intractable impaired awareness due to hypoglycemic events. Although current immunosuppressive protocols effectively prevent the acute rejection associated with initial T cell activation in recipients, chronic rejection has remained an obstacle for achieving long-term allogeneic islet engraftment. The development of donor-specific immune tolerance to the allograft is the ultimate goal given its potential ability to overcome chronic rejection and disregard the need for maintenance immunosuppression, which may be toxic to islet grafts. Recently, a breakthrough in tolerance induction during allogeneic islet transplantation using apoptotic donor lymphocytes (ADLs) in a non-human primate model had been reported. Several studies have suggested that the clonal depletion, anergy, and expansion of the antigen-specific regulatory immune network are the mechanisms for donor-specific tolerance with ADLs, which act synergistically to induce robust transplant tolerance. This achievement represents a huge step forward toward the clinical application of immune tolerance induction. We herein summarize the reported operational induction therapies in islet transplantation using the ADLs. Moreover, a few obstacles for the engraftment of transplanted islets, such as islet immunogenicity and instant blood-mediated response, which need to be resolved in the future, are also discussed.

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.

Innate Immune Cells in Immune Tolerance After Liver Transplantation.

Currently, liver transplantation is the most effective treatment for end-stage liver disease. Immunosuppressive agents are required to be taken after the operations, which have significantly reduced rejection rates and improved the short-term (<1 year) survival rates. However, post-transplant complications related to the immunosuppressive therapy have led to the development of new protocols aimed at protecting renal function and preventing de novo cancer and dysmetabolic syndrome. Donor specific immune tolerance, which means the mature immune systems of recipients will not attack the grafts under the conditions without any immunosuppression therapies, is considered the optimal state after liver transplantation. There have been studies that have shown that some patients can reach this immune tolerance state after liver transplantation. The intrahepatic immune system is quite different from that in other solid organs, especially the innate immune system. It contains a variety of liver specific cells, such as liver-derived dendritic cells, Kupffer cells, liver sinusoidal endothelial cells, liver-derived natural killer (NK) cells, natural killer T (NKT) cells, and so on. Depending on their specific structures and functions, these intrahepatic innate immune cells play important roles in the development of intrahepatic immune tolerance. In this article, in order to have a deeper understanding of the tolerogenic functions of liver, we summarized the molecular mechanisms of immune tolerance induced by intrahepatic innate immune cells after liver transplantation.

Prolongation of Allograft Survival by Intrathymic Introduction of Allogeneic Thymic Epithelial Cells

Thymic epithelial cells (TECs), the primary stromal population in the thymus, are critical to establish the microenvironments required for generating self-tolerant, pathogen-responsive T cells. However, due to the inaccessibility of the thoracic cavity and the fragility of TECs, manipulating the thymus to modulate adaptive immune responses remains challenging. We developed a novel surgical procedure that allowed access to thymus glands for intrathymic injection (ITI), without the need to cut the manubrium of the sternum bone. Horizontal cuts of the muscle layers between the first and second bones of the rib cage expose the thymus with minimal side effects. This novel ITI procedure enabled us to implant 16.5-day embryonic TECs into an adult syngeneic recipients’ thymus. Long-term survival and colonization of the implanted TECs was observed via live imaging, flow cytometry, and immunohistochemistry. We further investigated if introduction of allogeneic TECs in the thymus promote negative selection against allo-reactive T cells and induce donor-specific immune tolerance. We engrafted B6 islets under the kidney capsules of chemically induced diabetic Balb/C mice, preconditioned with T-cell depletion sera and intrathymic injection of B6 16.5-day embryonic TECs. Prolonged survival of the transplanted islets was observed compared to sham-operated controls. Mixed lymphocyte reaction experiments further showed dampened T cell proliferation responses against donor B6 antigens. Moreover, increased levels of Foxp3+ T-regulatory cells were detected in Balb/C recipients with ITI of B6 TECs, suggesting that implanted TECs could also promote the generation of alloantigen-specific T regulatory cells in addition to facilitating negative selection of donor antigen-reactive T cells. Our results suggest that intrathymic transplantation of TECs could be an effective way to induce donor-specific immune tolerance with limited general immune suppression. Disclosure I. Pradhan: None. S. Wong-Noonan: None. S. Bertera: None. R.J. Lakomy: None. H. Cohen: None. D. Wells: None. M. Trucco: None. Y. Fan: None.

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.

Enhanced Allogeneic Engraftment Achieved Via Prenatal Tolerance Induction and Postnatal Anti-CD45 Immunotoxin Conditioned Hematopoietic Cell Transplantation

INTRODUCTION: In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative, non-immunosuppressive transplant approach that takes advantage of the immunologic immaturity of the fetus to engraft donor cells across immune barriers and induce donor specific immune tolerance (DST). Though it has the potential to treat many congenital hematologic disorders, IUHCT is limited by levels of engraftment below those anticipated to be therapeutic for most target diseases, including sickle cell anemia. Thus, a likely initial clinical application of IUHCT is to induce DST to allow for non-myeloablative, non-immunosuppressive postnatal "booster" transplants to increase engraftment. We have previously validated this approach in murine sickle cell, beta-thalassemia, and non-diseased models using low dose total body irradiation and busulfan prior to postnatal bone marrow transplantation (BMT). Although encouraging, less toxic approaches that specifically target the hematopoietic system would make IUHCT + postnatal BMT more applicable. CD45 is a membrane glycoprotein expressed by all leukocytes and hematopoietic stem cells. Recent studies have demonstrated enhanced engraftment in a congenic postnatal transplant mouse model following pre-transplant conditioning with an anti-CD45-saporin (SAP) immunotoxin (Palchaudhuri et al., Nat Biotech 2016; 34:738-45). We hypothesized that mixed allogeneic chimerism achieved by IUHCT can be enhanced after birth by conditioning with an anti-CD45-SAP immunotoxin followed by a same-donor allogeneic BMT.METHODS: 5x106 BM mononuclear cells from C57Bl/6 mice (H2Kb, CD45.2+) were injected intravenously into gestational day 14 (E14) Balb/c fetuses (H2Kd, CD45.2+). Donor cell peripheral blood (PB) chimerism was confirmed at 4 weeks of age, at which time chimeric mice were injected via tail vein with PBS (control) or CD45.2-SAP. Six days following treatment, chimeric animals were intravenously injected with 30x106 B6Pep3b (H2Kb, CD45.1+) BM cells. Additional control groups consisted of recipients of IUHCT alone without a postnatal BMT and naïve 5 week-old Balb/c mice which did not undergo IUHCT and only received CD45.2-SAP + postnatal BMT. Following postnatal BMT, PB was assessed bi-weekly by flow cytometry for total donor chimerism and the contribution of prenatal and postnatal donor cells to engraftment. Mice were weighed before each bleeding and evaluated for signs of GVHD. Statistical comparison was performed using the Student’s t-test for 2 samples at each time point assuming unequal variances.RESULTS: There was no significant difference in initial donor cell chimerism levels at 4 weeks of age between treatment (IUHCT+CD45.2-SAP+BMT, 12.4±2.2%, n=5) and control (IUHCT alone, 9.3±2.7%, n=18; IUHCT+BMT, 10.0±2.6%, n=7) groups prior to therapy. In contrast to naïve non-chimeric Balb/c mice undergoing a postnatal BMT (n=10) which demonstrated no donor cell engraftment, recipients of IUHCT and a postnatal BMT (both PBS and CD45.2-SAP treated groups) demonstrated postnatal donor cell engraftment, supporting DST achieved by IUHCT. While postnatal BMT after IUHCT failed to demonstrate a significant enhancement of engraftment compared to IUHCT alone (4.4±1.3% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p = 0.7), low-level allogeneic chimerism following IUHCT was significantly enhanced to high-level chimerism following conditioning with CD45.2-SAP+BMT (55.7±9.8% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p=0.0001) (Fig. 1). The majority of donor cell chimerism resulted from the postnatal donor population in recipients of CD45.2-SAP, supporting a mechanism of enhanced engraftment via a competitive advantage following recipient CD45.2-SAP treatment (Fig. 2). Chimerism was stable and multilineage at 22 weeks indicating enhanced stem cell/early progenitor engraftment. All mice demonstrated appropriate weight gain without GVHD.CONCLUSIONS: Prenatal tolerance induction achieved by IUHCT followed by postnatal CD45-SAP conditioning and same-donor BMT results in enhanced stable multilineage allogeneic engraftment at therapeutic levels for many target diseases. This work supports a potentially less toxic approach for the strategy of prenatal tolerance induction to facilitate non-myeloablative, non-immunosuppressive postnatal BMT and has the potential to significantly expand the applicability of IUHCT. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Alloantigen gene transfer to hepatocytes promotes tolerance to pancreatic islet graft by inducing CD8+ regulatory T cells

Induction of donor-specific immune tolerance is a good alternative to chronic life-long immunosuppression for transplant patients. Donor major histocompatibility complex (MHC) molecules represent the main targets of the allogeneic immune response of transplant recipients. Liver targeted gene transfer with viral vectors induces tolerance toward the encoded antigen. The aim of this work was to determine whether alloantigen gene transfer to hepatocytes induces tolerance and promotes graft acceptance. C57BL/6 (H-2b) mice were treated with adeno-associated viral (AAV) vector targeting the expression of the MHC class I molecule H-2Kd to hepatocytes, before transplantation with fully allogeneic pancreatic islet from BALB/c mice (H-2d). AAV H-2Kd treated mice were tolerant to the alloantigen, as demonstrated by its long-term expression by the hepatocytes, even after a highly immunogenic challenge with an adenoviral vector. After chemical induction of diabetes, the AAV treated mice had significantly delayed rejection of fully allogeneic pancreatic islet grafts, with more than 40% of recipients tolerant (>100days). AAV-mediated expression of H-2Kd in the liver induced the local expansion of CD8+ T lymphocytes with allo-specific suppressive properties. The adoptive transfer of these liver-generated CD8+ Tregs into naive diabetic mice promoted the long-term survival of allogeneic pancreatic islet grafts. AAV-mediated long-term expression of a single MHC class I molecule in the liver induces the generation of a subset of allo-specific CD8+ Treg cells, which promote tolerance toward fully allogeneic graft. Liver gene transfer represents a promising strategy for in vivo induction of donor-specific tolerance. The liver has a special immune system, biased toward tolerance. In this study, we investigated the possibility of harnessing this property of the liver to induce tolerance to an allogeneic transplantation. We demonstrate for the first time that the in vivo gene transfer of an allogeneic antigen with an adeno-associated viral vector to mouse hepatocytes induces the expansion of a population of CD8+ regulatory T lymphocytes. These Tregs are then instrumental in preventing the rejection of allogeneic pancreatic islets transplanted in these animals. Allogeneic transplantation is the main treatment for the end-stage diseases of a number of organs. Life-long immunosuppressive treatments are still required to limit graft rejection, and these treatments exhibit serious side effects. Our present findings open a new avenue for promoting allo-specific tolerance via in vivo induction of CD8+ Treg expansion.

The Use of Eicosanoids to Enhance Donor Cell Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)

IntroductionIn utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive allogeneic transplant approach that has the potential to treat a number of congenital disorders, including hemoglobinopathies and immunodeficiencies. Donor cell engraftment at levels high enough to induce donor specific immune tolerance or to treat a target disease has been elusive and remains a major limitation to the clinical application of IUHCT. One of the most significant barriers to high levels of donor cell engraftment is competition with endogenous fetal hematopoietic stem cells (HSCs) for limited hematopoietic niches following transplant. 16,16-dimethyl-prostaglandin E2 (PGE2) and the epoxyeicosatrienoic acids (EETs) 11,12-EET and 14,15-EET have been shown to enhance donor HSC homing, survival, and cell cycling in postnatal murine and zebrafish models of HSC transplantation. We hypothesized that a single ex vivo treatment of donor cells with these eicosanoids would improve donor cell homing and survival following IUHCT,resulting in higher levels of postnatal donor engraftment.MethodsTen million bone marrow (BM) mononuclear cells from C57Bl/6-GFP mice (H2Kb) were injected intravenously into embryonic day (E14) Balb/c fetuses (H2Kd) via the vitelline vein. Donor cells were treated with vehicle, PGE2, 11,12-EET, or 14,15-EET immediately prior to IUHCT. Early homing to and engraftment of the fetal liver (FL) and spleen (FS) of PGE2 and vehicle treated BM cells at 4, 24 and 72 hours after IUHCT was assessed by flow cytometry. Donor cell survival and apoptosis was also assessed in the FL 96 hours post-IUHCT in these two treatment groups by flow cytometric analysis of intracellular expression of survivin (anti-apoptotic) and anti-caspase 3 (pro-apoptotic). Long-term peripheral blood donor cell engraftment was assessed monthly up to 6 months of age and multilineage engraftment (donor T cells, B cells, granulocytes, and macrophages) was determined at 6 months of age in recipients of all donor cell treatment groups. Statistical analysis was performed using ANOVA with BonferroniÕs multiple comparison test or Kruskal-Wallis with DunnÕs multiple comparison test for normal and non-normal data, respectively. Data reported as mean +/- SEM.ResultsPGE2 pre-treatment produced a significant increase in FL and FS engraftment at 72 hours post-IUHCT compared to vehicle treated donor cells (FL: 37.5 +/- 3.1% vs 21.6 +/- 1.3%; FS: 52.2 +/- 3.7% vs 39.2 +/- 1.8%; p < 0.05). There was no significant increase in donor cell engraftment in the FL at earlier time points associated with PGE2 treatment. PGE2 treatment was also associated with increased survival of donor cells compared to vehicle treated cells as indicated by increased donor cell expression of survivin (19.8±5.6% vs. 4.6±1.3%; p<0.05) and decreased donor cell anti-caspase 3 staining (9.3±1.2% vs. 64.1±10.6%; p<0.05) at 96 hours post-IUHCT. These findings of increased FL/FS engraftment and increased survival at 72 to 96 hours post IUHCT following donor cell treatment with PGE2 translated into significantly increased long-term donor cell engraftment up to 6 months of age. Similar to PGE2, ex vivo treatment of donor cells with 11,12-EET or 14,15-EET resulted in enhanced long-term allogeneic donor cell engraftment (Figure 1). Balanced multilineage engraftment was seen for all treatments suggesting enhanced engraftment at the level of the HSC. No deleterious effects were observed on murine growth or overall health.ConclusionsThe ex vivo treatment of donor cells with eicosanoids including PGE2, 11,12-EET, and 14,15 EET prior to IUHCT results in enhanced long-term multilineage allogeneic donor cell engraftment. Detailed studies of early homing to fetal hematopoietic organs and survival at 96 hours following IUHCT of PGE2 treated donor cells suggest the mechanism of enhanced engraftment is mainly do to a pro-survival effect of PGE2 and related eicosanoids. This work represents a novel application of these eicosanoids in an allogeneic model of IUHCT and highlights their potential to assist in future clinical applications of IUHCT. [Display omitted] DisclosuresZon:FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor-Specific Immune Tolerance to Allografts

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.

In utero hematopoietic cell transplantation: induction of donor specific immune tolerance and postnatal transplants.

In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative non-immunosuppressive transplant approach that allows for donor cell engraftment across immunologic barriers. Successful engraftment is associated with donor-specific tolerance. IUHCT has the potential to treat a large number of congenital hematologic, immunologic, and genetic diseases either by achieving high enough engraftment levels following a single IUHCT or by inducing donor specific tolerance to allow for non-toxic same-donor postnatal transplants. This review evaluates donor specific tolerance induction achieved by IUHCT. Specifically it addresses the need to achieve threshold levels of donor cell engraftment following IUHCT to consistently obtain immunologic tolerance. The mechanisms of tolerance induction including partial deletion of donor reactive host T cells by direct and indirect antigen presentation and the role of regulatory T cells in maintaining tolerance are reviewed. Finally, this review highlights the promising clinical potential of in utero tolerance induction to provide a platform on which postnatal cellular and organ transplants can be performed without myeloablative or immunosuppressive conditioning.

Hematopoietic chimerisms: friends or foes?

In humans, chimerisms (Chs) can occur naturally or be induced through artificial means. Feto-maternal Chs are natural and result from the spontaneous trafficking of hematopoietic or other types of cells across the placenta during pregnancy. These Chs can be transient or persist for many years and even decades. Mixed hematopoietic Chs (MHChs) can also be artificially induced, and they might have profound effects on the modulation of the immune system, which can be used for inducing donor-specific tolerance in recipients of allogeneic organ transplantation. Nonetheless, the main obstacle for the establishment of such Chs is that they require the engraftment of donor hematopoietic cells, which at present can only be accomplished using relatively toxic regimens that preclude its widespread use and currently restricts its application to some special patients, in which both a solid organ (e.g. a renal allograft) and a marrow transplantation are necessary. However, it is likely that less toxic strategies are developed that can be clinically applicable in the next decade to induce tolerance in organ transplantation. A variant of Chs is the molecular Chs, in which a proportion of the hematopoietic cells would be transduced to express a transgene (e.g. encoding a therapeutic protein, an auto-antigen, or an allergen), so that specific tolerance to these molecules is induced. This might have therapeutic applications in fields such as replacement and genetic therapies, autoimmune diseases, or allergy. Keywords: Mixed hematopoietic chimerism; feto-maternal chimerism; immune tolerance; hematopoietic transplantation; organ transplantation; molecular chimerism (Published: 25 September 2014) Citation: Advances in Regenerative Biology 2014, 1 : 24429 - http://dx.doi.org/10.3402/arb.v1.24429 Abstract in popular science Chimeras are mythological creatures composed of a lion, a snake, and a goat. In biology, chimeras refer to individuals composed of two or more types of genetically distinct cells that derive from different fertilized eggs. Some chimerisms (Chs) occur spontaneously, such as the feto-maternal Chs, which are very common and are due to the bidirectional pass of blood cells through the placenta. These Chs can be transient or they may persist for decades and contribute to the generation of different tissues. Although these Chs have been reported to be associated with a higher risk of certain diseases (e.g. fetal Ch and scleroderma in the mother), the biological role and the clinical significance of feto-maternal Chs are uncertain. When the Chs are caused only by blood cells (e.g. hematopoietic stem cells), they are called mixed hematopoietic Chs (MHChs). These MHChs can be created artificially as occurs in patients with leukemia or non-malignant diseases undergoing hematopoietic transplantation. Indeed, these patients usually become full quimeras (which means that 100% of their blood cells will be donor-derived for the rest of their lives). However, for the donor stem cells to engraft, the recipients must receive a therapy that eliminates (ablates) their own hematopoietic cells. A main limitation of these preparatory therapies (conditioning) is their high toxicity. However, since the creation of MHChs only requires a partial ablation, it can be done using less toxic conditioning regimens. The main clinical interest of MHChs is that they result in donor-specific immune tolerance, as both types of immune cells (donor and recipient) will be educated in a peaceful coexistence. So, although the conditioning regimens still need to be refined, such MHChs can be used to induce tolerance in recipients of organ transplantation, which no longer will require immunosuppressive drugs. In an analogous manner, Chs can be created using autologous hematopoietic cells that have been genetically modified with gene therapy vectors to produce a therapeutic protein, an autoantigen, or an allergen, so that a very robust tolerance to these molecules can be induced. This has potential applications in fields such as replacement and genetic therapies, autoimmune diseases, or allergy.

Resistance of Spontaneously Diabetic Ins2akita Mice to Allograft Tolerance Induced by Anti-CD154 Therapy

BackgroundDespite ongoing advances in the clinical islet transplant field, progressive decline in graft function continues to reduce the long-term success of islet transplantation for restoring euglycemia in type 1 diabetic recipients. To preserve graft function and avoid the use of chronic immunosuppressive drug therapy, a key goal is to induce donor-specific immune tolerance to islet transplants. Preclinical rodent studies of islet transplantation largely utilize models of diabetes either induced experimentally with beta cell toxins or spontaneously occurring in strains genetically prone to autoimmune diabetes. In this study, we sought to determine if chronic, severe hyperglycemia itself, independent of both beta cell toxins and host autoimmunity, influenced acute allograft rejection and/or the capacity to induce allograft tolerance. MethodTo this end, we studied the response to islet allografts in severely diabetic, non-autoimmune C57Bl/6 Ins2akita recipients. ResultsResults indicate that diabetic Ins2akita mice display higher levels of blood glucose, show more rapid acute islet allograft rejection, and are resistant to allograft prolongation induced with anti-CD154 therapy relative to wild-type littermates rendered diabetic with streptozotocin. As such, results suggest that severe hyperglycemia may be an independent risk factor impacting the capacity to induce tolerance to islet allografts. Thus, Ins2akita mice represent a stringent model for evaluating anti-rejection strategies in the setting of severe metabolic demand on islet transplants.

Either Abatacept or Belatacept in Combination With Donor Bone Marrow Infusion Induces Immune Tolerance Across a Full MHC Barrier in a Translational Large Animal Vascularized Composite Allotransplantation Model.

BACKGROUND: The establishment of tolerance protocols would avoid the debilitating effects of chronic immunosuppression and render Vascularized Composite Allotransplantation (VCA) a routine procedure. We investigated graft survival and tolerance induction using co-stimulation blockade with either abatacept or the higher-affinity equivalent drug belatacept. METHODS: Fully MHC- and gender mismatched MGH miniature swine underwent heterotopic hind-limb transplantation. Recipient animals received a short course (30 days) of tacrolimus monotherapy, +/- donor BM infusion, and CTLA4Ig (either abatacept or belatacept). Tacrolimus only and untreated animals served as controls. Recipients were assessed for: chimerism (SRY-gene PCR analysis), alloreactivity against donor antigens (in vitro CFSE-MLR assays), and donor-specific antibodies (DSA) production (serum flow cytometry analysis). Robust immune tolerance in vivo was assessed by skin grafting. Tolerance maintenance upon allograft removal was tested by CFSE-MLR. RESULTS: Both regimens using abatacept or belatacept plus donor BM infusion resulted in indefinite graft survival (>200 days). While only transient peripheral chimerism was observed, stable micro-chimerism in various graft and recipient tissues was achieved. An increase in intragraft Tregs was seen in tolerant animals as compared to controls. Tolerant animals displayed unresponsiveness to donor but not to third party allogeneic controls in CFSE-MLRs. Donor-matched skin grafts were accepted while third party grafts where quickly rejected indicating donor-specific tolerance. No signs of chronic rejection or donor-specific antibodies were seen in tolerant recipients in either group (abatacept vs belatacept). Graft removal, however, was associated with loss of donor-unresponsiveness 5 weeks post-graftectomy. CONCLUSIONS: Combined costimulation blockade and donor BM cell infusion induced robust donor-specific immune tolerance in a translational fully MHC-mismatched hind limb transplant model.

Editorial introductions

Editorial introductions

Combined Co-Stimulatory Blockade and Donor Bone Marrow Cells Induce Robust Immune Tolerance in a Fully MHC-Mismatched Swine Hind Limb Transplant Model

INTRODUCTION: Vascularized Composite Allografts (VCA), such as hand and upper extremity transplants, contain vascularized bone marrow (BM) and a BM niche representing a constant source of donor-derived stem cells and hence can favor chimerism and tolerance induction.1,2 This study investigates the immunological effects of vascularized BM within VCA under co-stimulation blockade-based regimen and its impact on allograft survival and tolerance induction. METHODS: Fully MHC- and gender mismatched MGH miniature swine (n=3-5 per group) underwent heterotopic hind-limb transplantation containing intact vascularized BM component (Figure 1).Figure 1: Schematic diagram of swine heterotopic hind limb transplant. Osteomyocutaneous flap is transplanted to a subcutaneous pocket with externalized skin component.Recipient animals received a short course (30 days) of tacrolimus monotherapy with or without donor BM infusion (60x106 cells/kg), and CTLA4Ig. Short course tacrolimus only and untreated animals served as controls. Chimerism was assessed by SRY PCR analysis. Sequential skin and muscle biopsies were performed for histology. Alloreactivity against donor antigens was assessed in vitro using mixed lymphocyte reaction (MLR) assays. Challenge with secondary skin grafts was utilized to demonstrate robust immune tolerance in vivo. RESULTS: The co-stimulation blockade based immunomodulatory protocol resulted in indefinite graft survival (>150 days) in 3 out of 5 animals whereas control and tacrolimus only groups rejected allografts at days 7+/-1 and 29+/-2 respectively (Figure 2).Figure 2: Kaplan-Meier survival curve demonstrating rejection free survival of skin component of hind limb allograft.Combined costimulation blockade with augmented donor BM infusion resulted in indefinite graft survival in 2 out of 3 animals (>150 days). Long-term survivors demonstrated stable micro-chimerism in various graft and recipient tissues including skin, lymph node, bone marrow, and spleen. MLR data showed unresponsiveness to donor but not to third party allogeneic controls. Secondary skin grafting demonstrated advanced rejection of third party grafts on day 7 while donor-matched grafts were accepted indicating donor-specific immune tolerance. There was no evidence of donor specific antibody formation in long-term survivors. Donor unresponsiveness in MLR was lost five weeks post-graftectomy, which demonstrated that persistent antigenic stimulation was required for operational tolerance. CONCLUSION: Combined costimulation blockade and donor BM cell infusion can induce robust immune tolerance in a fully MHC mismatched hind limb transplant model. Such targeted immunomodulatory protocols might eliminate the need for long-term multi-drug immunosuppression after reconstructive transplantation.

Effect of nonmuscle myosin-derived peptide on cutaneous burn wound injury

3 out of 5 animals whereas control and tacrolimus only groups rejected allografts at days 7 1 and 29 2 respectively. Long-term survivors demonstrated durable micro-chimerism in various graft and recipient tissues including skin, lymph node, bone marrow, and spleen. CFSE-MLR data showed unresponsiveness to donor but not to third party allogeneic controls. Secondary skin grafting demonstrated advanced rejection of third party grafts while donormatched grafts were accepted indicating donor-specific immune tolerance.