Bone marrow-derived dendritic cells (DCs) can be genetically engineered using adenoviral (Ad) vectors to express immunosuppressive molecules that promote T cell unresponsiveness. The success of these DCs for therapy of allograft rejection has been limited in part by the potential of the adenovirus to promote DC maturation and the inherent ability of the DC to undergo maturation following in vivo administration. DC maturation occurs via NF-κB-dependent mechanisms, which can be blocked by double-stranded “decoy” oligodeoxyribonucleotides (ODNs) containing binding sites for NF-κB. Herein, we describe the combined use of NF-κB ODNs and rAd vectors encoding CTLA4-Ig (Ad CTLA4-Ig) to generate stably immature murine myeloid DCs that secrete the potent costimulation blocking agent. These Ad CTLA4-Ig-transduced ODN DCs exhibit markedly impaired allostimulatory ability and promote apoptosis of activated T cells. Furthermore, administration of Ad CTLA4-Ig ODN-treated donor DCs (C57BL10; B10(H-2b)) before transplant significantly prolongs MHC-mismatched (C3HHeJ; C3H(H-2k)) vascularized heart allograft survival, with long-term (>100 days) donor-specific graft survival in 40% of recipients. The mechanism(s) responsible for DC tolerogenicity, which may involve activation-induced apoptosis of alloreactive T cells, do not lead to skewing of intragraft Th cytokine responses. Use of NF-κB antisense decoys in conjunction with rAd encoding a potent costimulation blocking agent offers promise for therapy of allograft rejection or autoimmune disease with minimization of systemic immunosuppression.

Background CD47, the integrin-related protein, plays an important role in immune resistance and escape of tumor cells. Antibodies blocking the CD47/SIRPα signal pathway can effectively stimulate macrophage-mediated phagocytosis of tumor cells, which becomes a promising approach for tumor immunotherapy. Nanobodies (Nbs) derived from camelid animals are emerging as a new force in antibody therapy. Results HuNb1-IgG4, an innovative anti-CD47 nanobody, was developed with high affinity and specificity. It effectively enhanced macrophage-mediated phagocytosis of tumor cells in vitro and showed potent anti-ovarian and anti-lymphoma activity in vivo. Importantly, HuNb1-IgG4 did not induce the agglutination of human red blood cells (RBCs) in vitro and exhibited high safety for hematopoietic system in cynomolgus monkey. In addition, HuNb1-IgG4 could be produced on a large scale in CHO-S cells with high activity and good stability. Also, we established anti-CD47/CD20 bispecific antibody (BsAb) consisted of HuNb1 and Rituximab, showing more preference binding to tumor cells and more potent anti-lymphoma activity compared to HuNb1-IgG4. Conclusions Both of HuNb1-IgG4 and anti-CD47/CD20 BsAb are potent antagonists of CD47/SIRPα pathway and promising candidates for clinical trials.

Abnormalities of dendritic cells (DCs) have been identified in type 1 diabetic patients and in nonobese diabetic (NOD) mice that are associated with augmented nuclear transcription factor (NF)-κB activity. An imbalance that favors development of the immunogenic DCs may predispose to the disease, and restoration of the balance by administration of DCs deficient in NF-κB activity may prevent diabetes. DCs propagated from NOD mouse bone marrow and treated with NF-κB–specific oligodeoxyribonucleotide (ODN) in vitro (NF-κB ODN DC) were assessed for efficacy in prevention of diabetes development in vivo. Gel shift assay with DC nuclear extracts confirmed specific inhibition of NF-κB DNA binding by NF-κB ODN. The costimulatory molecule expression, interleukin (IL)-12 production, and immunostimulatory capacity in presenting allo- and islet-associated antigens by NF-κB ODN DC were significantly suppressed. NF-κB ODN renders DCs resistant to lipopolysaccharide stimulation. Administration of 2 × 106 NF-κB ODN DCs into NOD mice aged 6–7 weeks effectively prevented the onset of diabetes. T-cells from pancreatic lymph nodes of NF-κB ODN DC–treated animals exhibited hyporesponsiveness to islet antigens with low production of interferon-γ and IL-2. These findings provide novel insights into the mechanisms of autoimmune diabetes and may lead to development of novel preventive strategies.

Glucosamine is a naturally occurring derivative of glucose and is an essential component of glycoproteins and proteoglycans, important constituents of many eukaryotic proteins. In cells, glucosamine is produced enzymatically by the amidation of glucose 6-phosphate and can then be further modified by acetylation to result in N-acetylglucosamine. Commercially, glucosamine is sold over-the-counter to relieve arthritis. Although there is evidence in favor of the beneficial effects of glucosamine, the mechanism is unknown. Our data demonstrate that glucosamine suppresses the activation of T-lymphoblasts and dendritic cells in vitro as well as allogeneic mixed leukocyte reactivity in a dosedependent manner. There was no inherent cellular toxicity involved in the inhibition, and the activity was not reproducible with other amine sugars. More importantly, glucosamine administration prolonged allogeneic cardiac allograft survival in vivo. We conclude that, despite its documented effects on insulin sensitivity, glucosamine possesses immunosuppressive activity and could be beneficial as an immunosuppressive agent.Glucosamine is a naturally occurring sugar that is synthesized by virtually all cells. Upon uptake, glucose is immediately phosphorylated and enzymatically converted into a series of substrates that will be either converted into glycogen, lipids, and proteins or used to generate ATP and CO 2 . 2-3% of glucose 6-phosphate, the immediate intracellular glucose derivative following uptake, is diverted into a pathway known as the hexosamine biosynthesis pathway (1, 2). The rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase is responsible for the commitment of glucose derivatives to the pathway, ultimately resulting in the formation of glycoprotein precursors (3). Glucosamine is not secreted outside of cells, but exogenously added glucosamine is taken up by glucose transporters (GLUT-2 and GLUT-4) and then phosphorylated (4, 5).In 1953, Quastel and Cantero (6) demonstrated that glucosamine possesses tumor-inhibiting activity. Since then, a number of reports have confirmed the tumoricidal activity of glucosamine (7-16). Glucosamine has been shown to inhibit nucleic acid and protein biosynthesis and irreversible damage to organelles in tumor cells, but not in normal cells (8 -15, 17-20).In addition, glucosamine also inhibits platelet aggregation and ATP release induced by Staphylococcus aureus, ADP, epinephrine, and collagen (21). The mechanisms by which glucosamine acts are not completely clear; however, it has been shown to alter the ultrastructure of plasma and intracellular membranes (9, 22), to inhibit membrane transport of nucleosides (14, 15), and reportedly to shift its distribution from glycoproteins to glycolipids (22).O'Neill and Parish (23) demonstrated that monosaccharides, especially amino sugars, are able to inhibit cytotoxic T-lymphocyte function in culture, preventing target lysis in a cytotoxic T-lymphocyte clone-specific manner. Yagita et al. further demonstrated that free hexosamines ...