Indefinite Graft Survival Research Articles

“FIRST-IN-HUMAN” CLINICAL TRIAL EMPLOYING ADOPTIVE TRANSFER OF AUTOLOGOUS THYMUS-DERIVED TREG CELLS (THYTREG) TO PREVENT GRAFT REJECTION IN HEART-TRANSPLANTED CHILDREN

Introduction: Immune allograft rejection remains the main obstacle to successful transplants. Due to their suppressive capacity, adoptive transfer of regulatory T cells (Treg) has acquired growing interest in achieving indefinite graft survival. Limited Treg recovery and reduced quality remain the main obstacles in current protocols where Treg are obtained from adult peripheral blood. We have developed a novel GMP-compatible protocol using the thymus routinely discarded during pediatric cardiac surgeries as Treg source, obtaining massive Treg counts with very high purity and suppressive capacity (Figure 1). Our Treg product has been approved by the Spanish Drug Agency (AEMPS) and is being used to treat transplanted children. Materials and Methods: We initiated the recruitment of patients last February for the first clinical trial worldwide (phase I/IIa) that evaluates the safety and efficacy of employing autologous thyTreg in 10 children (< 2 years old) undergoing heart transplantation. A single dose of thyTreg, manufactured under GMP conditions, is infused back into the patient at day +7 post-transplant and the remaining cells are cryopreserved for potential future reinfusions. The children are receiving as immunosuppressive therapy: tacrolimus, mofetil mycophenolate and short-term corticoids; without induction therapy with basiliximab (Figure 2). As a control cohort, we have performed a prospective study of 7 heart-transplanted children that were also thymectomiced and receive the same immunosuppressive therapy but not the thyTreg therapy, performing a comprehensive immune analysis at different time points before and up to 2 years post-transplant. Results and Discussion: The prospective study of our control cohort showed that the Treg to T effector CD4+ and CD8+ ratio significantly decreased after transplant until reaching values of -89,47% and -93,15% lower, respectively, at 2 years post-transplant (Figure 3). The marked decrease in the Treg/Teff ratio reveals that tolerance mechanisms are compromised in heart-transplanted children, just in the period with higher incidence of acute rejection. Due to this fact, the main parameters of efficacy in the clinical trial are to evaluate the recovery of circulating Treg, the Treg/Teff ratio, and determine changes in the incidence of acute rejection during the 2-year follow-up. Preliminary results about the evolution of patients treated with thyTreg will be available at the date of the congress. Conclusion: The clinical use of a cell therapy with thyTreg is a new therapeutic strategy to induce graft tolerance that could establish a new paradigm in the context of solid organ transplantation. The improved quality and amount of thyTreg obtained with this protocol allow to prepare hundreds of doses that could be administered periodically to induce indefinite immunological tolerance and thus achieve the indefinite survival of the transplanted organ.This work was supported by grants from Fundación Familia Alonso (FFA-19), grants from ISCIII co-founded by FEDER funds (PI18/00495; PI18/00506; DTS18/00038) and EXOHEP-CM Program (B2017/BMD3727).

IN VIVO ANTI-CD45RB ANTIBODY-INDUCED REGULATORY T CELLS DEMONSTRATE SPECIES-SPECIFIC TOLERANCE TO DISCORDANT HUMAN XENOGENIC ISLETS

Organ transplantation-Sichuan Youth Science and Technology Innovation Research Team. Background: Although islet xenotransplantation has been proven to be a promising strategy for treatment of type 1 diabetes, it’s application in clinical setting would have to overcome acute cellular rejection and challenging species barrier. The aim of this study was to determine the role of Foxp3+ regulatory T cells (Treg) in anti-CD45RB induced dominant and specific tolerance to human islet cells in mice. Methods: One thousand and five hundred IEQ human islets were isolated from deceased donors and transplanted under the kidney capsule of B6 diabetic mice, B6µMT-/- mice and thymectomized µMT-/- mice. The recipients were then temporarily treated with low dose anti-CD45RB. CD4+Foxp3+Tregs were identified in the blood, spleen, draining lymph nodes and within the graft of tolerant and rejecting mice by FCM and immunohistochemistry. Additional anti-CD25 mAb was applied in the tolerant B6µMT-/-mice, which received another human islets simultaneously. The CD4+CD25+ Tregs sorted from the tolerant mice were adoptively transferred to diabetic Scid mice which were grafted with another human islets to assess the suppressive function. Results: In vitro, CD4+CD25-Foxp3-T cells which were activated with anti-CD3/anti-CD28 beads and cultured with anti-CD45RB demonstrated significant increase in Foxp3+ expression. In vivo, an increase of Foxp3+ T cells was observed in treated mice and more significant increase in tolerant mice, compared with a negligible increase in rejected mice. Anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice and 88.9% in µMT-/- mice, whereas none of xenografts were accepted in these mice treated simultaneously with anti-CD25. µMT-/-mice which were thymectomized and treated by anti-CD45 yielded 100% of grafts with long-term survival, which indicated that the transplant tolerance was thymus-derived Tregs independent. Moreover, the µMT-/-mice which tolerated to the first human islets accepted the re-transplanted second party of islets from another human, whereas the tolerance was hampered by anti-CD25 treatment. Adoptive cell transfer model showed the CD4+CD25+ Tregs from the mice tolerant to the first human islets prolonged survival of the second human islets, compared to natural Tregs (p < 0.05). Conclusions: These data suggest that anti-CD45RB-induced tolerance in islet xenotransplantation is mediated by Tregs expansion. These tolerant Tregs had the potent capacity to transfer dominant and species-specific tolerance. The National Natural Science Foundation of China (No. 81172832, 81771723). Sichuan Youth Science and Technology Foundation (2013JQ0020). Special Program for Sichuan Youth Science and Technology Innovation Research Team (2014TD0010).

Vascularized composite allotransplantation combined with costimulation blockade induces mixed chimerism and reveals intrinsic tolerogenic potential.

Vascularized composite allotransplantation (VCA) has become a valid therapeutic option to restore form and function after devastating tissue loss. However, the need for high-dose multidrug immunosuppression to maintain allograft survival is still hampering more widespread application of VCA. In this study, we investigated the immunoregulatory potential of costimulation blockade (CoB; CTLA4-Ig and anti-CD154 mAb) combined with nonmyeoablative total body irradiation (TBI) to promote allograft survival of VCA in a fully MHC-mismatched mouse model of orthotopic hind limb transplantation. Compared with untreated controls (median survival time [MST] 8 days) and CTLA4-Ig treatment alone (MST 17 days), CoB treatment increased graft survival (MST 82 days), and the addition of nonmyeloablative TBI led to indefinite graft survival (MST > 210 days). Our analysis suggests that VCA-derived BM induced mixed chimerism in animals treated with CoB and TBI + CoB, promoting gradual deletion of alloreactive T cells as the underlying mechanism of long-term allograft survival. Acceptance of donor-matched secondary skin grafts, decreased ex vivo T cell responsiveness, and increased graft-infiltrating Tregs further indicated donor-specific tolerance induced by TBI + CoB. In summary, our data suggest that vascularized BM-containing VCAs are immunologically favorable grafts promoting chimerism induction and long-term allograft survival in the context of CoB.

Peri-graft porcine-specific CD4+ FoxP3+ regulatory T cells by CD40-CD154 blockade prevented the rejection of porcine islet graft in diabetic mice.

Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Treg s) in xeno-immune tolerance. However, the characteristics of Treg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction. Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n=18). Immunohistochemical staining showed significantly higher ratio of CD4+ Foxp3+ Treg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of Treg s evoked graft rejection, and adoptive transfer of Treg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These Treg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific. In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4+ Foxp3+ Treg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation.

Delivery of Pancreatic Islets and Single Dose Local Immune Suppression Into Subcutaneous Space Using Injectable Hydrogel Provides Indefinite Survival of the Graft in Mouse Model of Diabetes

Introduction Pancreatic islet transplantation is a promising technique to treat type 1 diabetes. Long-term survival of the graft is required for a successful islet transplantation. Repeated use of immunosuppressive drugs after organ/cell transplantation often leads to severe adverse effects including nephrotoxicity, hepatotoxicity, and opportunistic infections. Thus, development of a local immunosuppression protocol is necessary to improve the islet graft survival in clinics. Materials and Methods Pancreatic islets from Sprague-Dawley rats were transplanted into the subcutaneous space of B6 mice using injectable hydrogel. Three major groups were prepared for transplantation. (1) Islets transplanted FK506-loaded poly(lactic-co-glycolic acid) microspheres (10 mg/kg), (2) Islets transplanted with clodronate liposomes (6.25 mg/kg), and (3) Islet transplanted with the combination of both immune suppressants. The suspension of islets and immune suppressants in Matrigel was then injected into the subcutaneous space over the flanks of streptozocin-induced diabetic mice. Results Islets transplanted without any immunosuppression were rejected within two weeks. In contrast, the islets transplanted with the single immunosuppressive regimen of FK506 or clodronate improved survival rate compared with that of the control mice. More interestingly, the graft transplanted using the combination of both immune suppressants survived indefinitely. Immunological studies revealed that the immunosuppressive cocktail inhibited the proliferation of immune cells residing at the peripheral lymph nodes. Interestingly, the systemic immune system of the transplanted mice remained unaffected. Furthermore, histochemical analysis revealed the intact morphology of the islets at the transplanted site when codelivered with the immunosuppressant. Discussion Antigen presenting cells and T-cells orchestrate the immune rejection cascade. Macrophage depletion by the liposomal clodronate and the inhibition of T-cell activation by FK506 completely blocked the immune rejection cascade in the immune competent mice. The inhibition of immune stimulation in the peripheral lymph nodes improved the islet grafted into the subcutaneous space. Thus, the use of local immune suppression is an effective approach to enhance the survival of the transplanted islets. Conclusion We developed a protocol for the local codelivery of pancreatic islets and immune suppressive agents. Indefinite graft survival was obtained with the use of macrophage depleting agent and T-cell inhibitor. The single dose of local immune suppression during transplantation may avoid toxic effects associated with a long-term use of immune suppressive agents in clinics. National Research Foundation of Korea (NRF) Grant nos: 2015R1A5A2009124 and 2017R1D1A1B03027831. Korea Health Industry Development Institute (KHIDI) Grant no: HI16C1767.

Development of a Highly Effective Protocol using Local Immune Suppression Strategy in Pancreatic Islet Transplantation

Introduction Pancreatic islet transplantation is a promising technique to treat type 1 diabetes. Long-term survival of the graft is required for a successful islet transplantation. Repeated use of immunosuppressive drugs after organ/cell transplantation often leads to severe adverse effects including nephrotoxicity, hepatotoxicity, and opportunistic infections. Thus, development of a local immunosuppression protocol is necessary to improve the islet graft survival in clinics. Materials and Methods Pancreatic islets from Sprague-Dawley rats were transplanted into the subcutaneous space of B6 mice using injectable hydrogel. Briefly, 1000 islet equivalents were suspended in Matrigel premixed with FK506-loaded poly(lactic-co-glycolic acid) microspheres (10 mg/kg) and clodronate liposomes (6.25 mg/kg) and injected into the subcutaneous space over the flanks of streptozocin-induced diabetic mice. Blood glucose level was monitored using a potable glucometer. Results Islets transplanted without any immunosuppression were rejected within two weeks. In contrast, the islets transplanted with the immunosuppressive regimen of FK506 and clodronate survived indefinitely. Immunological studies revealed that the immunosuppressive cocktail inhibited the proliferation of immune cells residing at the peripheral lymph nodes. Interestingly, the systemic immune system of the transplanted mice remained unaffected. Furthermore, histochemical analysis revealed the intact morphology of the islets at the transplanted site when codelivered with the immunosuppressant. Discussion Antigen presenting cells and T-cells orchestrate the immune rejection cascade. Macrophage depletion by the liposomal clodronate and the inhibition of T-cell activation by FK506 completely blocked the immune rejection cascade in the immune competent mice. The inhibition of immune stimulation in the peripheral lymph nodes improved the islet grafted into the subcutaneous space. Thus, the use of local immune suppression is an effective approach to enhance the survival of the transplanted islets. Conclusion We developed a protocol for the local codelivery of pancreatic islets and immune suppressive agents. Indefinite graft survival was obtained with the use of macrophage depleting agent and T-cell inhibitor. The single dose of local immune suppression during transplantation may avoid toxic effects associated with a long-term use of immune suppressive agents in clinics. National Research Foundation of Korea (NRF) Grant nos: 2015R1A5A2009124 and 2017R1D1A1B03027831. Korea Health Industry Development Institute (KHIDI) Grant no: HI16C1767.

Split Tolerance in a Murine Model of Heterotopic En Bloc Chest Wall Transplantation.

Background:Congenital and acquired chest wall deformities represent a significant challenge to functional reconstruction and may impact feasibility of heart transplantation for patients with end-stage organ failure. In the recent past, the concept of replacing like-with-like tissue by using vascularized composite allografts (VCA) has been enthusiastically employed for reconstruction of complex tissue defects.Methods:In this study, we introduce a novel murine model for en bloc chest wall, heart, and thymus transplantation and thereby the use of complex tissue allografts for reconstruction of both chest wall defects and also end-stage organ failure. Additionally, this model allows us to study the features of combined vascularized bone marrow (VBM), thymus, and heart transplantation on allograft survival and function. Heterotopic chest wall, thymus, and heart transplants were performed in untreated syngeneic and allogeneic combinations and in allogeneic combinations treated with costimulation blockade (CTLA4-Ig and MR-1).Results:Indefinite (ie, 150 d, N = 3) graft survival was observed in syngeneic controls. In untreated recipients of allogeneic grafts, the skin component was rejected after 10 (±1) days, whereas rejection of the heart occurred after 13 (± 1) days (N = 3). Costimulation blockade treatment prolonged survival of the heart and chest wall component (130 d, N = 3) as well as the VBM niche as evidenced by donor-specific chimerism (average: 2.35 ± 1.44%), whereas interestingly, the skin component was rejected after 13 (±1) days.Conclusion:Thus, this novel microsurgical model of VCA combined with solid organ transplantation is technically feasible and results in split tolerance when treated with costimulatory blockade.

Thymus-Derived Treg Infusion to Prevent Graft Rejection in Heart-Transplanted Children

Introduction: Immune graft rejection is the main obstacle to successful transplants. Immunosuppressive drugs fail to prevent chronic rejection and also impair the immune system, resulting in reduced lifetime survival rates. Transfer of regulatory T cells (Treg) has acquired growing interest in attempts to achieve indefinite graft survival [1]. The limited Treg numbers that can be purified from peripheral blood, along with low survival and limited suppressive capacity of expanded Treg obtained from adults, constitute important drawbacks for the effectiveness of this strategy. Methods: We established a collaboration amongst several teams working in the field to overcome current barriers and make Treg therapy a reality equipped to achieve indefinite graft survival. The major innovation is the employment of thymus, the site of Treg generation, as a new source of Treg [2]. Our goal is to perform a first-in-human trial treating heart-transplanted children with autologous thymus-derived Treg (thy-Treg) to prevent graft rejection. Results: We purified Treg from thymuses discarded routinely during cardiac surgeries in children with congenital diseases, obtaining massive quantities of thy-Treg with >98% purity. The final thy-Treg product shows high suppressive capacity, decreasing the proliferation of CD4+ and CD8+ T cells in co-cultures by more than 70%. Next, we propose to conduct a clinical trial transferring thy-Treg to heart-transplanted children. In the first instance, thymuses removed during transplant surgery will be processed in GMP-conditions, and autologous thy-Treg will be infused back to the patient at day +10, when immunosuppressant doses are routinely reduced. Due to the high quality and quantity of these thy-Treg and the favorable features of the pediatric immune system, we are confident that this strategy will boost Treg-mediated tolerance and reduce the incidence of graft rejection. Conclusion: Thymic tissue constitutes an alternative source of Treg that could be employed as cellular immunotherapy to prevent rejection. The massive quantity of Treg obtained from thymus, their high suppressive capacity, stability and survival, could overcome the drawbacks of employing Treg from peripheral blood. We propose that thy-Treg will constitute a therapeutic arsenal with infinite possibilities for the treatment of immune-related diseases, including graft rejection or autoimmune disorders.FIGURE 1: Purification and enrichment of thymus-derived Treg. Thymus tissue from one 5-months old chridren. Left: frequency of Treg cells in the total of thymocytes before pirification. Right: Highty Treg obtained after purification. Right: pure thynus-derived Treg obtained after pirification and 10-day culture.This work was supported by grants from Instituto de Salud Carlos III (ISCIII) co-financed by FEDER funds (PI15/00011; ICI14/00282).FIGURE 2: Suppressive capacity of thymus-derived Treg. Allogenic PBMCs were stained with CFSE and then co-cultured alone or with thymus derived Treg. (ratioPBMC:Treg 21). Proliferation ofCD4+ and CD8 T cells was analysed after 3 bays culture.

Abstract 44: Tolerance Induction with Post Transplantation High-Dose Cyclophosphamide relies on Recipient-derived Immune Regulation and Donor Bone Marrow in a Murine Model of Vascularized Composite Allotransplantation

PURPOSE: Developing novel treatment concepts to minimize/avoid immunosuppression by induction of immune tolerance represents the prime task in the field of transplantation. In the clinic to date, this was achieved in highly selected patients in clinical trials of kidney transplantation combined with concomitant stem cell or bone marrow transplantation. Vascularized composite allotransplantation (VCA) is evolving as viable treatment modality for patients suffering from functional and esthetic defects to craniofacial structures, extremities and urogenital tissues. In certain VCAs transplantation of vascularized bone marrow may be inherently part of the allograft designs (i.e. hands and faces) and thereby representing a unique opportunity in combining bone marrow and VCA. METHODS: Murine skin, heart, and hind limb (VCA) transplants were performed across a full MHC mismatch barrier. Recipients treatment comprised non-myeloablative TBI and T-cell depletion and a single dose of post-transplant cyclophosphamide (PTCy). Donor BM and splenocytes (DBM) were injected at the time of transplantation. Post-transplant multi-lineage and, Foxp3 chimerism as well as Vβ-TCR staining was performed. Donor-specific unresponsiveness was tested by MLR and by 2° skin and solid organ transplantation (SOT). Mechanistic studies were undertaken using transgenic mice (DEREG and Foxp3-DTR) to investigate the role of regulatory T cells (Tregs). VCA was performed in animals thymectomized prior to receiving PTCy and the transplant to highlight the role of the thymus in induction and maintenance of tolerance in this murine model. RESULTS: Untreated animals rejected skin grafts, SOT and VCA acutely within 14 ± 1 days, 9 ± 2 days, and 8 ± 1 days, respectively. The treatment regimen extended skin and SOT graft survival (32 ± 8; 65 ± 4, respectively). Additional DBM augmentation lead to allograft survival of >150 days in skin and SOT. However, indefinite graft survival of >150 days was observed in all animals receiving the induction regimen and a VCA ± DBM. In groups receiving a VCA ± DBM, donor chimerism was detected at 22.51% ± 5.96% and 30.17% ± 8.72%, respectively. Prior or post transplantation depletion of recipient-derived Tregs in VCA recipients did not lead to abrogation of tolerance. In recipients of skin graft + DBM, chimerism was not achieved after recipient-derived Treg depletion prior to transplantation (POD-2) and consequently the allografts were rejected (MST = 23.5 days, N=4). In-vitro, Vβ-T cell receptor staining indicates clonal deletion as an additional central tolerance mechanism. However, transplantation into a thymectomized host did not abrogate long-term allograft survival (N=6). All long-term survivors showed donor-specific T cell unresponsiveness in-vitro (MLR) while demonstrated proliferation against 3rd party stimulators. In-vivo, tolerant animals accepted donor-matched secondary skin, while 3rd party FVB/N skin was acutely rejected. Donor-matched hearts were accepted long-term. CONCLUSION: Taken together, robust tolerance and immunosuppression-free long-term allograft survival can be achieved with PTCy in stringent fully MHC mismatched murine models of skin, heart, and vascularized composite allotransplantation. Stable multi lineage chimerism and recipient derived regulatory T cells play a critical role in maintaining tolerance in this model, in particular in the peritransplant time period.

Transplantation tolerance: don't forget about the B cells.

Establishing a state of transplantation tolerance that leads to indefinite graft survival without the need for lifelong immunosuppression has been achieved successfully in limited numbers of transplant recipients in the clinic. These successes led to studies aimed at identifying potential biomarkers that diagnose allograft tolerance and identify the patients most amenable to drug minimization, and implicated an enriched B cell signature of tolerance. The emergence of a specialized subset of regulatory B cell (Bregs ), that possess immune-modulatory function in inflammation and autoimmune disease, raised the possibility that Bregs play critical roles in the promotion of transplantation tolerance and that Bregs are the underlying explanation for the B cell signature of tolerance. However, B cells are best known to play a key role in humoral immunity, and excessive production of donor specific antibodies has clear deleterious effects in transplantation. Thus, for tolerance to be persistent, alloantibody responses must also be curtailed, either through the suppression of T cell help or the induction of B cell-intrinsic dysfunction. Recent findings indicate a unique subset of follicular regulatory T cells (Tfr) that can suppress B cell function and induce epigenetic modifications that result in sustained defects in B cell differentiation and function. In this review, we summarize studies in animals and humans that suggest roles for Bregs and dysfunctional B cells in transplantation tolerance, and discuss how these insights may provide a roadmap for new approaches to diagnose, and new therapies to induce allograft tolerance.

Computational immuno-biology for organ transplantation and regenerative medicine.

Organ transplantation and regenerative medicine are adopted platforms that provide replacement tissues and organs from natural or engineered sources. Acceptance, tolerance and rejection depend greatly on the proper control of the immune response against graft antigens, motivating the development of immunological and genetical therapies that prevent organ failure. They rely on a complete, or partial, understanding of the immune system. Ultimately, they are innovative technologies that ensure permanent graft tolerance and indefinite graft survival through the modulation of the immune system. Computational immunology has arisen as a tool towards a mechanistic understanding of the biological and physicochemical processes surrounding an immune response. It comprehends theoretical and computational frameworks that simulate immuno-biological systems. The challenge is centered on the multi-scale character of the immune system that spans from atomistic scales, during peptide-epitope and protein interactions, to macroscopic scales, for lymph transport and organ-organ reactions. In this paper, we discuss, from an engineering perspective, the biological processes that are involved during the immune response of organ transplantation. Previous computational efforts, including their characteristics and visible limitations, are described. Finally, future perspectives and challenges are listed to motivate further developments.

Immunotoxin Against a Donor MHC Class II Molecule Induces Indefinite Survival of Murine Kidney Allografts.

Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II+ cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab′)2 fragment of a monoclonal antibody against the donor MHC class II molecule I‐Ak conjugated with the plant‐derived ribosomal inactivating protein gelonin. This anti–I‐Ak gelonin immunotoxin depletes I‐Ak expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3+ cells within donor grafts, diminished donor‐specific antibody formation, and delayed rejection of subsequent donor‐type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient‐orientated immunosuppression.

Cell therapy to induce allograft tolerance: time to switch to plan B?

Organ transplantation is widely acknowledged as the best option for end stage failure of vital organs. Long-term graft survival is however limited by graft rejection, a destructive process resulting from the response of recipient’s immune system against donor-specific alloantigens. Prevention of rejection currently relies exclusively on immunosuppressive drugs that lack antigen specificity and therefore increase the risk for infections and cancers. Induction of donor-specific tolerance would provide indefinite graft survival without morbidity and therefore represents the grail of transplant immunologists. Progresses in the comprehension of immunoregulatory mechanisms over the last decades have paved the way for cell therapies to induce allograft tolerance. The first part of the present article reviews the promising results obtained in experimental models with adoptive transfer of ex vivo-expanded regulatory CD4+ T cells (CD4+ Tregs) and discuss which source and specificity should be preferred for transferred CD4+ Tregs. Interestingly, B cells have recently emerged as potent regulatory cells, able to establish a privileged crosstalk with CD4+ T cells. The second part of the present article reviews the evidences demonstrating the crucial role of regulatory B cells in transplantation tolerance. We propose the possibility to harness B cell regulatory functions to improve cell-based therapies aiming at inducing allograft tolerance.

Enhancement of islet engraftment and achievement of long-term islet allograft survival by Toll-like receptor 4 blockade.

Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. In vitro TLR4 blockade decreased lipopolysaccharide-mediated β-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.

The regulatory T cell effector molecule fibrinogen-like protein 2 is necessary for the development of rapamycin-induced tolerance to fully MHC-mismatched murine cardiac allografts.

Therapies that promote tolerance in solid organ transplantation will improve patient outcomes by eliminating the need for long-term immunosuppression. To investigate mechanisms of rapamycin-induced tolerance, C3H/HeJ mice were heterotopically transplanted with MHC-mismatched hearts from BALB/cJ mice and were monitored for rejection after a short course of rapamycin treatment. Mice that had received rapamycin developed tolerance with indefinite graft survival, whereas untreated mice all rejected their grafts within 9 days. In vitro, splenic mononuclear cells from tolerant mice maintained primary CD4(+) and CD8(+) immune responses to donor antigens consistent with a mechanism that involves active suppression of immune responses. Furthermore, infection with lymphocytic choriomeningitis virus strain WE led to loss of tolerance suggesting that tolerance could be overcome by infection. Rapamycin-induced, donor-specific tolerance was associated with an expansion of regulatory T (Treg) cells in both the spleen and allograft and elevated plasma levels of fibrinogen-like protein 2 (FGL2). Depletion of Treg cells with anti-CD25 (PC61) and treatment with anti-FGL2 antibody both prevented tolerance induction. Tolerant allografts were populated with Treg cells that co-expressed FGL2 and FoxP3, whereas rejecting allografts and syngeneic grafts were nearly devoid of dual-staining cells. We examined the utility of an immunoregulatory gene panel to discriminate between tolerance and rejection. We observed that Treg-associated genes (foxp3, lag3, tgf-β and fgl2) had increased expression and pro-inflammatory genes (ifn-γ and gzmb) had decreased expression in tolerant compared with rejecting allografts. Taken together, these data strongly suggest that Treg cells expressing FGL2 mediate rapamycin-induced tolerance. Furthermore, a gene biomarker panel that includes fgl2 can distinguish between rejecting and tolerant grafts.

An immunosufficient murine model for the study of human islets.

For the sake of therapy of diabetes, it is critical to understand human beta cell function in detail in health and disease. Current studies of human beta cell physiology in vivo are mostly limited to immunodeficient mouse models, which possess significant technical limitations. This study aimed to create a new model for the study of human islets through induction of transplant tolerance in immunosufficient mice. B6 diabetic mice were transplanted with human islets and treated with anti-CD45RB. To assess whether anti-CD45RB-induced transplant tolerance requires B cells, B6 recipients received additional anti-CD20 or B6μMT-/- mice were used. For some anti-CD45RB-treated B6μMT-/- mice, additional anti-CD25 mAb was applied at the early or late stage post-transplant. Immunohistology was performed to show the Foxp3 cells in grafted anti-CD45RB/anti-CD20-treated Foxp3-GFP B6 mice. The results showed that anti-CD45RB alone allowed indefinite graft survival in 26.6% of B6 mice, however 100% of xenografts were accepted in mice treated simultaneously with anti-CD20, and 88.9% of xenografts accepted in anti-CD45RB-treated μMT-/- mice. These μMT-/- mice accepted the islets from another human donor but rejected the islets from baboon. Additional administration of anti-CD25 mAb at the time of transplantation resulted in 100% rejection, whereas 40% of grafts were rejected while the antibody was administrated at days 60 post-transplant. Immunohistologic examination showed Foxp3+ cells accumulated around grafts. We conclude that induction of tolerance to human islets in an immunosufficient mouse model could be generated by targeting murine CD45RB and CD20. This new system will facilitate study of human islets and accelerate the dissection of the critical mechanisms underlying islet health in human disease.

Autoantibody Responses to a Heart Allograft Augment Allograft Vasculopathy By Amplifying Conventional Cellular and Humoral Alloimmune Effector Mechanisms.

Introduction: Although transplant-related autoantibody responses are reported increasingly, their contribution to graft failure remains poorly understood. We used a MHC-mismatched murine heart transplant model in which passenger donor CD4 T cells trigger recipient autoantibody to examine the hypothesis that humoral autoimmunity contributes to allograft vasculopathy (AV) by amplifying conventional alloimmune responses. Methods: A new donor strain (bm12.Kd.IE) was created, to incorporate additional MHC class I (H-2Kd) and class II (I-E) target alloantigens. IgG allo- and autoantibody responses were measured weekly. Indirect-pathway CD4 T cell responses were assessed by transfer of CFSE-labelled TCR75 T cells, CD8 cytotoxic responses were measured by ELISPOT. Donor CD4 T cell were depleted with anti-CD4 antibody (YTS 191.1) AV was assessed morphometrically. Results: Heart allografts from unmodified donors provoked long-lasting indirect-pathway CD4 T cell responses and strong, class-switched alloantibody responses against the mismatched Kd class I alloantigen. Moderate CD8 T cellular cytotoxicity was also observed. These were associated with development of progressive allograft vasculopathy and eventual graft failure. In contrast, alloimmune responses were barely above background following grafting with CD4 T cell-depleted hearts that did not trigger autoimmunity, with only minimal vasculopathy development and indefinite graft survival. The contribution of donor CD4 T cells to graft rejection was not due to a direct effect on the graft, because they are killed within days after transplantation, but instead to the humoral immunity that they trigger; in the absence of recipient B cells, donor T cell responses remained vigorous, but host CD4 T cell alloimmunity was diminished and no longer influenced by the presence of donor CD4 T cells. Conclusions: Concurrent humoral autoimmunity contributes to allograft rejection by augmenting conventional alloimmunity, and is triggered by even transient donor T cell survival.

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.

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.

Blockade of T-Cell Homeostasis by Anti-Interleukin-7 Receptor Monoclonal Antibody Allows Indefinite Islet and Long-Term Skin Allograft Survival

Background: T cell depletion is followed by T cell homeostasis with increase in memory T cells which are more potent in mediating rejection. We demonstrate that IL-7R blockade with or without prior T cell depletion inhibits T cell homeostasis and prolongs graft survival. Methods: Tail skin from C57BL/6 was transplanted to Balb/c mice with 3 treatment protocols: protocol 1 (no treatment, n=10), 2 (T cell depletion by combining 2 depleting mAbs: anti-CD4 and anti-CD8 on day -3 and -1, n=10), and 3 (same as protocol 2, followed by a blocking anti-IL-7Rα mAb qod, n=11). Islets from C57BL/6 were transplanted to streptozotocin-induced diabetic Balb/c mice, divided into 3 groups: group A (no treatment, n=10), B (anti-IL-7Rα mAb qod from day 0 to rejection, n=5), and C (anti-IL-7Rα mAb qod from day -21 to PTD90, n=6). Results: Median skin graft survival with protocol 1, 2, and 3 was 9.5, 30, and 58 days, respectively (protocol 3 vs 2, p< 0.0001). IL-7R blockade following T cell depletion profoundly inhibited lymphocyte reconstitution. At PTD35, the mean absolute numbers of CD3+, CD3+CD4+, CD3+CD8+, and CD19+ cells in the spleen were 6.7, 4.1, 0.8, and 119 in protocol 3 vs 57.5, 43.6, 7.3, and 276 ×105 cells in protocol 2, respectively (all p< 0.05). Importantly, the mean absolute number of CD44hiCD62Llomemory T cells was 4-fold lower in protocol 3 vs protocol 2: 3.8 vs 14.3 ×105 cells, and the percentage of CD4+CD25+FOXP3+ Treg to total CD4+ T cells was slightly increased: 13 vs 10% (all p< 0.05). Similar results were found in the lymph node and in the blood. Moreover, thymic cellularity was markedly reduced in protocol 3 vs 2: 0.7 vs 142 ×105 cells, p< 0.05. IL-7R blockade abrogated cellular immune responses as shown by direct IFNγ ELISPOT (41.3 vs 193.5 spots/105 T cells) and by MLR-3H thymidine (238 vs 10510 CPM) and inhibited humoral immune responses as shown by a reduction in DSA (310 vs 996 MFI) (protocol 3 vs 2, all p< 0.05). Histology revealed abundant subcutaneous and dermal leukocyte infiltrates at PTD21 in protocol 2, whereas few infiltrates were seen in protocol 3 at PTD21 and PTD35. The addition of low-dose tacrolimus 0.5 mg/kg/d to protocol 2 did not improve median graft survival (32.5 days, n=6, p=NS), whereas the same dose of tacrolimus added to protocol 3 further prolonged skin graft survival to >90 days in 4 of 6 recipients (p< 0.05). Median islet graft survival was not significantly prolonged when anti-IL-7Rα was given from day 0 (group B vs A: 29 vs 21 days, p=NS). However, when treatment was started 21 days before islet graft, 5 of 6 recipients had indefinite graft survival >180 days (group C vs A, p< 0.005), associated with an abrogation of DSA (group C vs A: 260 vs 976 MFI, p< 0.01). Conclusion: IL-7R blockade following with T cell depletion is a potent immunosuppressive regimen which abrogates both cellular and humoral allo-immune responses and may be used synergistically with low-dose tacrolimus to avoid nephrotoxity.