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IL-7 is an important cytokine for T cell lymphopoiesis. Blockade of the IL-7 signaling pathway has been shown to induce long-term graft survival or graft tolerance in murine transplant models through inhibiting T cell homeostasis and favoring immunoregulation. In this study, we assessed for the first time the effects of a blocking anti-human cluster of differentiation 127 (CD127) mAb administered in combination with low-dose tacrolimus or thymoglobulin in a life-sustaining kidney allograft model in baboons. Contrary to our expectation, the addition of an anti-CD127 mAb to the treatment protocols did not prolong graft survival compared to low-dose tacrolimus alone or thymoglobulin alone. Anti-CD127 mAb administration led to full CD127 receptor occupancy during the follow-up period. However, all treated animals lost their kidney graft between 1 week and 2 weeks after transplantation. Unlike in rodents, in nonhuman primates, anti-CD127 mAb treatment does not decrease the absolute numbers of lymphocyte and lymphocyte subsets and does not effectively inhibit postdepletional T cell proliferation and homeostasis, suggesting that IL-7 is not a limiting factor for T cell homeostasis in primates., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature hematopoietic precursors known to suppress immune responses. Interaction of SIRP alpha (SIRPα), expressed by myeloid cells, with the ubiquitous receptor CD47 is an important immune checkpoint of the innate response regulating macrophages and dendritic cells functions. We previously described that MDSC expressing SIRPα accumulated after transplantation and maintained kidney allograft tolerance. However, the role of the SIRPα/CD47 axis on MDSC function remained unknown. Here, we found that blocking SIRPα or CD47 with monoclonal antibodies (mAbs) induced differentiation of MDSC into myeloid cells overexpressing MHC class II, CD86 costimulatory molecule and increased secretion of macrophage-recruiting chemokines (eg, MCP-1). Using a model of long-term kidney allograft tolerance sustained by MDSC, we observed that administration of blocking anti-SIRPα or CD47 mAbs induced graft dysfunction and rejection. Loss of tolerance came along with significant decrease of MDSC and increase in MCP-1 concentration in the periphery. Graft histological and transcriptomic analyses revealed an inflammatory (M1) macrophagic signature at rejection associated with overexpression of MCP-1 mRNA and protein in the graft. These findings indicate that the SIRPα-CD47 axis regulates the immature phenotype and chemokine secretion of MDSC and contributes to the induction and the active maintenance of peripheral acquired immune tolerance., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Nephrotoxicity of calcineurin inhibitors and uncontrolled effector function of alloreactive T lymphocytes are main drivers of transplant dysfunctions. T lymphocytes either directly damage tissues or indirectly promote inflammation and antibody responses. Beside inhibitors of calcium-dependent pathways and antimetabolites, modulators of T-cell costimulation are elected pharmacological tools to enable interference with immune-mediated transplant dysfunctions. CD28 and CTLA-4 are major costimulatory and coinhibitory cell surface signaling molecules interacting with CD80/86, known to be critically important for immune response of committed T cells and regulation. Initial bench to beside translation, 2 decades ago, resulted in the development of belatacept CTLA-4 fused with an immunoglobulin Fc domain, a biologic inhibiting interaction of both CD28 and CTLA-4 with CD80/86. Despite proven effectiveness in inhibiting alloimmune responses, clinical use of belatacept in kidney transplantation revealed a substantially high incidence of acute, cell-mediated rejection. The cause of belatacept-resistant graft rejection was allocated to elevated pretransplant frequencies of CD28 memory T cells. Owing to different requirements in CD28 costimulatory and CTLA-4 coinhibitory signals to control naive and memory T cells, selective antagonists of CD28-CD80/86 interactions have been developed on the rationale that preservation of CTLA-4-mediated regulatory mechanisms would result in a better control of alloreactivity and would represent a regulatory T-cell-compatible immunosuppression. After the successful testing of selective CD28 antagonists in First In Human studies, this review delineates how this shift in paradigm performed in preclinical transplantation models and evaluates its clinical potential.

FR104 is a monovalent pegylated Fab' Ab, antagonist of CD28, under development for treatment of transplant rejection and autoimmune diseases. In contrast to CD80/86 antagonists (CTLA4-Ig), FR104 selectively blunts CD28 costimulation while sparing CTLA-4 and PD-L1 coinhibitory signals. In the present work, FR104 has been evaluated in a first-in-human study to evaluate the safety, pharmacokinetics, pharmacodynamics, and potency of i.v. administrations in healthy subjects. Sixty-four subjects were randomly assigned to four single ascending dose groups, two double dose groups and four single ascending dose groups challenged with keyhole limpet hemocyanin. Subjects were followed up over a maximum of 113 d. Overall, the pharmacokinetics of FR104 after a single and double infusions was approximately linear at doses ≥0.200 mg/kg. CD28 receptor occupancy by FR104 was saturated at the first sampling time point (0.5 h) at doses above 0.02 mg/kg and returned to 50% in a dose-dependent manner, by day 15 (0.020 mg/kg) to 85 (1.500 mg/kg). FR104 was well tolerated, with no evidence of cytokine-release syndrome and no impact on blood lymphocyte subsets. Inhibition of anti-keyhole limpet hemocyanin Ab response was dose-dependent in FR104 recipients and was already apparent at a dose of 0.02 mg/kg. Abs to FR104 were detected in 22/46 (48%) of FR104 recipients and only 1/46 (2.2%) was detected during drug exposure. In conclusion, selective blockade of CD28 with FR104 was safe and well tolerated at the doses tested. The observed immunosuppressive activity indicated that FR104 has potential to show clinical activity in the treatment of immune-mediated diseases., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation., (Copyright © 2016 by the American Society of Nephrology.)

Selective targeting of CD28 might represent an effective immunomodulation strategy by preventing T cell costimulation, while favoring coinhibition since inhibitory signals transmitted through CTLA-4; PD-L1 and B7 would not be affected. We previously showed in vitro and in vivo that anti-CD28 antagonists suppress effector T cells while enhancing regulatory T cell (Treg) suppression and immune tolerance. Here, we evaluate FR104, a novel antagonist pegylated anti-CD28 Fab' antibody fragment, in nonhuman primate renal allotransplantation. FR104, in association with low doses of tacrolimus or with rapamycin in a steroid-free therapy, prevents acute rejection and alloantibody development and prolongs allograft survival. However, when FR104 was associated with mycophenolate mofetil and steroids, half of the recipients rejected their grafts prematurely. Finally, we observed an accumulation of Helios-negative Tregs in the blood and within the graft after FR104 therapy, confirmed by Treg-specific demethylated region DNA analysis. In conclusion, FR104 reinforces immunosuppression in calcineurin inhibitor (CNI)-low or CNI-free protocols, without the need of steroids. Accumulation of intragraft Tregs suggested the promotion of immunoregulatory mechanisms. Selective CD28 antagonists might become an alternative CNI-sparing strategy to B7 antagonists for kidney transplant recipients., (© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors., Methods: In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model., Results: Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals., Conclusions: Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression., (© 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.)

Background: Blockade of costimulation signaling required for immune response, such as CD40/CD40L and CD28/B7, is a reasonable strategy to prevent rejection and in defined combinations may allow donor specific tolerance. Indeed, in rodents, costimulation blockade with CD28/B7 antagonists or with CD40Ig was able to induce regulatory T cells and transplant tolerance whereas in primates, anti-CD40 antibodies, anti-CD40L antibodies or CTLA4Ig, used as monotherapy, significantly delayed graft rejection., Methods: Using an adeno-associated virus (AAV) vector mediated gene transfer of a human CD40Ig fusion protein (hCD40Ig) in primates, we evaluated the capacity of this costimulation blockade molecule interfering with CD40/CD40L signaling in prolonging kidney transplants in cynomolgus monkeys., Results: This gene transfer strategy allowed for maintaining a plateau of hCD40Ig production within two months and avoided a high-scale production phase of this molecule. Although the hCD40Ig was able to bind efficiently to human and macaque CD40L and high (>200 μg/ml) transgene expression was obtained, no effect on graft survival was observed. In addition, there was no inhibition of humoral response to vaccination. In vitro, hCD40Ig strongly increased mixed lymphocyte reaction, and when compared to the anti-CD40L antibody h5C8, was not as potent to induce complement-dependent cytotoxicity., Conclusion: These data suggest that CD40/CD40L blockade using a non-depleting CD40Ig fusion protein, a therapeutic strategy that showed efficacy in rodents, is not able to modulate the immune response in primates. These data highlight important biological differences between rodent and primate models to evaluate therapeutic strategies at the preclinical level., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In the early 1990s, Linsley and colleagues produced a soluble fusion protein, comprising of the extracellular domain of cytotoxic T lymphocyte antigen (CTLA)4 and the human IgG1 Fc domain. Since then, several hundreds of scientific publications have demonstrated that CTLA4-Ig blocks CD28-mediated co-stimulation and suppresses unwanted T cell-mediated responses in animal models of transplantation, autoimmunity and inflammation. In the past two decades, Bristol-Myers Squibb Co. has developed abatacept, a CTLA4-Ig molecule for treating psoriasis and rheumatoid arthritis, and belatacept, a second-generation, higher affinity CTLA4-Ig molecule for use in kidney transplantation. Belatacept represents a new class of transplantation immunosuppressants and potentially offers clinicians a breakthrough therapy to preserve kidney function in the long term and reduce the side effects of current immunosuppressive therapies.

Polyclonal anti-lymphoyctes antibodies were first successfully used in the 1970 in organ transplantation, but ten years later, monoclonal antibodies (mAb) emerged as a new class of immunosuppressive agents in transplantation with the potential to target highly specifically immune cells responsible for acute rejection. Some have proved their efficacy, such as mAb recognizing CD3- and CD25-positive T cells and have been extensively studied in clinical trials. Others such as mAb against CD52 and CD20, are still under investigation; finally, the next challenge is, based on our improved understanding of the mechanisms of immune recognition and allograft rejection, to use these mAb either alone or in combination with standard immunosuppressive regimens to manipulate the allogenic response to reach antigen-specific tolerance desired in solid-organ transplantation.

Drugs targeting memory lymphocytes may allow for a better control of rejection in transplantation, particularly in immunized patients. In this article the rationale of targeting interleukin 7 receptor alpha (IL-7Ralpha), a molecule expressed by both memory and naive T cells, is reviewed in the context of transplantation. Whereas naive T cells are partly responsible for acute rejection and are targeted by current immunosuppressive drugs that block costimulatory signals (cyclosporine A, anti-CD3 antibody, anti-CD52 antibody, anti-thymocyte globulin, etc.), memory T cells are resistant to costimulation blockade. As such, memory cells are an obstacle to experimental tolerance induction and may be involved in chronic rejection. There is thus much scientific interest in developing molecules able to target these cells. The role of the IL-7/IL-7Ralpha pathway in transplantation rejection has been suggested by the effect of an anti-IL-7 monoclonal antibody which, when associated with costimulation blockade, prolonged heart allograft survival in mice. Here the hypothesis that targeting IL-7Ralpha would preserve effector T cells that are less dependent on IL-7 for survival while sparing regulatory CD4+ CD25high IL-7Ralpha(low) T cells is discussed. An anti-IL-7Ralpha antibody could also help achieve allograft tolerance by reducing alloreactive cells.

Blockade of CD40-CD40 ligand (CD40L) costimulation has been shown to synergize with that of CTLA4/CD28-B7 to promote transplant tolerance. To date, however, CD28-B7 interactions have been prevented using B7-blocking reagents like CTLA4-Ig that inhibit CD28-B7 together with CTLA4-B7 interactions. In this study, we have tested anti-CD28 Abs to prevent selectively CD28-B7 interactions while preserving CTLA4-B7 in addition to CD40-CD40L blockade. In the LEW.1W to LEW.1A rat combination, interfering with CD40-CD40L interactions by CD40Ig administration through gene transfer resulted in indefinite heart allograft survival due to the appearance of clonotypic CD8+CD45RClow regulatory T cells that were capable of transferring the tolerant state to naive animals. However, cardiac transplants in these recipients systematically developed chronic rejection lesions. Whereas anti-CD28 Ab monotherapy only delayed acute rejection and failed to induce tolerance, coadministration of anti-CD28 Abs and CD40Ig resulted in the long-term acceptation of allografts without chronic rejection lesions in 60% of the recipients, reduced the level of intragraft mRNA transcripts for cytokines and immune factors, and fully abrogated alloantibody production. In addition, the nature of regulatory cells was modified: the CD8+CD45RClow clonotypic T cells described in the CD40Ig-treated animals could not be found in cotreated animals, and the other CD8+CD45RClow cells had no regulatory activity and a different cytokine expression profile. Instead, in cotreated recipients we found IDO-dependent non-T cells with regulatory activity in vitro. Thus, the addition of a short-term anti-CD28 treatment with CD40Ig resulted in decreased heart allograft chronic rejection lesions, complete inhibition of Ab production, and modified regulatory mechanisms.

Background: Lymphocyte-activated gene-3 (LAG-3, CD223) is upregulated during the early stages of T-cell activation and could be the target of cytotoxic antibodies for induction therapy in transplantation., Methods: Fully vascularized heterotopic allogeneic heart transplantation was performed in rats across a full major histocompatibility complex-mismatch barrier (LEW.1W into LEW.1A). Recipients received two injections (day 0 and 3) of cytotoxic antibodies directed to the extra-loop of LAG-3 immunoglobulin (Ig)-like N-terminal domain or control antibodies., Results: LAG-3 mRNA transcripts accumulated in cardiac allografts undergoing rejection, but not in peripheral lymphoid organs. Administration of anti-LAG-3 antibodies on the day of transplantation did not modify alloreactivity of T lymphocytes from the spleen and did not change the alloantibody response. However, it inhibited graft infiltration by effector mononuclear cells, reduced intragraft levels of interferon-gamma mRNA and prolonged allograft survival from 6 days in controls to a median of 27 days. Anti-LAG-3 antibodies were also active in prolonging survival when administered in a delayed manner, after rejection onset. LAG-3 being also expressed by activated regulatory T (Treg) cells, we tested the effect of anti-LAG-3 antibodies on graft acceptance after donor blood transfusions, a Treg-dependent tolerance induction model. We found that tolerance induction was prevented by anti-LAG-3 antibodies., Conclusions: Targeting LAG-3-positive cells with cytotoxic antibodies is immunosuppressive in transplantation by depleting effectors T cells and therefore may represent a treatment for rejection episodes focused only on pathogenic cells. However, it might not be compatible with tolerance-induction strategies.

Background: In heart allograft in the rat, a sustained costimulation blockade with CTLA4Ig prevents alloreactive T-cell activation and promotes a long-term graft survival through the action of tolerogeneic dendritic cells. It is unclear whether similar mechanisms might occur after xenotransplantation. To test that hypothesis, we have analyzed the action of CTLA4Ig in a model of CD4(+)T cell-mediated xenograft rejection., Methods: Hamster hearts were transplanted into LEW.1A rats receiving an accommodation-inducing treatment consisting of a short course administration of LF15-0195 and a daily administration of cyclosporine A (CSA). To achieve long-term delivery of CTLA4Ig, an intravenous administration of an adenovirus vector coding for mouse CTLA4Ig (Ad-CTLA4Ig) was added to the accommodation induction protocol. On day 40 post-transplantation, rejection was induced by CSA withdrawal. In other xenograft recipients, CD28/B7 costimulation was inhibited at that time only by injections of CTLA4Ig or anti-CD28 antibodies. Graft survival, immunohistology, as well as development of antibodies and regulatory cells were examined., Results: Xenografts survived 6 days after CSA withdrawal in controls and were rejected, as previously described, through the action of CD4(+) xenoreactive T cells. Interfering with CD28/B7 costimulation inhibited this xenoreactive T cell response and delayed rejection to day 10. In recipients that had received Ad-CTLA4Ig, survival was prolonged to day 19 and this was accompanied by the appearance of regulatory cells exhibiting non-donor-specific suppressive activity dependent on IL-2, NO, and IDO. These regulatory cells were different from those previously identified after Ad-CTLA4Ig administration in heart allograft in the rat. In these recipients, rejection occurred as a consequence of an evoked anti-donor IgM response and complement activation and not of a cellular rejection as complement inhibition with cobra venom factor further prolonged xenograft survival., Conclusion: CD28/B7 blockade delays CD4(+) T cell-mediated rejection after CSA withdrawal in accommodated recipients of hamster heart xenografts. In addition, a sustained expression of CTLA4Ig has the potential of inducing cellular regulatory mechanisms. However, such treatment does not prevent the development of xenoreactive IgM antibodies that participate in vascular rejection processes in a complement-dependent manner.

Bristol-Myers Squibb is developing belatacept, a soluble fusion protein of the B7-binding domain of CTLA4 with amino acid changes A29Y and L104E and an Ig tail, which inhibits lymphocyte co-stimulation through CD28, for the potential treatment of solid organ transplant rejection. Belatacept is currently undergoing phase III clinical trials.

T cells are considered to play a major indirect role in the pathogenesis of xenograft vascular rejection, by promoting the induction of anti-donor antibodies that trigger complement- and antibody-dependent cell cytotoxicity. However, how vigorous the T cell xenoresponse is in vivo, and whether, besides their helper function, T cells are capable of directly affecting the graft is still unclear. We have previously shown that cyclosporine A (CsA) withdrawal in accommodated cardiac xenograft recipient allows for a rapid and dense T-cell infiltration, concomitant to an acute graft rejection. In this paper we further characterize the role of T cells in this rejection process and we demonstrate that adoptive transfer of CD4+ T cells in irradiated recipients of long-term cardiac xenografts is sufficient to trigger acute rejection, in the absence of any detectable induced anti-hamster antibody response. Therefore, our data suggest that unusually strong T-cell response will be another major barrier to xenotransplantation, even if antibody-mediated vascular rejection is controlled.

Abstract: Galactosyl-transferase knockout (GT-KO) pigs represent the latest major progress to reduce immune reactions in xenotransplantation. However, their organs are still subject to rapid humoral rejection involving complement activation requiring the ongoing development of further genetic modifications in the pig. In a pig-to-baboon renal transplantation setting, we have used donor pigs that are not only GT-KO, but also transgenic for human CD55 (hCD55), hCD59, hCD39, and fucosyl-transferase (hHT). We studied kidney xenograft survival, physiological and immunologic parameters, xenogeneic rejection characteristics, as well as viral transmission aspects among two groups of baboons: control animals (n = 2), versus those (n = 4) treated with a cocktail of cyclophosphamide, tacrolimus, mycophenolate mofetil, steroids, and a recombinant human C1 inhibitor. Whereas control animals showed clear acute humoral rejection at around day 4, the treated animals showed moderately improved graft survival with rejection at around 2 weeks posttransplantation. Biopsies showed signs of acute vascular rejection (interstitial hemorrhage, glomerular thrombi, and acute tubular necrosis) as well as immunoglobulin (Ig)M and complement deposition in the glomerular and peritubular capillaries. The low level of preformed non-Gal-α1.3Gal IgM detected prior to transplantation increased at 6 days posttransplantation, whereas induced IgG appeared after day 6. No porcine endogenous retrovirus (PERV) transmission was detected in any transplanted baboon. Thus, surprisingly, organs from the GT-KO, hCD55, hCD59, hCD39, and hHT transgenic donors did not appear to convey significant protection against baboon anti-pig antibodies and complement activation, which obviously continue to be significant factors under a suboptimal immunosuppression regimen. The association, timing, and doses of immunosuppressive drugs remain critical. They will have to be optimized to achieve longer graft survivals. [Copyright &y& Elsevier]