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Objectives Loss of vaccine‐induced antibodies (Abs) after chemotherapy against paediatric acute lymphoblastic leukaemia (ALL) is common and often necessitates re‐immunisation after cessation of treatment. Even so, some ALL survivors fail to mount or to maintain protective Abs. Germinal centres (GCs) are clusters of proliferating B cells in follicles of secondary lymphoid tissues (SLTs) formed during adaptive immune responses and the origins of long‐lived memory B and plasma cells that are the source of Abs. Furthermore, productive GC reactions depend on T follicular helper (TFH) cells. To understand why chemotherapy induces deficits in Ab responses, we examined how SLTs were affected by chemotherapy. Methods Rhesus macaques were infused with either three cycles of the anthracycline doxorubicin or saline, followed by immunisation with a de novo and booster antigen. Spleen and lymph nodes were removed, and memory B, bulk T and TFH cells were examined. Results Despite adequate GC morphology, a diminished memory and IgG+ B‐cell population along with diminished total and booster vaccine‐specific IgG‐producing memory B cells were noted in the spleens of macaques with past doxorubicin exposure compared to the saline‐treated controls (P, In this study, we found that the recovery of IgG‐producing memory B cells in the spleen lags behind after cessation of anthracycline treatment, a widely used drug in acute lymphoblastic leukaemia protocols. This finding was accompanied by impaired splenic B‐cell response to booster antigen, raising the possibility of residual damage to serological memory after treatment of blood cancers.

Tuberculosis (TB) still is the principal cause of death from infectious disease and improved vaccination strategies are required to reduce the disease burden and break TB transmission. Here, we investigated different routes of administration of vectored subunit vaccines based on chimpanzee-derived adenovirus serotype-3 (ChAd3) for homologous prime-boosting and modified vaccinia virus Ankara (MVA) for heterologous boosting with both vaccine vectors expressing the same antigens from Mycobacterium tuberculosis (Ag85B, ESAT6, Rv2626, Rv1733, RpfD). Prime-boost strategies were evaluated for immunogenicity and protective efficacy in highly susceptible rhesus macaques. A fully parenteral administration regimen was compared to exclusive respiratory mucosal administration, while parenteral ChAd3-5Ag prime-boosting and mucosal MVA-5Ag boosting were applied as a push-and-pull strategy from the periphery to the lung. Immune analyses corroborated compartmentalized responses induced by parenteral versus mucosal vaccination. Despite eliciting TB-specific immune responses, none of the investigational regimes conferred a protective effect by standard readouts of TB compared to non-vaccinated controls, while lack of protection by BCG underpinned the stringency of this non-human primate test modality. Yet, TB manifestation after full parenteral vaccination was significantly less compared to exclusive mucosal vaccination.

While tuberculosis continues to afflict mankind, the immunological mechanisms underlying TB disease development are still incompletely understood. Advanced preclinical models for TB research include both rhesus and cynomolgus macaques (Macaca mulatta and Macaca fascicularis, respectively), with rhesus typically being more susceptible to acute progressive TB disease than cynomolgus macaques. To determine which immune mechanisms are responsible for this dissimilar disease development, we profiled a broad range of innate and adaptive responses, both local and peripheral, following experimental pulmonary Mycobacterium tuberculosis (Mtb) infection of both species. While T-cell and antibody responses appeared indistinguishable, we identified anti-inflammatory skewing of peripheral monocytes in rhesus and a more prominent local pro-inflammatory cytokine release profile in cynomolgus macaques associated with divergent TB disease outcome. Importantly, these differences were detectable both before and early after infection. This work shows that inflammatory and innate immune status prior to and at early stages after infection, critically affects outcome of TB infection.

Summary BCG vaccination can strengthen protection against pathogens through the induction of epigenetic and metabolic reprogramming of innate immune cells, a process called trained immunity. We and others recently demonstrated that mucosal or intravenous BCG better protects rhesus macaques from Mycobacterium tuberculosis infection and TB disease than standard intradermal vaccination, correlating with local adaptive immune signatures. In line with prior mouse data, here, we show in rhesus macaques that intravenous BCG enhances innate cytokine production associated with changes in H3K27 acetylation typical of trained immunity. Alternative delivery of BCG does not alter the cytokine production of unfractionated bronchial lavage cells. However, mucosal but not intradermal vaccination, either with BCG or the M. tuberculosis-derived candidate MTBVAC, enhances innate cytokine production by blood- and bone marrow-derived monocytes associated with metabolic rewiring, typical of trained immunity. These results provide support to strategies for improving TB vaccination and, more broadly, modulating innate immunity via mucosal surfaces., Graphical Abstract, Highlights Nonhuman primates recapitulate trained immunity upon live attenuated TB vaccination Intravenous BCG induces changes in H3K27 acetylation and enhances cytokine production Mucosal BCG improves induction of trained immunity of monocytes over intradermal BCG The M. tuberculosis-derived candidate vaccine MTBVAC appears equally potent as BCG, Vierboom et al. demonstrate the induction of trained immunity in blood and bone marrow monocytes after vaccination with live attenuated TB vaccines in nonhuman primates. Mucosal respiratory delivery of BCG or MTBVAC induces trained immunity more efficiently compared to standard intradermal vaccination.

Kidney allograft biopsies were analyzed for the presence of B cell clusters/aggregates using CD20 staining. Few B cells were found in the diffuse interstitial infiltrates, but clusters of B cells were found in nodular infiltrates. These nodular infiltrates were smaller shortly after transplantation, and their size increased over time. At the time of clinical rejection, the nodules often presented as tertiary lymphoid structures (TLS) with lymphoid-like follicles. The presence of small B cell clusters during the first 2 months after transplantation was not associated with early rejection. Even in animals that did not reject their allograft, TLS-like structures were present and could disappear over time. Although TLS were more often found in samples with interstitial fibrosis and tubular atrophy (IFTA), TLS were also present in samples without IFTA. The presence and density of clusters resembling tertiary lymphoid structures most likely reflect an ongoing immune response inside the graft and do not necessarily signify a poor graft outcome or IFTA.

Costimulatory and coinhibitory receptor–ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28–CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4–CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab′ Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the β-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys.

Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

The experimental autoimmune encephalitis (EAE) model is used for preclinical research into the pathogenesis of multiple sclerosis (MS), mostly in inbred, specific pathogen free (SPF)-raised laboratory mice. However, the naive state of the laboratory mouse immune system is considered a major hurdle in the translation of principles from the EAE model to the MS patient. Non-human primates (NHP) have an immune system harboring T- and B-cell memory against environmental antigens, similar as in humans. We sought to further refine existing NHP EAE models, which may help to bridge the gab between mouse EAE models and MS. We report here on new EAE models in three NHP species: rhesus monkeys (Macaca mulatta), cynomolgus monkeys (Macaca fascicularis) and common marmosets (Callithrix jacchus). EAE was induced with recombinant human myelin oligodendrocyte glycoprotein extracellular domain (1-125) (rhMOG) formulated in incomplete Freund's adjuvant (IFA). IFA lacks the bacterial antigens that are present in complete Freund's adjuvant (CFA), which are notorious for the induction of discomforting side effects. Clinically evident EAE could be induced in two out of five rhesus monkeys, six out of six cynomolgus monkeys and six out of six common marmosets. In each of these species, the presence of an early, high anti-rhMOG IgM response is correlated with EAE with an earlier onset and more severe disease course. Animals without an early high IgM response either did not develop disease (rhesus monkeys) or developed only mild signs of neurological deficit (marmoset and cynomolgus monkeys).

The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the alpha(4) integrins expressed by T lymphocytes. The alpha(4)beta(1) integrin mediates migration of memory T lymphocytes into the CNS, whereas the alpha(4)beta(7) integrin mediates migration preferentially into gastrointestinal tissue. This paradigm was established primarily from investigations in rodents; thus, the objective of this investigation was to determine if blocking the alpha(4)beta(7) integrin exclusively would affect migration of T lymphocytes into the CNS of primates. The effects of the dual alpha(4)beta(1) and alpha(4)beta(7) antagonist natalizumab were compared with those of the alpha(4)beta(7) antagonist vedolizumab on experimental autoimmune encephalomyelitis in the rhesus monkey. Animals received an initial i.v. bolus of placebo, natalizumab (30 mg/kg), or vedolizumab (30 mg/kg) before intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein and then Ab once weekly thereafter. Natalizumab prevented CNS inflammation and demyelination significantly (p

Background. Translational research in a relevant preclinical model is recommended before Treg-inducing protocols can be implemented in humans. We have characterized rhesus monkey CD25(+) cells phenotypically and functionally. Methods. The phenotype of CD4(+)CD25(high) cells was determined by FACS, focusing on established markers of mouse and human Treg cells. Percentages of cells positive for CD45RA, CD62L, and intracellular CTLA-4 and FOXP3 were compared between CD4(+)CD25(high) and CD4(+)CD25(-) cells. CD25(-) cells stimulated with anti-CD3, ConA, and/or allogeneic peripheral blood mononuclear cells were mixed with freshly isolated CD25(+) cells. The suppressive activity of the CD25(+) cells in vitro was assessed using several experimental conditions. Results. Rhesus monkey CD4(+)CD25(high) cells expressed high intracellular levels of CTLA-4 and FOXP3, whereas expression was negligible in CD4(+)CD25(-) cells. The CD25(high) population was mostly CD45RA(-), indicative of a memory phenotype. The CD25(+) cells were anergic, because they showed low proliferative responses, no interleukin-2 production, and some interferon-gamma and interleukin-10 production. Proliferation of CD4(+)CD25(-) cells stimulated by anti-CD3 or allogeneic cells was decreased when CD4(+)CD25(+) cells were added at a 1:1 ratio. In addition, we found that CD25(+) cells inhibited the interleukin-2 and interferon-gamma production by anti-CD3 -stimulated CD25(-) cells in a dose-dependent fashion, through a cell-cell contact-dependent mechanism. Conclusions. Rhesus monkey CD4(+)CD25(+) cells have similar phenotypic and functional characteristics as natural Tregs in humans. These findings allow testing of Treg expansion and induction protocols in a relevant preclinical model, the rhesus monkey.

The immune system plays a central role in the defense against environmental threats - such as infection with viruses, parasites or bacteria - but can also be a cause of disease, such as in the case of allergic or autoimmune disorders. In the past decades the impressive development of biotechnology has provided scientists with biological tools for the development of highly selective treatments for the different types of disorders. However, despite some clear successes the translation of scientific discoveries into effective treatments has remained challenging. The often-disappointing predictive validity of the preclinical animal models that are used in the selection of the most promising vaccine or drug candidates is the Achilles heel in the therapy development process. This publication summarizes the relevance and usage of non-human primates as pre-clinical model in infectious and autoimmune diseases, in particular for biologicals, which due to their high species-specificity are inactive in lower species. (C) 2015 Elsevier B.V. All rights reserved.

Background. Chronic allograft nephropathy (CAN) is a common late complication of kidney transplantation. Antibodies to both human leukocyte antigen and nonhuman leukocyte antigen antigens have been implicated in the development of this condition. Here we investigated the presence of antivimentin antibodies in nonhuman primate recipients of kidney allografts as a possible predictor of CAN and the effects of immunosuppression. Methods. Thirty seven rhesus monkeys received a kidney allograft to study the potency of several different immunosuppressive regimens (conventional immunosuppression, n=19, vs. costimulatory blockade, n=18). Monkeys were tested for antivimentin antibody by enzyme-linked immunosorbent assay and for anti-donor antibody by staining donor spleen cells with recipient serum. The appearance of antibodies was correlated with the graft pathology in biopsy and necropsy material. Results. Antivimentin antibodies were found in 31 of 37 animals, whereas only 15 of 32 animals made anti-donor antibodies. Conventional immunosuppression did not prevent antivimentin antibody formation. Costimulation blockade, in particular blocking CD40 and CD86, significantly delayed or prevented antivimentin antibody formation, but did not prevent CAN. Antivimentin antibodies were not significantly associated with development of CAN. Conclusions. We postulate that vimentin acts as an autoantigen after renal transplantation; it elicits an autoimmune response that is not regulated by cyclosporine. This autoimmune response may be part of the complex immunologic events occurring posttransplantation and may contribute to the development of CAN, but cannot be considered as a major cause of CAN because this condition also develops without antivimentin antibodies.

Costimulation blockade as a single immunosuppressive treatment modality is not sufficient to prevent graft rejection. Here, we report an induction therapy using antagonistic antibodies against CD40 and CD86, given twice weekly from day -1 until day 56, followed by a delayed 12-week course of low-dose cyclosporine A (CsA) treatment in the rhesus monkey kidney-allograft model. Low-dose CsA treatment was initiated on day 42 and tapered until total cessation of all treatment on day 126. Treatment with anti-CD40/86 alone resulted in graft survival of 61, 71, 75, 78, and 116 days. Costimulation blockade followed by CsA resulted in more than 3-year drug-free survival in two of four animals. None of the animals developed donor-specific alloantibodies. Transforming growth factor-beta producing cells are present in early as well as in late kidney-graft biopsies and could play a role in the observed long-term drug-free graft survival.

Objective The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys. Materials and methods Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 μg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Results Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, p ≤0.02), 6- (4358 vs 748, p ≤0.02), and 5-fold (884 vs 168, p ≤0.04), respectively. We then evaluated the effects of a 2-day defibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, p ≤0.05), 8- (5361 vs 660, p ≤0.01), and 8-fold (954 vs 119, p ≤0.01), respectively. Conclusions Our data demonstrate that in nonhuman primates: 1) defibrotide enhances rhG-CSF–elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

Background Transplantation of islets of Langerhans can restore insulin production in diabetic patients. Because of the shortage of human donor organs, transplantation of porcine islets may be an alternative solution. The present study was aimed at the characterization of rejection mechanisms of porcine islets transplanted into eight nondiabetic monkeys under the kidney capsule. Methods Cultured adult pig islets were used, which showed no expression of the galactose(alpha1,3)galactose epitope, major histocompatibility complex class II, or CD45, and no binding of antibodies or complement after exposure to monkey serum. Immunosuppression consisted of cyclophosphamide, cyclosporine A (CsA), and steroids (group 1); or antithymocyte globulin, anti-interleukin-2 receptor antibody, CsA, and steroids (group 2). In three animals of group 2, islets were also transplanted in the portal vein. Results Although all monkeys had preformed anti-pig antibodies, no correlation was found between antibody titers and rejection and no deposition of antibodies or complement was observed in the grafts. Group 1 showed islets up to day 11, followed by T-cell infiltration and rejection at approximately day 14. In group 2, two monkeys showed infiltrates consisting predominantly of T cells starting at approximately day 29, whereas two monkeys showed well-preserved islets without infiltration up to day 53. In the livers of the three monkeys that also received islets intraportally and were resectioned on days 21, 33, and 49, no islets could be detected. Conclusions This study demonstrates that cultured adult pig islets can survive in the monkey for more than 53 days without signs of rejection under standard immunosuppression.

Background. Costimulation blockade has been proposed to induce allograft tolerance. We combined an antagonist anti-CD40 monoclonal antibody (mAb) with an antagonist anti-CD86 mAb in a rhesus monkey kidney allograft model. We chose this combination because it leaves CD80-CD152 signaling unimpaired, allowing for the down-regulatory effect of CD152 signaling to take place through this pathway. Methods. Rhesus monkeys underwent transplantation with a major histocompatibility complex-mismatched kidney. One group of animals received anti-CD40 alone, and a second group received the combination of anti-CD40 and anti-CD86, twice weekly for 56 days. Results. Three animals with low levels of anti-CD40 rejected the transplanted kidney while still receiving treatment. Three animals with high levels of anti-CD40 rejected at days 91, 134, and 217 with signs of chronic rejection. Animals treated with the combination of anti-CD40 and anti-CD86 mAbs rejected their kidneys at days 61, 75, and 78, shortly after cessation of treatment. Two animals were killed on days 71 and 116 with a blocked ureter. These animals developed virtually no signs of tubulitis or infiltration during treatment and no donor-specific alloantibodies. Conclusions. Both treatment protocols prevented rejection for the duration of the treatment in most animals. Blocking costimulation by anti-CD40 or by anti-CD40 plus anti-CD86 may be an effective method to prevent graft rejection and may obviate the need for other immunosuppressive drugs, especially in the immediate posttransplantation period.

Background Tolerance to organ allografts in primates including man has been elusive, although in rodents and pigs tolerance can be achieved to organ allografts with relatively short courses of immunosuppressive treatment. In all varieties of graft acceptance that do not require full-dose maintenance immunosuppression, immunological engagement of donor and recipient and an early unstable period have been observed. On the basis of the hypothesis that elimination of aggressive T cell function should tip the balance in favor of an operationally tolerant state, experiments have been performed in monkeys allowing recipient-donor interaction before T-cell ablation and a short course of immunosuppression. Methods Rhesus monkeys received an allogeneic kidney graft from a MHC-mismatched donor. The animals either received anti-CD3 immunotoxin (FN18-CRM9) alone, started 2 days after transplantation, or in combination with a short course of cyclosporine (CsA) and/or rapamycin (RAPA), started at 5 days after transplantation. Kidney function was followed by monitoring serum creatinine levels and regular biopsies. Humoral and cellular antidonor immunity was tested in vitro before and at several time points after transplantation. Results Graft survival of monkeys that received CsA alone (mean survival time (MST)=29.3) was significantly prolonged compared with the controls (MST=6). FN18-CRM9 treatment alone also resulted in prolonged graft survival (MST=29.4). The combined treatment of FN18-CRM9 and CsA and/or RAPA resulted in prolonged graft survival after all immunosuppression was stopped (MST=207.8). Conclusions It seems feasible to postpone immunosuppression posttransplantation and yet prevent allograft rejection without the need of permanent immunosuppression.

Background. In rodents it has been demonstrated that blockade of the CD40-CD154 (CD40L) pathway at the time of donor-specific blood transfusion (DST) can result in indefinite graft survival. Because it has been reported in the past that DST in monkeys can have a favorable effect on graft outcome and that blockade of the CD40-CD154 pathway can lead to prolonged kidney graft survival in monkeys, we have combined anti-CD154 treatment with DST in a monkey kidney graft model. The aim of this study was to investigate the immunosuppressive potential of blocking the CD40-CD154 interaction at the time of a DST in rhesus monkeys. Methods. One donor-derived blood transfusion was given on day -15 after the first anti-CD 154 injection. The anti-CD154 antibody was given on days -15, -13, -11, -9, and -7. The kidney was transplanted on day 0. Cyclosporine was given after kidney transplantation. Results. No major difference in graft survival was observed between the groups. The animals died due to grade 11 acute rejection. At the time of transplantation, no antibody response could be detected directed against donor antigens. After transplantation, all animals surviving for more than 3 weeks had antidonor antibodies. There were no differences in the intragraft events analyzed by real time reverse transcriptase-polymerase chain reaction. Conclusions. DST under the cover of relatively high levels of anti-CD154 failed to result in prolonged graft survival or prevent the formation of antidonor antibodies, when cyclosporine was given after transplantation.

The remarkable clinical efficacy of anti-CD20 monoclonal antibodies (mAb) in relapsing-remitting multiple sclerosis points at the critical involvement of B cells in the disease. However, the exact pathogenic contribution of B cells is poorly understood. In this publication we review new data on the role of CD20+ B cells in a unique experimental autoimmune encephalomyelitis (EAE) model in common marmosets (Callithrix jacchus), a small-bodied neotropical primate. We will also discuss the relevance of these data for MS. Different from rodent EAE models, but similar to MS, disease progression in marmosets can develop independent of autoantibodies. Progressive disease is mediated by MHC class lb (Caja-E) restricted cytotoxic T cells, which are activated by gamma-herpesvirus-infected B cells and cause widespread demyelination of cortical gray matter. B-cell directed monoclonal antibody therapies (anti-CD20 versus anti-BLyS and anti-APRIL) have a variable effect on EAE progression, which we found associated with variable depletion of the Epstein Barr virus (EBV)-like gamma-herpesvirus CalHV3 from lymphoid organs. These findings support an important pathogenic role of CD20+ B cell in MS, especially of the subset infected with EBV.

The classical role of the immune system is to detect and eliminate foreign organisms, such as microbial infection, while at the same time the system should not respond against the body itself (Mills, Immunology 11:807–822, 2011). This implies that the antigen-sensing and antigen-presenting cells (APC) should have the capacity to detect a few foreign antigens within a sea of self-antigens. For this vital task, APC are equipped with conserved pattern-recognition receptor families (PRR), such as Toll-like receptors (TLR). TLR detect conserved molecular structures on pathogens, named pathogen-associated molecular patterns (PAMPS), and tissue-damage signals associated with self-antigens, named danger-associated molecular patterns (DAMPS, or alarmins). Nonresponsiveness is at least, in part, regulated by the interaction of C-type lectin receptors (CLR) with carbohydrates expressed on self-antigens.

MT204 is a humanized IgG1 antibody specific for interleukin-2 (IL-2) of human and rhesus monkey origin. It potently antagonizes IL-2 signaling in both CD25+ and CD25− cells by a unique mode of action. MT204 can not only prevent soluble IL-2 from binding to the intermediate affinity IL-2 receptors but can also antagonize IL-2 that is already bound to the CD25 subunit of high affinity IL-2 receptors on the cell surface. As initial steps toward development of a therapeutic antibody, pharmacokinetics (PK) and tolerability of MT204, as well as efficacy were investigated in rhesus monkeys. MT204 was infused by single escalating (2, 10 and 50 mg/kg) or repeat dose administrations (50 mg/kg, 1×/week for 3 weeks). Over the course of the experiment, the animals were regularly observed for clinical adverse reaction and bled for laboratory investigations (PK, hematology, chemical chemistry and leukocyte subset analysis). For the efficacy study, six rhesus monkeys were grafted with MHC-mismatched rhesus skin and infused with MT204 (50 mg/kg), on the day of transplantation and again on days 5 and 12 post grafting. Efficacy was determined by assessment of graft necrosis at different time-points. No obvious adverse effects were observed clinically after single infusion, or after three repeated infusions of 50 mg/kg and no MT204-related toxic effects were revealed by hematology or clinical chemistry. In the efficacy study, MT204-treated animals showed a significant delay in graft rejection versus control animals (p = 0.025 by Log-rank test). The characteristics of MT204, displaying linear pharmacokinetics, half-life in the range expected for a human IgG, benign safety profile and signs of efficacy, warrant further development of this antibody for therapy.

SummaryBlockade of co-stimulation signals between T cells and antigen-presenting cells could be an important approach for treatment of autoimmune diseases and transplant rejection. Recently a series of small compound inhibitors which bind human CD80 (B7-1) and inhibit T cell co-stimulation has been described. To investigate their potency for clinical use, one of these compounds, RhuDex™, was evaluated for reactivity with rhesus monkey CD80. The in vitro biological effect on rhesus monkey lymphocytes, the potency for suppression of an inflammatory recall response and the protein-induced delayed type hypersensitivity (DTH) response in the skin were studied. In a rhesus monkey T cell co-stimulation assay RhuDex™ inhibited proinflammatory cytokine release and cellular proliferation with micromolar potency. Systemic administration of RhuDex™ to rhesus monkeys inhibited the DTH response significantly, indicating that this compound may inhibit autoimmune mediated inflammatory processes where the target, CD80, is up-regulated.

The development of an in vitro assay predicting the chances of graft survival after treatment with immunoregulatory agents is a major topic in transplantation. Antibodies (Abs) interfering in the costimulatory pathway are promising candidates for the induction of tolerance. To evaluate these antibodies for clinical use studies non-human primates are the only feasible option due to species specificity of the antibodies. Peripheral blood mononuclear cells, isolated from a large panel of rhesus monkeys, were used in a unidirectional mixed lymphocyte reaction to evaluate the ability of antibodies blocking the costimulatory pathway, to affect both primary and secondary proliferative and cytolytic allospecific immune responses in vitro. These blocking antibodies were also used in protocols prolonging allograft survival in a life-supporting kidney allotransplant model in rhesus macaques. The ultimate aim is to establish a correlation between parameters obtained in vitro and the success of transplantation in vivo. The combination of anti-CD80 and anti-CD86 resulted in a complete abrogation of the primary alloresponse as measured in a proliferation assay. Adding anti-CD40 significantly reduced this inhibitory effect although the in vivo effects of this antibody have been shown to be beneficial. The secondary response was most prominently inhibited by the combination of anti-CD80/86. Paradoxically, anti-CD40 alone markedly inhibited the secondary proliferative response, but did not add to the inhibitory effect of the combination of anti-CD80/86. The cytolytic response was inhibited maximally only when CsA was added to the combination of anti-CD80/86. Treatment with monoclonal antibodies alone without immunosuppressive drugs was sufficient to maintain graft survival during the time of treatment in most animals. However, rejection was initiated as soon as the treatment ceased and no tolerance, resulting in long-term graft and patient survival, was established. The complete inhibition of primary alloresponses and the partial inhibition of secondary proliferative alloresponses correlate with prolonged graft survival during treatment, but have no predictive value for the success of tolerance induction for kidney allografts in rhesus monkeys.

In rodents it has been demonstrated that blockade of the CD40-CD154 (CD40L) pathway at the time of donor-specific blood transfusion (DST) can result in indefinite graft survival. Because it has been reported in the past that DST in monkeys can have a favorable effect on graft outcome and that blockade of the CD40-CD154 pathway can lead to prolonged kidney graft survival in monkeys, we have combined anti-CD154 treatment with DST in a monkey kidney graft model. The aim of this study was to investigate the immunosuppressive potential of blocking the CD40-CD154 interaction at the time of a DST in rhesus monkeys.One donor-derived blood transfusion was given on day -15 after the first anti-CD154 injection. The anti-CD154 antibody was given on days -15, -13, -11, -9, and -7. The kidney was transplanted on day 0. Cyclosporine was given after kidney trans-plantation.No major difference in graft survival was observed between the groups. The animals died due to grade II acute rejection. At the time of transplantation, no antibody response could be detected directed against donor antigens. After transplantation, all animals surviving for more than 3 weeks had antidonor antibodies. There were no differences in the intragraft events analyzed by real time reverse transcriptase-polymerase chain reaction.DST under the cover of relatively high levels of anti-CD154 failed to result in prolonged graft survival or prevent the formation of antidonor antibodies, when cyclosporine was given after transplantation.

IN a previous study we demonstrated that porcine islets of Langerhans transplanted to monkeys will not be hyperacutely rejected. In immunosuppressed monkeys (cycplophosphamide, cyclosporine [CyA], and prednisolone) rejection occurred in the second week posttransplantation. Rejection infiltrates were dominated by T cells. We therefore wanted to investigate whether a more T-cell-directed immunosuppressive regimen could improve graft survival. Our aim was to deplete recipients of T cells prior to transplantation (using ATG) and prevent rejection posttransplantation using a combination of CyA, prednisolone, and anti-IL-2R antibody (Zenapax).

Experiments were performed in accordance with the “Principles of Laboratory Animal Care” (NIH publication No. 85-23, revised 1985) and institutional guidelines. Four outbred cynomolgous monkeys (Macaca fascicularis; Biomedical Primate Research Centre, Rijswijk, The Netherlands) weighing 5 to 12 kg were used. Pancreata from large sows were obtained from a local slaughterhouse. Highly purified (.95%) islets were cultured 1 to 2 weeks in RPMI with 20 mmol/L HEPES and 10% porcine serum at 37°C in ambient air. These islets were grafted under the capsule of both kidneys of the monkeys, which received cyclophosphamide (day 21 40 mg/kg, day 11 10 mg/kg), cyclosporine (CyA) intramuscularly (IM) and orally, reaching at least 600 ng/mL trough levels, and prednisolone (1 mg/kg/d). Monkey anti-pig antibodies were assessed by a hemolytic complement-dependent assay.