Combination of Anti-CD40 and Anti-CD40L Antibodies as Co-Stimulation Blockade in Preclinical Cardiac Xenotransplantation.

The blockade of the CD40/CD40L immune checkpoint is considered essential for cardiac xenotransplantation. However, it is still unclear which single antibody directed against CD40 or CD40L (CD154), or which combination of antibodies, is better at preventing organ rejection. For example, the high doses of antibody administered in previous experiments might not be feasible for the treatment of humans, while thrombotic side effects were described for first-generation anti-CD40L antibodies. To address these issues, we conducted six orthotopic pig-to-baboon cardiac xenotransplantation experiments, combining a chimeric anti-CD40 antibody with an investigational long-acting PASylated anti-CD40L Fab fragment. The combination therapy effectively resulted in animal survival with a rate comparable to a previous study that utilized anti-CD40 monotherapy. Importantly, no incidence of thromboembolic events associated with the administration of the anti-CD40L PAS-Fab was observed. Two experiments failed early because of technical reasons, two were terminated deliberately after 90 days with the baboons in excellent condition and two were extended to 120 and 170 days, respectively. Unexpectedly, and despite the absence of any clinical signs, histopathology revealed fungal infections in all four recipients. This study provides, for the first time, insights into a combination therapy with anti-CD40/anti-CD40L antibodies to block this immune checkpoint.

Corrigendum: Impaired antigen-specific B-cell responses after Influenza vaccination in kidney transplant recipients receiving co-stimulation blockade with Belatacept.

[This corrects the article DOI: 10.3389/fimmu.2022.918887.].

Conversion from calcineurin inhibitors to belatacept-based immunosuppressive therapy skews terminal proliferation of non-classical monocytes and lowers lymphocyte counts.

Belatacept, a modified form of CTLA-Ig that blocks CD28-mediated co-stimulation of T cells, is an immune-suppressant that can be used as an alternative to calcineurin inhibitors (CNIs). In kidney transplant recipients, belatacept has been associated with improved renal function and reduced cardiovascular toxicity. Monocytes as well as T-lymphocytes play causal roles in the pathophysiology of atherosclerotic disease. We hypothesized that the beneficial impact of the use of belatacept over CNIs on cardiovascular risk could be partly explained by the impact of belatacept therapy on these circulating leukocytes. Hence, we phenotyped circulating leukocytes in transplanted patients with a stable renal function that were randomized between either continuation of CNI or conversion to belatacept in two international studies in which we participated. In 41 patients, we found that belatacept-treated patients consistently showed lower numbers of B-lymphocytes, T-lymphocytes as well as CD14-negative monocytes (CD14NM), especially in non-diabetic patients. Our observation that this decrease was associated to plasma concentrations of TNFα is consistent with a model where CD14NM-production of TNFα is diminished by belatacept-treatment, due to effects on the antigen-presenting cell compartment.

Impaired antigen-specific B-cell responses after Influenza vaccination in kidney transplant recipients receiving co-stimulation blockade with Belatacept.

Emerging data suggest that costimulation blockade with belatacept effectively controls humoral alloimmune responses. However, whether this effect may be deleterious for protective anti-infectious immunity remains poorly understood. We performed a mechanistic exploratory study in 23 kidney transplant recipients receiving either the calcineurin-inhibitor tacrolimus (Tac, n=14) or belatacept (n=9) evaluating different cellular immune responses after influenza vaccination such as activated T follicular Helper (Tfh), plasmablasts and H1N1 hemagglutinin (HA)-specific memory B cells (HA+mBC) by flow-cytometry, and anti-influenza antibodies by hemagglutination inhibition test (HI), at baseline and days 10, 30 and 90 post-vaccination. The proportion of CD4+CD54RA-CXCR5+ Tfh was lower in belatacept than Tac patients at baseline (1.86%[1.25-3.03] vs 4.88%[2.40-8.27], p=0.01) and remained stable post-vaccination. At M3, HA+mBc were significantly higher in Tac-treated patients (0.56%[0.32-1.49] vs 0.27%[0.13-0.44], p=0.04) and correlated with activated Tfh numbers. When stratifying patients according to baseline HA+mBc frequencies, belatacept patients with low HA+mBC displayed significantly lower HA+mBc increases after vaccination than Tac patients (1.28[0.94-2.4] vs 2.54[1.73-5.70], p=0.04). Also, belatacept patients displayed significantly lower seroprotection rates against H1N1 at baseline than Tac-treated patients (44.4% vs 84.6%) as well as lower seroconversion rates at days 10, 30 and 90 after vaccination (50% vs 0%, 63.6% vs 0%, and 63.6% vs 0%, respectively). We show the efficacy of belatacept inhibiting T-dependent antigen-specific humoral immune responses, active immunization should be highly encouraged before starting belatacept therapy.

Traumatic brain injury does not disrupt costimulatory blockade-induced immunological tolerance to glial-restricted progenitor allografts.

BACKGROUND: Cell transplantation-based treatments for neurological disease are promising, yet graft rejection remains a major barrier to successful regenerative therapies. Our group and others have shown that long-lasting tolerance of transplanted stem cells can be achieved in the brain with systemic application of monoclonal antibodies blocking co-stimulation signaling. However, it is unknown if subsequent injury and the blood-brain barrier breach could expose the transplanted cells to systemic immune system spurring fulminant rejection and fatal encephalitis. Therefore, we investigated whether delayed traumatic brain injury (TBI) could trigger graft rejection. METHODS: Glial-restricted precursor cells (GRPs) were intracerebroventricularly transplanted in immunocompetent neonatal mice and co-stimulation blockade (CoB) was applied 0, 2, 4, and 6 days post-grafting. Bioluminescence imaging (BLI) was performed to monitor the grafted cell survival. Mice were subjected to TBI 12 weeks post-transplantation. MRI and open-field test were performed to assess the brain damage and behavioral change, respectively. The animals were decapitated at week 16 post-transplantation, and the brains were harvested. The survival and distribution of grafted cells were verified from brain sections. Hematoxylin and eosin staining (HE) was performed to observe TBI-induced brain legion, and neuroinflammation was evaluated immunohistochemically. RESULTS: BLI showed that grafted GRPs were rejected within 4 weeks after transplantation without CoB, while CoB administration resulted in long-term survival of allografts. BLI signal had a steep rise following TBI and subsequently declined but remained higher than the preinjury level. Open-field test showed TBI-induced anxiety for all animals but neither CoB nor GRP transplantation intensified the symptom. HE and MRI demonstrated a reduction in TBI-induced lesion volume in GRP-transplanted mice compared with non-transplanted mice. Brain sections further validated the survival of grafted GRPs and showed more GRPs surrounding the injured tissue. Furthermore, the brains of post-TBI shiverer mice had increased activation of microglia and astrocytes compared to post-TBI wildtype mice, but infiltration of CD45+ leukocytes remained low. CONCLUSIONS: CoB induces sustained immunological tolerance towards allografted cerebral GRPs which is not disrupted following TBI, and unexpectedly TBI may enhance GRPs engraftment and contribute to post-injury brain tissue repair.

Myeloid-derived suppressor cells expand after transplantation and their augmentation increases graft survival.

Myeloid-derived suppressor cells (MDSCs) expand in an inflammatory microenvironment such as cancer and autoimmunity. To study if transplantation induces MDSCs and these cells regulate allograft survival, C57BL/6 donor hearts were transplanted into BALB/c recipients and endogenous MDSCs were characterized. The effects of adoptive transfer of transplant (tx), tumor (tm), and granulocyte-colony stimulating factor (g-csf)-expanded MDSCs or depletion of MDSC were assessed. MDSCs expanded after transplantation (1.7-4.6-fold) in the absence of immunosuppression, homed to allografts, and suppressed proliferation of CD4 T cells in vitro. Tx-MDSCs differed phenotypically from tm-MDSCs and g-csf-MDSCs. Among various surface markers, Rae-1 expression was notably low and TGF-ß receptor II was high in tx-MDSCs when compared to tm-MDSCs and g-csf-MDSCs. Adoptive transfer of these three MDSCs led to differential graft survival: control (6 days), tx-MDSCs (7.5 days), tm-MDSCs (9.5 days), and g-csf-MDSCs (19.5 days). In combination with anti-CD154 mAb, MDSCs synergistically extended graft survival from 40 days (anti-CD154 alone) to 86 days with tm-MDSCs and 132 days with g-csf-MDSCs. Early MDSC depletion (day 0 or 20), however, abrogated graft survival, but late depletion (day 25) did not. In conclusion, MDSCs expanded following transplantation, migrated to cardiac allografts, prolonged graft survival, and were synergistic with anti-CD154 mAb.

Induction of immunological tolerance to myelinogenic glial-restricted progenitor allografts.

The immunological barrier currently precludes the clinical utilization of allogeneic stem cells. Although glial-restricted progenitors have become attractive candidates to treat a wide variety of neurological diseases, their survival in immunocompetent recipients is limited. In this study, we adopted a short-term, systemically applicable co-stimulation blockade-based strategy using CTLA4-Ig and anti-CD154 antibodies to modulate T-cell activation in the context of allogeneic glial-restricted progenitor transplantation. We found that co-stimulation blockade successfully prevented rejection of allogeneic glial-restricted progenitors from immunocompetent mouse brains. The long-term engrafted glial-restricted progenitors myelinated dysmyelinated adult mouse brains within one month. Furthermore, we identified a set of plasma miRNAs whose levels specifically correlated to the dynamic changes of immunoreactivity and as such could serve as biomarkers for graft rejection or tolerance. We put forward a successful strategy to induce alloantigen-specific hyporesponsiveness towards stem cells in the CNS, which will foster effective therapeutic application of allogeneic stem cells.

Impact of infection on transplantation tolerance.

Allograft tolerance is the ultimate goal of organ transplantation. Current strategies for tolerance induction mainly focus on inhibiting alloreactive T cells while promoting regulatory immune cells. Pathogenic infections may have direct impact on both effector and regulatory cell populations, therefore can alter host susceptibility to transplantation tolerance induction as well as impair the quality and stability of tolerance once induced. In this review, we will discuss existing data demonstrating the effect of infections on transplantation tolerance, with particular emphasis on the role of the stage of infection (acute, chronic, or latent) and the stage of tolerance (induction or maintenance) in this infection-tolerance interaction. While the deleterious effect of acute infection on tolerance is mainly driven by proinflammatory cytokines induced shortly after the infection, chronic infection may generate exhausted T cells that could in fact facilitate transplantation tolerance. In addition to pathogenic infections, commensal intestinal microbiota also has numerous significant immunomodulatory effects that can shape the host alloimmunity following transplantation. A comprehensive understanding of these mechanisms is crucial for the development of therapeutic strategies for robustly inducing and stably maintaining transplantation tolerance while preserving host anti-pathogen immunity in clinically relevant scenarios.

Donor-Derived Regulatory Dendritic Cell Infusion Maintains Donor-Reactive CD4+CTLA4hi T Cells in Non-Human Primate Renal Allograft Recipients Treated with CD28 Co-Stimulation Blockade.

Donor-derived regulatory dendritic cell (DCreg) infusion before transplantation, significantly prolongs renal allograft survival in non-human primates. This is associated with enhanced expression of the immunoregulatory molecules cytotoxic T-lymphocyte-associated antigen (Ag) 4 (CTLA4) and programmed cell death protein 1 (PD1) by host donor-reactive T cells. In rodents and humans, CD28 co-stimulatory pathway blockade with the fusion protein CTLA4:Ig (CTLA4Ig) is associated with reduced differentiation and development of regulatory T cells (Treg). We hypothesized that upregulation of CTLA4 by donor-reactive CD4+ T cells in DCreg-infused recipients treated with CTLA4Ig, might be associated with higher incidences of donor-reactive CD4+ T cells with a Treg phenotype. In normal rhesus monkeys, allo-stimulated CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells exhibited a regulatory phenotype, irrespective of PD1 expression. CTLA4Ig significantly reduced the incidence of CD4+CTLA4hi, but not CD4+CTLA4med/lo T cells following allo-stimulation, associated with a significant reduction in the CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio. In CTLA4Ig-treated renal allograft recipient monkeys, there was a marked reduction in circulating donor-reactive CD4+CTLA4hi T cells. In contrast, in CTLA4Ig-treated monkeys with DCreg infusion, no such reduction was observed. In parallel, the donor-reactive CD4+CTLA4hi/CD4+CTLA4med/lo T cell ratio was reduced significantly in graft recipients without DCreg infusion, but increased in those given DCreg. These observations suggest that pre-transplant DCreg infusion promotes and maintains donor-reactive CD4+CTLA4hi T cells with a regulatory phenotype after transplantation, even in the presence of CD28 co-stimulation blockade.

Treatment with Biologicals in Rheumatoid Arthritis: An Overview.

Management and therapy of rheumatoid arthritis (RA) has been revolutionized by the development and approval of the first biological disease-modifying antirheumatic drugs (bDMARDs) targeting tumor necrosis factor (TNF) α at the end of the last century. Today, numerous efficacious agents with different modes of action are available and achievement of clinical remission or, at least, low disease activity is the target of therapy. Early therapeutic interventions aiming at a defined goal of therapy (treat to target) are supposed to halt inflammation, improving symptoms and signs, and preserving structural integrity of the joints in RA. Up to now, bDMARDs approved for therapy in RA include agents with five different modes of action: TNF inhibition, T cell co-stimulation blockade, IL-6 receptor inhibition, B cell depletion, and interleukin 1 inhibition. Furthermore, targeted synthetic DMARDs (tsDMARDs) inhibiting Janus kinase (JAK) and biosimilars also are approved for RA. The present review focuses on bDMARDs and tsDMARDS regarding similarities and possible drug-specific advantages in the treatment of RA. Furthermore, compounds not yet approved in RA and biosimilars are discussed. Following the American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) recommendations, specific treatment of the disease will be discussed with respect to safety and efficacy. In particular, we discuss the question of favoring specific bDMARDs or tsDMARDs in the two settings of insufficient response to methotrexate and to the first bDMARD, respectively.

Anti-Interleukin-6 Promotes Allogeneic Bone Marrow Engraftment and Prolonged Graft Survival in an Irradiation-Free Murine Transplant Model.

Transfer of recipient regulatory T cells (Tregs) induces mixed chimerism and tolerance in an irradiation-free bone marrow (BM) transplantation (BMT) model involving short-course co-stimulation blockade and mTOR inhibition. Boosting endogenous Tregs pharmacologically in vivo would be an attractive alternative avoiding the current limitations of performing adoptive cell therapy in the routine clinical setting. Interleukin-6 (IL-6) potently inhibits Treg differentiation and its blockade was shown to increase Treg numbers in vivo. Therefore, we investigated whether IL-6 blockade can replace adoptive Treg transfer in irradiation-free allogeneic BMT. Treatment with anti-IL-6 instead of Treg transfer led to multi-lineage chimerism (persisting for ~12 weeks) in recipients of fully mismatched BM and significantly prolonged donor skin (MST 58 days) and heart (MST > 100 days) graft survival. Endogenous Foxp3+ Tregs expanded in anti-IL-6-treated BMT recipients, while dendritic cell (DC) activation and memory CD8+ T cell development were inhibited. Adding anti-IL-17 to anti-IL-6 treatment increased Treg frequencies, but did not further prolong donor skin graft survival significantly. These results demonstrate that IL-6 blockade promotes BM engraftment and donor graft survival in non-irradiated recipients and might provide an alternative to Treg cell therapy in the clinical setting.

Advances in targeting co-inhibitory and co-stimulatory pathways in transplantation settings: the Yin to the Yang of cancer immunotherapy.

In the past decade, the power of harnessing T-cell co-signaling pathways has become increasingly understood to have significant clinical importance. In cancer immunotherapy, the field has concentrated on two related modalities: First, targeting cancer antigens through highly activated chimeric antigen T cells (CAR-Ts) and second, re-animating endogenous quiescent T cells through checkpoint blockade. In each of these strategies, the therapeutic goal is to re-ignite T-cell immunity, in order to eradicate tumors. In transplantation, there is also great interest in targeting T-cell co-signaling, but with the opposite goal: in this field, we seek the Yin to cancer immunotherapy's Yang, and focus on manipulating T-cell co-signaling to induce tolerance rather than activation. In this review, we discuss the major T-cell signaling pathways that are being investigated for tolerance induction, detailing preclinical studies and the path to the clinic for many of these molecules. These include blockade of co-stimulation pathways and agonism of coinhibitory pathways, in order to achieve the delicate state of balance that is transplant tolerance: a state which guarantees lifelong transplant acceptance without ongoing immunosuppression, and with preservation of protective immune responses. In the context of the clinical translation of immune tolerance strategies, we discuss the significant challenge that is embodied by the fact that targeted pathway modulators may have opposing effects on tolerance based on their impact on effector vs regulatory T-cell biology. Achieving this delicate balance holds the key to the major challenge of transplantation: lifelong control of alloreactivity while maintaining an otherwise intact immune system.

Molecular and Cellular Characterization of Human CD8 T Suppressor Cells.

Bidirectional interactions between dendritic cells and Ag-experienced T cells initiate either a tolerogenic or immunogenic pathway. The outcome of these interactions is of crucial importance in malignancy, transplantation, and autoimmune diseases. Blockade of costimulation results in the induction of T helper cell anergy and subsequent differentiation of antigen-specific CD8+ T suppressor/regulatory cells (Ts). Ts, primed in the presence of inhibitory signals, exert their inhibitory function in an antigen-specific manner, a feature with tremendous clinical potential. In transplantation or autoimmunity, antigen-specific Ts can enforce tolerance to auto- or allo-antigens, while otherwise leaving the immune response to pathogens uninhibited. Alternatively, blockade of inhibitory receptors results in the generation of cytolytic CD8+ T cells, which is vital toward defense against tumors and viral diseases. Because CD8+ T cells are MHC Class I restricted, they are able to recognize HLA-bound antigenic peptides presented not only by APC but also on parenchymal cells, thus eliciting or suppressing auto- or allo-immune reactions.

Should belatacept be the centrepiece of renal transplantation?

Belatacept was developed to minimize cardiovascular risk and nephrotoxicity associated with calcineurin inhibitor (CNI)-based immunosuppression. Recently, 7-year data from the Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial (BENEFIT), a phase III study comparing belatacept with cyclosporine, have been published. While during the first year of belatacept the risk of acute rejection episodes was elevated, this seemingly had marginal consequences for long-term graft survival and function as well as patient survival.For patients at a low-immunological risk, this drug seems to be a safe and effective alternative to CNI-based immunosuppression. Whether the higher rates of acute rejection episodes in the first year outweigh the gain in long-term graft function is still debated. In particular, the lower incidence of donor-specific antibodies indicates that belatacept should not be considered as lower intensity immunosuppression over the long term.Therefore, should belatacept be the centrepiece of immunosuppression for renal patients?All randomized trials so far have focussed on patients at a low immunological risk. Furthermore, cyclosporine A (CsA), the comparator of belatacept in BENEFIT and the Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial-EXTended criteria donors (BENEFIT-EXT), is not the CNI of choice in modern transplantation. Furthermore, while at Year 7 the rate of cancer and infections was comparable with the CsA group, long-term data are missing on safety issues for a large number of patients. Thus, currently belatacept may be the drug of choice for a select group of patients, but not for everyone.This review highlights the benefits and uncertainties of the use of belatacept in kidney transplantation.

Clinical efficacy of a new CD28-targeting antagonist of T cell co-stimulation in a non-human primate model of collagen-induced arthritis.

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co-stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen-induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA-4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C-reactive protein (CRP) and interleukin (IL)-6 and anti-collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII-induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.

B7-H4 as a protective shield for pancreatic islet beta cells.

Auto- and alloreactive T cells are major culprits that damage ß-cells in type 1 diabetes (T1D) and islet transplantation. Current immunosuppressive drugs can alleviate immune-mediated attacks on islets. T cell co-stimulation blockade has shown great promise in autoimmunity and transplantation as it solely targets activated T cells, and therefore avoids toxicity of current immunosuppressive drugs. An attractive approach is offered by the newly-identified negative T cell co-signaling molecule B7-H4 which is expressed in normal human islets, and its expression co-localizes with insulin. A concomitant decrease in B7-H4/insulin co-localization is observed in human type 1 diabetic islets. B7-H4 may play protective roles in the pancreatic islets, preserving their function and survival. In this review we outline the protective effect of B7-H4 in the contexts of T1D, islet cell transplantation, and potentially type 2 diabetes. Current evidence offers encouraging data regarding the role of B7-H4 in reversal of autoimmune diabetes and donor-specific islet allograft tolerance. Additionally, unique expression of B7-H4 may serve as a potential biomarker for the development of T1D. Future studies should continue to focus on the islet-specific effects of B7-H4 with emphasis on mechanistic pathways in order to promote B7-H4 as a potential therapy and cure for T1D.

ASKP1240, a fully human anti-CD40 monoclonal antibody, prolongs pancreatic islet allograft survival in nonhuman primates.

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti-CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti-CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201-12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post-PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti-donor cellular responses and development of anti-donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.