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Effective immune responses require antigen presentation by major histocompatibility complexes with cognate T-cell receptor and antigen-independent costimulatory signaling for T-cell activation, proliferation, and differentiation. Among several costimulatory signals, CD40-CD40L is of special interest to the transplantation community because it plays a vital role in controlling or regulating humoral and cellular immunity. Blockade of this pathway has demonstrated inhibition of donor-reactive T-cell responses and prolonged the survival of transplanted organs. Several anti-CD154 and anti-CD40 antibodies have been used in the transplantation model and demonstrated the potential of extending allograft and xenograft rejection-free survival. The wide use of anti-CD154 antibodies was hampered because of thromboembolic complications in transplant recipients. These antibodies have been modified to overcome the thromboembolic complications by altering the antibody binding fragment (Fab) and Fc (fragment, crystallizable) receptor region for therapeutic purposes. Here, we review recent preclinical advances to target the CD40-CD40L pair in transplantation.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with six gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (three gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted in the baboons. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the requirement of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective "multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

There are 5.7 million people in the United States with heart failure, which is life-limiting in 20% of patients.1 While data is most robust in the United States for this cohort, it is known to be a global problem with over 23 million people carrying the diagnosis.1 For end-stage heart failure, many require a heart transplantation, however, there is a shortage in the supply of organ donors. Cardiac xenotransplantation has been proposed to “bridge the gap” in supply for these patients requiring transplantation. Recent pre-clinical success using genetically modified pig donors in baboon recipients has demonstrated survival greater than 6 months.2–5 First-in-human transplantation of a genetically modified pig kidney demonstrated 54 hour rejection-free function when perfused by a deceased human recipient, demonstrating the feasibility of cross-species transplantation and invigorating enthusiasm further to utilize this new organ source for a population that would otherwise die waiting for a human organ.6 While this human study demonstrated proof-of-principle of overcoming hyperacute rejection, further regulatory oversight by Food and Drug Administration (FDA) may be required with pre-clinical trials in large animal models of xenotransplantation with long-term survival. These studies not only require a multi-disciplinary team and expertise in orthotopic transplantation (cardiac surgery, anesthesia and cardiopulmonary bypass), immunology and genetic engineering; but also, specifically handling large animal recipients that cannot communicate their symptoms. Here we detail our approach to pig-to-primate large animal model of orthotopic cardiac xenotransplantation perioperatively and in the months thereafter in long-term surviving animals. We also detail xenograft surveillance methods and common issues that arise in the postoperative period specific to this model and ways to overcome them.

We report orthotopic (life-supporting) survival of genetically engineered porcine cardiac xenografts (with 3-9 progressive gene modifications) for almost 9 months in baboon recipients. This work builds on our previously reported heterotopic cardiac xenograft (3 gene modifications) survival up to 945 days with an anti-CD40 monoclonal antibody-based immunosuppression. In this current study, life-supporting xenografts containing multiple human complement regulatory, thromboregulatory, and anti-inflammatory proteins, in addition to growth hormone receptor knockout (KO) and carbohydrate antigen KOs, were transplanted. Selective "multi-gene" xenografts demonstrate survival greater than 8 months without the use of adjunctive medications and without evidence of abnormal xenograft thickness or rejection. These data demonstrate that selective “multi-gene" modifications improve cardiac xenograft survival significantly and may be foundational for paving the way to bridge transplantation in humans.

Heterotopic cardiac transplantation in the intra-abdominal position in a large animal model has been essential in the progression of the field of cardiac transplantation. Our group has over 10 years of experience in cardiac xenotransplantation with pig to baboon models, the longest xenograft of which survived over 900 days, with rejection only after reducing immunosuppression. This article aims to clarify our approach to this model in order to allow others to share success in long-term survival. Here, we demonstrate the approach to implantation of a cardiac graft into the intra-abdominal position in a baboon recipient for the study of transplantation and briefly highlight our model’s ability to provide insight into not only xenotransplantation but across disciplines. We include details that have provided us with consistent success in this model; performance of the anastomoses, de-airing of the graft, implantation of a long-term telemetry device for invasive graft monitoring, and ideal geometric positioning of the heart and telemetry device in the limited space of the recipient abdomen. We additionally detail surveillance techniques to assess long-term graft function.

Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway.Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized αCD154, n = 9), 5C8H1 (mouse-human chimeric αCD154, n = 5), or 2C10R4 (mouse-rhesus chimeric αCD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant.Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P0.002) and 2C10R4-treated (124 ± 37, P0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20 lymphocytes from baseline at day 14 after transplant (-457 ± 152 cells/μL) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype.In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.

Anti-CD154 monotherapy is associated with antidonor allo-antibody (Ab) elaboration, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. In the context of calcineurin inhibitors (CNI), these pathogenic phenomena are delayed by preemptive "induction" B cell depletion.αCD154 (IDEC-131)-treated cynomolgus monkey heart allograft recipients were given peritransplant rituximab (αCD20) alone or with rabbit antihuman thymocyte globulin.Relative to previously reported reference groups, αCD20 significantly prolonged survival, delayed Ab detection, and attenuated CAV within 3 months in αCD154-treated recipients (αCD154 + αCD20 graft median survival time90 days, n = 7, vs 28 days for αCD154 alone (IDEC-131), n = 21; P = 0.05). Addition of rabbit antihuman thymocyte globulin to αCD154 (n = 6) or αCD154 + αCD20 (n = 10) improved graft protection from graft rejection and failure during treatment but was associated with significant morbidity in 8 of 16 recipients (6 infections, 2 drug-related complications). In αCD20-treated animals, detection of antidonor Ab and relatively severe CAV were anticipated by appearance of CD20 cells (1% of lymphocytes) in peripheral blood and were associated with low αCD154 trough levels (below 100 μg/mL).These observations support the hypothesis that efficient preemptive "induction" CD20 B cell depletion consistently modulates pathogenic alloimmunity and attenuates CAV in this translational model, extending our prior findings with calcineurin inhibitors to the context of CD154 blockade.

Purpose Transplantation of organs from animals to humans (xenotransplantation) has been proposed to address the critical shortage of organs for transplantation. The use of genetically engineered organ xenografts, including knock-out (KO) of alpha 1-3 galactosyltransferase (GTKO) along with insertion of a human complement regulator genes (hCD46, hDAF) and other cell surface carbohydrate KOs, has been helpful in circumventing acute and subacute rejection. However, thrombotic complications still remain. Xenografts with additional human transgenes, including hTBM, have been generated to address this barrier. Here, we studied the importance of hTBM in preventing coagulation by comparing two transplanted groups utilizing GE donors with or without hTBM. Methods Specific pathogen-free baboons of either sex weighing 15-30 kg (2-3 years of age) were used as recipients. 6 to 8-week-old genetically modified pigs with hTBM (GTKO.hCD46.hTBM) or without hTBM (triple knock out-GTKO.B4KO.CMAHKO, with or without hCD46 and hDAF) were used as donors, provided by Revivicor. Expression of transgenes were consistent and high level across all pigs. All animals were used in compliance with guidelines provided by the Institutional Animal Care and Use Committee (IACUC). Results GTKO.hCD46.hTBM donors (n=3) were found to have no intracardiac thrombus with survival up to 30 days post-transplant. However, all non-hTBM donors (n=3) were found to have a large intracardiac thrombus burden with propagation into major vascular structures such as the aorta, pulmonary artery and coronary sinus. Gross examination of thrombi indicated acute and subacute components, suggesting early formation of the thrombus postoperatively. Additionally, early clinical signs of thrombus formation included profound hypocalcemia requiring continuous calcium infusions in the first 48-72 hours postoperatively. These phenomena were not prevented by continuous heparin infusion. Histologic examination revealed no signs of rejection. Conclusion hTBM prevents intracardiac thrombus in this model. While immunologic rejection has been circumvented with novel immunosuppression regimens and GTKO, it is likely that hTBM or other transgenes involved in regulation of coagulation may be needed for prevention of thrombotic complications.

Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

Supplemental digital content is available in the text., Background Inducible costimulator (ICOS) is rapidly upregulated with T-cell stimulation and may represent an escape pathway for T-cell costimulation in the setting of CD40/CD154 costimulation blockade. Induction treatment exhibited no efficacy in a primate renal allograft model, but rodent transplant models suggest that the addition of delayed ICOS/ICOS-L blockade may prolong allograft survival and prevent chronic rejection. Here, we ask whether ICOS-Ig treatment, timed to anticipate ICOS upregulation, prolongs NHP cardiac allograft survival or attenuates pathogenic alloimmunity. Methods Cynomolgus monkey heterotopic cardiac allograft recipients were treated with αCD40 (2C10R4, d0-90) either alone or with the addition of delayed ICOS-Ig (d63-110). Results Median allograft survival was similar between ICOS-Ig + αCD40 (120 days, 120-125 days) and αCD40 (124 days, 89-178 days) treated animals, and delayed ICOS-Ig treatment did not prevent allograft rejection in animals with complete CD40 receptor coverage. Although CD4+ TEM cells were decreased in peripheral blood (115 ± 24) and mLNs (49 ± 1.9%) during ICOS-Ig treatment compared with monotherapy (214 ± 27%, P = 0.01; 72 ± 9.9%, P = 0.01, respectively), acute and chronic rejection scores and kinetics of alloAb elaboration were similar between groups. Conclusions Delayed ICOS-Ig treatment with the reagent tested is probably ineffective in modulating pathogenic primate alloimmunity in this model.

Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.

© 2016 the American Physiological Society. Airway remodeling is a hallmark feature of asthma and chronic obstructive pulmonary disease. Clinical studies and animal models have demonstrated increased airway smooth muscle (ASM) mass, and ASM thickness is correlated with severity of the disease. Current medications control inflammation and reverse airway obstruction effectively but have limited effect on remodeling. Recently we identified the expression of bitter taste receptors (TAS2R) on ASM cells, and activation with known TAS2R agonists resulted in ASM relaxation and bronchodilation. These studies suggest that TAS2R can be used as new therapeutic targets in the treatment of obstructive lung diseases. To further establish their effectiveness, in this study we aimed to determine the effects of TAS2R agonists on ASM growth and promitogenic signaling. Pretreatment of healthy and asthmatic human ASM cells with TAS2R agonists resulted in a dose-dependent inhibition of ASM proliferation. The antimitogenic effect of TAS2R ligands was not dependent on activation of protein kinase A, protein kinase C, or high/intermediate-conductance calcium-activated K+ channels. Immunoblot analyses revealed that TAS2R agonists inhibit growth factor-activated protein kinase B phosphorylation without affecting the availability of phosphatidylinositol 3,4,5-trisphosphate, suggesting TAS2R agonists block signaling downstream of phosphatidylinositol 3-kinase. Furthermore, the antimitogenic effect of TAS2R agonists involved inhibition of induced transcription factors (activator protein-1, signal transducer and activator of transcription-3, E2 factor, nuclear factor of activated T cells) and inhibition of expression of multiple cell cycle regulatory genes, suggesting a direct inhibition of cell cycle progression. Collectively, these findings establish the antimitogenic effect of TAS2R agonists and identify a novel class of receptors and signaling pathways that can be targeted to reduce or prevent airway remodeling as well as bronchoconstriction in obstructive airway disease.