Unmethylated Cytosine-guanine Dinucleotides Research Articles

Malignant Plasma Cell Derived Mitochondrial Damps Activate Macrophages Via Sting Signalling to Promote Myeloma Development

Mitochondrial damage-associated patterns (mtDAMPs) include proteins, lipids, metabolites, and DNA and have various context specific immunoregulatory functions. Cell free mitochondrial DNA (mtDNA) contains islands of unmethylated cytosine-guanine dinucleotide (CpG) motifs which are recognised via pattern recognition receptors and are potent activators of the innate immune system. Cell free mtDNA is elevated in the circulation of trauma and cancer patients, however the functional consequences of elevated mtDNA are largely undefined. Multiple myeloma (MM) relies upon cellular interactions within the bone marrow (BM) microenvironment for survival and progression. A key feature of myeloma is the homing and retention of MM cells to the BM, and it can be assumed that this homing process is mediated by chemotactic signals provided by the BM microenvironment. Here, using in-vivo models, we describe the role of MM cell derived mtDAMPs in the pro-tumoral BM microenvironment, and the mechanism and functional consequence of mtDAMPs in myeloma disease progression. Initially, we identified elevated levels of mtDNA in the peripheral blood serum of myeloma patients compared to healthy controls. This was also consistently observed in the serum of mice, using two distinct myeloma mouse models: KaLwRij-5TGM1 and NSG-MM1S. Using the MM xenograft model, we established that mtDNA was derived from myeloma cells. Blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA in the serum of engrafted mice and correlated to disease progression. Since macrophages sense and respond to mtDAMPs we isolated BM macrophages from myeloma engrafted mice and showed they had an increased gene expression signature indicative of STING pathway activation. The same signature was observed when bone marrow-derived macrophages (BMDMs) were treated with mtDAMPs in culture, suggesting that this activation was mediated via myeloma-derived mtDAMPs. To understand the role of macrophage STING signalling in MM, we engrafted KaLwRij mice with murine 5TGM1(GFP+ Luci+) cells. After tumour was established, we treated the mice with STING inhibitor H-151 (750nM). Using live imaging, we observed that tumour progression was reduced in the STING inhibited group compared to the non-treated group. Flow cytometry of the bone marrow confirmed a significant reduction in 5TGM1(GFP+ Luci+) cell burden in the STING inhibited group. Furthermore, cytokine array analysis of BMDMs treated with mtDAMPs revealed an upregulation of chemokines involved in chemotaxis, suggesting that mtDAMPs induce a migratory signature in BM macrophages. To investigate this, we performed a transwell assay to assess the chemotaxis of 5TGM1 cells towards conditioned media from BMDMs treated with mtDAMPs. We found that 5TGM1 migration was enhanced with mtDAMPs treatment and reduced with H-151 treatment. Furthermore, RT-qPCR analysis of FACs isolated macrophages from KaLwRij-5TGM1 mice showed increase expression of genes associated with chemotaxis. Moreover, the expression of the chemotaxis gene signature was attenuated with H-151 inhibitor treatment. Finally, using our syngeneic 5TGM1-KaLwRij mouse model we found that depletion of macrophages via clodronate-liposome treatment resulted in egress of myeloma cells from the BM and into the peripheral blood. Here, we demonstrate that malignant plasma cells release mtDNA, a form of mtDAMPs, into the myeloma BM microenvironment, which in turn activates macrophages via STING signalling. We establish the functional role of these mtDAMPs activated macrophages in reducing disease progression and retaining MM cells in the pro-tumoral BM microenvironment.

Comparison of Therapeutic Antibiotics, Probiotics, and Synthetic CpG-ODNs for Protective Efficacy Against Escherichia coli Lethal Infection and Impact on the Immune System in Neonatal Broiler Chickens.

The poultry industry needs alternatives to antibiotics, as there are growing public concerns about the emergence of antimicrobial resistance owing to antimicrobial use in animal production. We have reported that the administration of neonatal chicks with synthetic DNA oligodeoxynucleotides containing unmethylated cytosine guanine dinucleotide (CpG) motifs (CpG-ODN) can protect against bacterial pathogens in chickens. The objective of this study was to compare the immunoprotective effects of CpG-ODN and probiotics against Escherichia coli infection vs. commonly used therapeutic antibiotics. Day-old broiler chicks were divided into five groups (n = 35/group; 30 for the challenge experiment and 5 for the flow cytometry analysis). The chicks in Group 1 received a single dose of CpG-ODN by the intramuscular route on day 4 (D4) posthatch (PH), and Group 2 received drinking water (DW) with a probiotic product (D1-D15 PH, DW). The Group 3 chicks received tetracycline antibiotics during D9-D13 in DW; the Group 4 chicks got sodium sulfamethazine on D9, D10, and D15 PH in DW; and the Group 5 chicks were administered intramuscular (IM) saline D4 PH, DW. We challenged all the groups (n = 30/group) with E. coli (1 × 105 or 1 × 106 colony-forming units/bird) on D8 PH through the subcutaneous route. Our data demonstrated that the CpG-ODNs, but not the probiotics, could protect neonatal broiler chickens against lethal E. coli septicemia, as would the tetracycline or sodium sulfamethazine. The flow cytometry analysis (n = 5/group) revealed enrichment of immune cells in the CpG-ODN group and a marked decrease in macrophages and T-cell numbers in antibiotics-treated groups, indicating immunosuppressive effects. Our data showed that, like therapeutic antibiotics, CpG-ODNs reduced clinical signs, decreased bacterial loads, and induced protection in chicks against E. coli septicemia. Unlike therapeutic antibiotics-induced immunosuppressive effects, CpG-ODN caused immune enrichment by increasing chicken immune cells recruitment. Furthermore, this study highlights that, although therapeutic antibiotics can treat bacterial infections, the ensuing immunosuppressive effects may negatively impact the overall chicken health.

CpG-C ODN M362 as an immunoadjuvant for HBV therapeutic vaccine reverses the systemic tolerance against HBV

Chronic Hepatitis B virus (CHB) infection is a global public health problem. Oligodeoxynucleotides (ODNs) containing class C unmethylated cytosine-guanine dinucleotide (CpG-C) motifs may provide potential adjuvants for the immunotherapeutic strategy against CHB, since CpG-C ODNs stimulate both B cell and dendritic cell (DC) activation. However, the efficacy of CpG-C ODN as an anti-HBV vaccine adjuvant remains unclear. In this study, we demonstrated that CpG M362 (CpG-C ODN) as an adjuvant in anti-HBV vaccine (cHBV-vaccine) successfully and safely eliminated the virus in HBV-carrier mice. The cHBV-vaccine enhanced DC maturation both in vivo and in vitro, overcame immune tolerance, and recovered exhausted T cells in HBV-carrier mice. Furthermore, the cHBV-vaccine elicited robust hepatic HBV-specific CD8+ and CD4+ T cell responses, with increased cellular proliferation and IFN-γ secretion. Additionally, the cHBV-vaccine invoked a long-lasting follicular CXCR5+ CD8+ T cell response following HBV re-challenge. Taken together, CpG M362 in combination with rHBVvac cleared persistent HBV and achieved long-term virological control, making it a promising candidate for treating CHB.

A study on the protective effects of CpG oligodeoxynucleotide-induced mucosal immunity against lung injury in a mouse acute respiratory distress syndrome model.

This study aims to determine the feasibility of using oligodeoxynucleotides with unmethylated cytosine-guanine dinucleotide sequences (CpG ODN) as an immunity protection strategy for a mouse model of acute respiratory distress syndrome (ARDS). This is a prospective laboratory animal investigation. Twenty-week-old BALB/c mice in Animal research laboratory were randomized into groups. An ARDS model was induced in mice using lipopolysaccharides (LPSs). CpG ODN was intranasally and transrectally immunized before or after the 3rd and 7th days of establishing the ARDS model. Mice were euthanized on Day 7 after the second immunization. Then, retroorbital bleeding was carried out and the chest was rapidly opened to collect the trachea and tissues from both lungs for testing. CpG ODN significantly improved the pathologic impairment in mice lung, especially after the intranasal administration of 50 μg. This resulted in the least severe lung tissue injury. Furthermore, interleukin-6 (IL-6) and IL-8 concentrations were lower, which was second to mice treated with the rectal administration of 20 µg CpG ODN. In contrast, the nasal and rectal administration of CpG ODN in BALB/c mice before LPS immunization did not appear to exhibit any significant protective effects. The intranasal administration of CpG ODN may bea potential treatment approach to ARDS. More studies are needed to further determine the protective mechanism of CpG ODN.

Nanovaccine based on a protein-delivering dendrimer for effective antigen cross-presentation and cancer immunotherapy

Cancer vaccines for prevention and treatment of tumors have attracted tremendous interests as a type of cancer immunotherapy strategy. A major challenge in achieving robust T-cell responses to destruct tumor cells after vaccination is the abilities of antigen cross-presentation for antigen-presenting cells (APCs) such as dendritic cells (DCs). Herein, we demonstrate that a polyamidoamine dendrimer modified with guanidinobenzoic acid (DGBA) could serve as an effective protein carrier to enable delivery of protein antigen, thereby leading to effective antigen cross-presentation by DCs. With ovalbumin (OVA) as the model antigen and unmethylated cytosine-guanine dinucleotides (CpG) as the adjuvant, a unique type of tumor vaccine is formulated. Importantly, such DGBA-OVA-CpG nanovaccine can induce robust antigen-specific cellular immunities and further demonstrates outstanding prophylactic efficacy against B16-OVA melanoma. More significantly, the nanovaccine shows excellent therapeutic effect to treat established B16-OVA melanoma when used in combination with the programmed cell death protein 1 (PD-1) checkpoint-blockade immunotherapy. This study presents the great promises of employing rationally engineered cytosolic protein carriers for the development of tumor vaccines to achieve effective cancer immunotherapy.

Unopposed IL-18 signaling leads to severe TLR9-induced macrophage activation syndrome in mice

AbstractThe term macrophage activation syndrome (MAS) defines a severe, potentially fatal disorder characterized by overwhelming inflammation and multiorgan involvement. Interleukin-18 (IL-18) is a proinflammatory cytokine belonging to the IL-1 family, the activity of which is regulated by its endogenous inhibitor IL-18 binding protein (IL-18BP). Elevated IL-18 levels have been reported in patients with MAS. Herein, we show that on repeated toll-like receptor 9 (TLR9) stimulation with unmethylated cytosine guanine dinucleotide containing single-stranded DNA (CpG), IL-18BP−/− mice display severe MAS manifestations, including increased weight loss, splenomegaly, anemia, thrombocytopenia, hyperferritinemia, and bone marrow hemophagocytosis as compared with wild-type mice. Serum-free IL-18 was detected in CpG-treated IL-18BP−/− mice only. Levels of interferon-γ (IFN-γ) and of IFN-γ signature genes, such as the chemokine Cxcl9 or the transcription factor CIIta, were significantly increased in IL-18BP−/− mice. Blocking IL-18 receptor signaling attenuated the severity of MAS and IFN-γ responses in IL-18BP−/− mice. Blocking IFN-γ had comparable effects to IL-18 inhibition on most MAS manifestations. Our data indicate that endogenous IL-18BP exerts a protective role in CpG-induced MAS and that IL-18, which acts upstream of IFN-γ, is involved in the severity of MAS.

Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector

Nonviral gene therapy is expected to become a regular treatment for a variety of difficult-to-treat diseases, such as cancer and virus infection. Plasmid DNA, which is used in most nonviral gene delivery systems, usually contains, unmethylated cytosine-guanine dinucleotides, so called CpG motifs. CpG motifs are recognized by immune cells as a danger signal, leading to an inflammatory response. Such inflammatory responses could affect the safety and effectiveness of nonviral gene therapy. Therefore, reducing the number of CpG motifs in plasmid DNA has been used to increase the potency of plasmid DNA-based gene therapy. Previous studies have demonstrated that CpG reduction can extend the time period of transgene expression from plasmid DNA after in vivo gene transfer. In this review, the biological functions of the CpG motif are briefly summarized. Then, safety issues of nonviral gene therapy are discussed from the viewpoint of the inflammatory response to the CpG motif in plasmid DNA, and the effects of the CpG motif in plasmid DNA on the transgene expression profile of nonviral gene transfer are reviewed.

Increased frequency of immunoglobulin (Ig)A-secreting cells following Toll-like receptor (TLR)-9 engagement in patients with Kawasaki disease

Kawasaki disease (KD) is an acute vasculitis affecting mainly infants and children. Human B cells express Toll-like receptor (TLR)-9, whose natural ligands are unmethylated cytosine-guanine dinucleotide (CpG) motifs characteristic of bacterial DNA. The aim of this study was to clarify the pathogenesis of KD analysing the activation status of peripheral blood mononuclear cells (PBMC), focusing on B lymphocyte activation and functions. Ten patients and 10 age-matched healthy donors were recruited from the Bambino Gesù Hospital of Rome, Italy and enrolled into this study. We determined phenotype profile and immunoglobulin (Ig) production of PBMC from KD patients and age-matched controls. We found that the frequency of CD19(+) B lymphocytes and CD19(+) /CD86(+) activated B lymphocytes from KD patients during the acute phase before therapy was increased significantly. Moreover, B lymphocytes of acute-phase KD patients were more prone to CpG oligodeoxynucleotide (ODN) activation compared with the age-matched controls, as assessed by a significant increase of the number of IgA-secreting cells (SC). In the same patients we found a marked increase of IgM, IgG, interleukin (IL)-6 and tumour necrosis factor (TNF)-α production compared with the control group. In addition, in two convalescent KD patients, conventional treatment with intravenous immunoglobulin (IVIG) restored the normal frequency of CD19(+) B cells, the number of IgA-, IgM- and IgG-SC and the production of IL-6 and TNF-α. Our findings indicate that the percentages of peripheral B lymphocytes of acute-phase KD patients are increased and are prone to bacterial activation in terms of increased numbers of IgA-SC and increased production of IL-6 and TNF-α inflammatory cytokines. Thus, our data support the hypothesis of an infectious triggering in KD.

Neonate Intestinal Immune Response to CpG Oligodeoxynucleotide Stimulation

BackgroundThe development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates.Methodology/Principal FindingsFirst, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice.Conclusions/SignificanceThis is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases.

Increased immunoglobulin A in alcoholic liver cirrhosis: exploring the response of B cells to Toll-like receptor 9 activation

Alcoholic liver cirrhosis (ALC) is characterized by increased circulating levels of immunoglobulins (Igs). ALC patients undergo bacterial translocation evidenced by the presence of bacterial DNA in peripheral blood. Bacterial pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN) and unmethylated cytosine-guanine dinucleotide (CpG) DNA are ligands of Toll-like receptor (TLR)-4, TLR-2 and TLR-9, respectively. Although TLR activation results generally in the secretion of proinflammatory cytokines, activation of B cells through TLR-7 or TLR-9 is involved in their maturation and Ig synthesis. The aim of the present study was to assess Ig synthesis by ALC B cells under PAMP activation in order to evaluate the possible involvement of TLR pathways in the increased Ig levels, and especially the hyper-IgA observed in ALC. CpG, in combination with interleukin (IL)-10 or IL-21, enhanced IgA, IgG and IgM synthesis by healthy donor (HD) PBMCs, but had only a weak effect on ALC PBMCs. Relative CpG-induced IgA production by purified ALC B cells was less important when compared to HD B cells, in accordance with the lower TLR-9 expression on ALC B cells compared to HD B cells, but the absolute IgA production by CpG-activated B cells was enhanced significantly for ALC when compared to HD, in agreement with their intrinsic ability to produce spontaneously more IgA than HD. LPS and PGN had no direct activity on B cells, whereas R848 also enhanced Ig synthesis, as reported recently. Taken together, these results suggest that TLR priming of B cells could account for the hyperimmunoglobulinaemia observed in ALC patients.

Dissociated Induction of Cytotoxicity and DTH by CFA and CpG

Vaccinations typically rely on immunization with live virus for eliciting protective CD8 T cell immunity. There is increasing interest to use subunit vaccination strategies to achieve such responses. Complete Freund's adjuvant (CFA) and unmethylated cytosine-guanine dinucleotide containing DNA are considered some of the most potent adjuvants for eliciting immunity. Whereas a wealth of information is available on how these adjuvants affect CD4 T cell responses, their effects on engaging CD8 T cell immunity are not completely understood. We immunized C57BL/6J mice with the class I restricted peptides Uty or SIINFEKL using these 2 adjuvants and tested for cytokine secretion, proliferation, in vivo cytotoxicity, and delayed-type hypersensitivity (DTH). Our data show that CFA-induced CD8 T cells to proliferate, mediate DTH, and to secrete interferon-gamma, interleukin (IL)-2 and IL-17. Despite these markers of CD8 T cell activation, CFA failed to induce an early cytotoxic CD8 T cell response. In contrast, unmethylated cytosine-guanine dinucleotide containing DNA promoted a vigorous cytolytic response without activating substantial cytokine production, proliferation or DTH. These data have implications for CD8 T cell subunit vaccine design in which cytotoxicity versus DTH plays a key role in host defense.

Immune modulatory oligonucleotides in the prevention and treatment of allergen-induced eustachian tube dysfunction in the animal model

This article reviews the current literature investigating the applications and success of immune modulatory oligonucleotides as immunotherapy to treat and prevent allergen-induced eustachian tube dysfunction in animal models. Synthetic DNA-based immunotherapy agents composed of unmethylated cytosine-guanine dinucleotides (CpG ODNs) that bind to Toll-like receptors have been found to have tremendous potential as therapeutic agents and adjuvants. CpG ODNs can induce a shift in the cytokine profile and immune response that favors the T-helper type 1 pathway and suppresses the T-helper type 2 pathway. This makes CpG ODNs promising candidates for treating allergic diseases. Current CpG ODN studies have demonstrated prevention and treatment of acute allergen inflammation of the eustachian tube in an animal model of otitis media. Immune modulatory oligonucleotides in immunotherapy, administered systemically or topically, have been shown to be safe and effective in the animal model.

Immunostimulatory sequences in immunotherapy

To review the current literature regarding immunostimulatory sequences of DNA for immunotherapy with respect to signaling mechanisms, cytokine profiles, structural characteristics and the applicability and success of this strategy to treat allergic disease. The binding of synthetic DNA-based immunotherapy agents composed of unmethylated cytosine-guanine dinucleotides (CpG ODN) to toll-like receptors have been found to be species-specific. CpG ODNs are capable of inducing a shift in the cytokine profile and immune response that favors the Th1 pathway and suppresses the Th2 pathway. This makes using CpG ODNs a promising candidate for the treatment of allergic diseases, which are known to be mediated by Th2-based response. Current CpG ODN studies have demonstrated prevention and reversal of acute allergen inflammation, airway hyper-reactivity and remodeling. Early animal and human trials of CpG ODNs have shown them to be both well tolerated and effective. The use of immunostimulatory sequences in immunotherapy, although still in the early stages of development, has thus far been shown to be both well tolerated and effective, and offers the potential for a better tolerated, more rapid, more efficacious and longer-lasting therapy over current immunotherapy protocols.

Immunotherapy of asthma using CpG oligodeoxynucleotides

Asthma and other atopic disorders have increased in prevalence and severity over the past three decades. Reduced risk of atopic disease associated with early life exposure to infections and microbes has raised the possibility that pathogen-associated molecular patterns (PAMPs) may confer protection against allergic disorders, a concept that has been named the "Hygiene Hypothesis". This relationship is most likely mediated through the induction of specific patterns of anti-atopic immune responses that follow engagement of innate immune mechanisms. Bacterial DNA is one such immunostimulatory microbe-associated ligand, whose properties can be mimicked by oligodeoxynucleotides (ODN) containing unmethylated cytosine-guanine dinucleotides in specific base sequences (CpG motifs), motifs characteristic of prokaryotic DNA that have been suppressed in eukaryotic DNA. Based initially on observations that CpG ODN induced Th1-type patterns of immune responses, we proposed that CpG ODN might represent a novel therapeutic strategy for the prevention and treatment of atopic disorders. Current understanding suggests multiple mechanisms of action of CpG ODN, but our initial hypothesis has been supported by extensive studies demonstrating, in animal models, efficacy in both incipient and established atopic asthma. These preclinical studies are now being translated into clinical trials exploring this new approach to immunotherapy for atopic disease.

A newly identified CpG oligodeoxynucleotide motif that stimulates rainbow trout ( Oncorhynchus mykiss) immune cells to produce immunomodulatory factors

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated cytosine–guanine dinucleotides (CpGs) have been shown to have immunostimulatory effects on B cells, natural killer cells and dendritic cells in mammals. However, little is known of the cellular basis of CpG immunostimulation in fish. We have addressed this question in the rainbow trout, Oncorhynchus mykiss. Prokaryotic DNA (from Escherichia coli), but not eukaryotic DNA (from calf), caused proliferation of spleen and head kidney cells, whereas CpG ODNs stimulated only head kidney cells. While spleen cells remained unresponsive to direct stimulation with CpG ODN, conditioned medium collected from spleen and head kidney cells both stimulated proliferation, suggesting CpG ODNs induce the secretion of immunomodulatory factors. Preliminary experiments to deplete B cells indicate that neither the source of the soluble factors or the proliferating responder cells are B cells. These data begin to define the cellular basis of CpG-mediated immunostimulation in trout.

Immunomodulation: the future of allergy and asthma treatment.

As the prevalence of asthma and allergic disease increases around the world, it is clear that more effective therapies and disease-modifying agents are needed. Treatment for allergic disease is evolving with an increase in understanding of the etiology. The first immunomodulatory treatment was recently approved for use in the United States when the Food and Drug Administration approved the use of a humanized monoclonal anti-IgE antibody in patients with allergic asthma. Another strategy that has proved effective in a murine model is the downregulation of the whole immune system by targeting adhesion molecules, which has been evaluated in a recent human trial. Other strategies for the treatment of allergic diseases concentrate on refocusing the immune system away from an allergic-type response. These include the use of targeted therapies towards specific cytokines, cytokine receptors or chemokine receptors, and the use of specific bacterial DNA sequences (unmethylated cytosine-guanine dinucleotides). Finally, attention is being focused on possible therapies that may tilt the immune response to a non-allergic response by interfering with signaling molecule pathways. Immunomodulation will play a key role in future therapies for allergic disease. These treatment modalities may not only treat allergic disease, but also be beneficial in reducing the morbidity and mortality for which it is responsible.

Synergism between cytosine-guanine oligodeoxynucleotides and monoclonal antibody in the treatment of lymphoma

Synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine dinucleotides (CpG ODN) are potent immunostimulatory agents that can activate various immune cell subsets. We have found that CpG ODN show a variety of effects that could be useful in enhancing the efficacy of antibody therapy of lymphoma. In a mouse model, CpG ODN alone had no effect on survival of animals inoculated with lymphoma. In contrast, CpG ODN plus monoclonal antibody (MAb) was more effective at inhibiting tumor growth than MAb alone or MAb plus control ODN. Cytosine-guanine ODN plus MAb cured mice with a large tumor burden that could not be cured with MAb therapy alone. We also evaluated the effects of CpG ODN on the phenotype of human malignant B cells. Cytosine-guanine ODN upregulated the expression of a number of antigens, including CD20. The upregulation of CD20 was most extensive in cells that had low baseline expression of this antigen. We conclude that CpG ODN enhances the efficacy of MAb in a murine lymphoma model, most likely by activating effector cells, and upregulates expression of CD20 on primary human malignant B cells. Given the effects of CpG ODN on both target antigen expression and effector-cell function, further evaluation of the combination of CpG ODN plus rituximab (Rituxan; Genentech, Inc, South San Francisco, CA, and IDEC Pharmaceuticals, San Diego, CA) and CpG ODN plus other MAbs is warranted. Semin Oncol 29 (suppl 2):93-97. Copyright © 2002 by W.B. Saunders Company.

CpG Oligodeoxynucleotides Enhance Monoclonal Antibody Therapy of a Murine Lymphoma

Bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine dinucleotides known as cytosine phosphorothioate guanine oligodeoxynucleotides (CpG ODN) can activate various immune-cell subsets, including cells that participate in antibody- dependent cell-mediated cytotoxicity (ADCC). Studies have shown that CpG ODN enhance the efficacy of antitumor monoclonal antibody (MoAb) therapy in the 38C13 murine B-cell lymphoma. We performed a series of in vivo experiments using this tumor model to better characterize combination therapy with MoAb and CpG ODN. CpG ODN enhanced the efficacy of MoAb therapy of lymphoma in a dose-dependent manner. This effect was seen whether the CpG ODN was given before or after the MoAb therapy, but was decreased when CpG ODN was given more than 2 days after MoAb therapy. Three doses of CpG ODN and MoAb were more effective than single doses. There was no obvious toxicity with multiple dosing. These studies confirm that immunostimulatory CpG ODN enhance the efficacy of MoAb therapy, and that multiple courses of combination therapy with CpG ODN can serve as an effective therapy for lymphoma. Further exploration of this potentially potent combination of treatments, including clinical evaluation, is indicated.

Microbial products induce autoimmune disease by an IL-12-dependent pathway.

The development and exacerbation of autoimmune diseases are associated with antecedent infectious illness. Microbial products such as LPS, bacterial DNA, or oligonucleotides containing an unmethylated cytosine-guanine dinucleotide have cytokine modulating properties. These products converted quiescent myelin basic protein-specific T cells into effector cells capable of transferring experimental allergic encephalomyelitis. The disease-promoting properties of the microbial products were solely dependent on their capacity to induce the production of IL-12.