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PubMed2003

Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene, Tact1/Actl7b, represses its expression in somatic cells.

Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5'-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5' upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.

Actins↗
PubMed2000

The covariation between TpA deficiency, CpG deficiency, and G+C content of human isochores is due to a mathematical artifact.

CpG and TpA dinucleotides are underrepresented in the human genome. The CpG deficiency is due to the high mutation rate from C to T in methylated CpG's. The TpA suppression was thought to reflect a counterselection against TpA's destabilizing effect in RNA. Unexpectedly, the TpA and CpG deficiencies vary according to the G+C contents of sequences. It has been proposed that the variation in CpG suppression was correlated with a particular chromatin organization in G+C-rich isochores. Here, we present an improved model of dinucleotide evolution accounting for the overlap between successive dinucleotides. We show that an increased mutation rate from CpG to TpG or CpA induces both an apparent TpA deficiency and a correlation between CpG and TpA deficiencies and G+C content. Moreover, this model shows that the ratio of observed over expected CpG frequency underestimates the real CpG deficiency in G+C-rich sequences. The predictions of our model fit well with observed frequencies in human genomic data. This study suggests that previously published selectionist interpretations of patterns of dinucleotide frequencies should be taken with caution. Moreover, we propose new criteria to identify unmethylated CpG islands taking into account this bias in the measure of CpG depletion.

Animals↗
PubMed2002

A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes.

DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.

Amino Acid Sequence↗
PubMed2002

Immunization with the Chlamydia trachomatis mouse pneumonitis major outer membrane protein by use of CpG oligodeoxynucleotides as an adjuvant induces a protective immune response against an intranasal chlamydial challenge.

Recently, we have shown that a vaccine consisting of a purified preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with C. trachomatis MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of the C. trachomatis MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to Chlamydia and significant lymphoproliferation of T-cells following stimulation with C. trachomatis EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the C. trachomatis MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory challenge.

Adjuvants, Immunologic↗
PubMed2003

Protection of chickens against Escherichia coli infections by DNA containing CpG motifs.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate of Escherichia coli was applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after the E. coli challenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN((2007)) had a survival rate of 15%. In contrast, groups that received CpG-ODN((2007)), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P < 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN((2007)) by the subcutaneous route (P < 0.01). A dose of as little as 3.16 micro g of CpG-ODN((2007)), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds against E. coli infection (P < 0.01). This study demonstrates that CpG-ODN((2007)) has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN((2007)) has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.

Adjuvants, Immunologic↗
PubMedDate not supplied

The influence of CpG motifs on a protein or DNA-based Th2-type immune response against major pollen allergens Bet v 1a, Phl p 2 and Escherichia coli-derived beta-galactosidase.

BACKGROUND: DNA immunization and protein immunization with CpG motifs as adjuvants represent promising approaches in allergen-specific immunotherapy. OBJECTIVE: We investigated the effect of coinjection or prepriming with CpG-ODN on Th2-type responses induced by gene gun and protein immunization. METHODS: BALB/c mice were immunized with the gene gun using plasmid DNA containing the cDNAs coding for the genes of Bet v 1a, Phl p 2 and beta-galactosidase or with the purified Al(OH)(3)-adsorbed proteins. In addition, CpG-ODN were applied by coinjection or by prepriming treatment. Antibody and cytokine responses were measured by ELISA, proliferative and cytotoxic responses were determined by standard labeling procedures. Furthermore, the allergenic activity of sera was measured by passive cutaneous anaphylaxis. RESULTS: Gene gun immunization and protein immunization induced a clear Th2-type response for all antigens. The Th1-promoting effect of CpG-ODN coinjection together with gene gun immunization was restricted to beta-galactosidase as indicated by the increase of IgG2a and a marked expression of IFN-gamma. CpG motifs also increased the specific cytotoxic response against beta-galactosidase. Prepriming with CpG-ODN and gene gun or protein immunization with Bet v 1a exhibited no significant difference to the non-CpG control group. However, sera from mice preprimed with CpG-ODN induced no anaphylaxis with gene gun immunization, but with protein immunization. CONCLUSIONS: The effect of CpG motifs in vivo depends on a variety of parameters like the nature of the antigen and the immunization modality. Furthermore, our studies indicate that a combination of CpG + DNA immunization may be more effective in antagonizing Th2 responses than the combination of CpG + protein immunization.

Adjuvants, Immunologic↗
PubMed2005

Local therapy with CpG motifs in a murine model of allergic airway inflammation in IFN-beta knock-out mice.

BACKGROUND: CpG oligodeoxynucleotides (CpG-ODN) are capable of inducing high amounts of type I IFNs with many immunomodulatory properties. Furthermore, type-I IFNs have been proposed to play a key role in mediating effects of CpG-ODN. The precise role of IFN-beta in the immunomodulatory effects of CpG-ODN is not known. OBJECTIVE: Here, we aimed to elucidate the role of IFN-beta in the anti-allergic effect of CpG motifs. METHODS: We assessed the immune response in OVA-primed/OVA-challenged IFN-beta knockout (-/-) mice compared to wild type (WT) control, after intranasal and systemic treatment with synthetic CpG motifs. RESULTS: Vaccination with CpG-ODN reduced the number of cells in airways of OVA-sensitized WT but not IFN-beta-/- mice. Although airway eosinophilia was reduced in both treated groups, they were significantly higher in IFN-beta-/- mice. Other inflammatory cells, such as lymphocytes and macrophages were enhanced in airways by CpG treatment in IFN-beta-/- mice. The ratio of IFN-gamma/IL-4 cytokines in airways was significantly skewed to a Th1 response in WT compared to IFN-beta-/- group. In contrast, IL-4 and IgE were reduced with no differences between groups. Ag-specific T-cell proliferation, Th1-cytokines such as IFN-gamma, IL-2 and also IL-12 were significantly lower in IFN-beta-/- mice. Surprisingly, we discovered that intranasal treatment of mice with CpG-ODN results in mild synovitis particularly in IFN-beta-/- mice. CONCLUSION: Our results indicate that induction of Th1 response by therapy with CpG-ODN is only slightly and partially dependent on IFN-beta, while IFN-beta is not an absolute requirement for suppression of airway eosinophilia and IgE. Furthermore, our finding of mild synovitis is a warning for possible negative effects of CpG-ODN vaccination.

Adjuvants, Immunologic↗
PubMed2002

Immunostimulatory CpG-DNA activates murine microglia.

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-DNA) triggers innate immune cells through the pattern recognition receptor Toll-like receptor 9 (TLR-9). CpG-DNA possesses potent immunostimulatory effects on macrophages, dendritic cells, and B lymphocytes. Therefore, CpG-DNA contributes to inflammation during the course of bacterial infections. In contrast to other TLR-dependent microbial patterns, CpG-DNA is a strong inductor of IL-12. Thus, it acts as a Th1-polarizing agent that can be utilized as potent vaccine adjuvant. To assess the role of CpG-DNA in immune reactions in the CNS, we analyzed the effects of CpG-DNA on microglial cells in vitro and in vivo. Primary microglial cells as well as microglial cell lines express TLR-9 mRNA. Consequently, CpG-DNA activated microglial cells in vitro and induced TNF-alpha, IL-12p40, IL-12p70, and NO. Furthermore, MHC class II, B7-1, B7-2, and CD40 molecules were up-regulated. In addition, phagocytic activity of microglia was enhanced. After intracerebroventricular injection of CpG-DNA, microglial cells were activated and produced TNF-alpha and IL-12p40 transcripts, as shown by in situ hybridization. These results indicate that microglia is sensitive to CpG-DNA. Thus, bacterial DNA containing CpG motifs could not only play an important role during infections of the CNS, but also might trigger and sustain Th1-dominated immunopathogenic reactions.

Adjuvants, Immunologic↗
PubMed2003

CpG-A oligonucleotides induce a monocyte-derived dendritic cell-like phenotype that preferentially activates CD8 T cells.

Human B cells and plasmacytoid dendritic cells recognize CpG motifs within microbial DNA via Toll-like receptor 9. Two functionally distinct types of CpG motif containing oligonucleotides (CpG ODN) have been described, CpG-A and CpG-B. In contrast to CpG-B, CpG-A induces high amounts of type I IFN (IFN-alpha and IFN-beta) in plasmacytoid dendritic cells. In the present study, we examined the effects of CpG-A on human primary monocytes. In PBMC stimulated with CpG-A and GM-CSF, monocytes showed excellent survival, increased in size and granularity, and within 3 days developed a dendritic cell-like phenotype that was characterized by down-regulation of CD14, partial up-regulation of CCR7, and an increased surface expression of costimulatory and Ag-presenting molecules. This effect could be inhibited by a combination of blocking Abs to type I IFN, and no such CpG-A-induced changes were observed in purified monocytes. Although IL-12 production by this dendritic cell-like phenotype required additional stimulation with CD40 ligand, this cell type spontaneously up-regulated IL-15 expression. Consistent with the known effect of IL-15 on effector and memory CD8 T cells, the frequency of CCR7(-)/CD45RA(-) CD8 T cells was selectively increased in allogeneic T cell assays. Furthermore, this dendritic cell type was more potent to support both the generation and the IFN-gamma production of autologous influenza matrix peptide-specific memory CD8 T cells as compared with dendritic cells generated in the presence of GM-CSF and IL-4. In conclusion, monocytes exposed to the cytokine milieu provided by CpG-A rapidly develop a dendritic cell-like phenotype that is well equipped to support CD8 T cell responses.

Adjuvants, Immunologic↗
PubMed2003

CpG oligodeoxynucleotides protect normal and SIV-infected macaques from Leishmania infection.

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of "human" CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania: Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania: Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.

Adjuvants, Immunologic↗
PubMed2004

Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-gamma synthesis.

Innate cellular production of IFN-gamma is suppressed after repeated exposure to LPS, whereas CpG-containing DNA potentiates IFN-gamma production. We compared the modulatory effects of LPS and CpG on specific cellular and cytokine responses necessary for NK-cell dependent IFN-gamma synthesis. C3H/HeN mice pretreated with LPS for 2 days generated 5-fold less circulating IL-12 p70 and IFN-gamma in response to subsequent LPS challenge than did challenged control mice. In contrast, CpG-pretreated mice produced 10-fold more circulating IFN-gamma without similar changes in IL-12 p70 levels, but with 10-fold increases in serum IL-18 relative to LPS-challenged control or endotoxin-tolerant mice. The role of IL-18 in CpG-induced immune potentiation was studied in splenocyte cultures from control, LPS-conditioned, or CpG-conditioned mice. These cultures produced similar amounts of IFN-gamma in response to rIL-12 and rIL-18. However, only CpG-conditioned cells produced IFN-gamma when cultured with LPS or CpG, and production was ablated in the presence of anti-IL-18R Ab. Anti-IL-18R Ab also reduced in vivo IFN-gamma production by >2-fold in CpG-pretreated mice. Finally, combined pretreatment of mice with LPS and CpG suppressed the production of circulating IFN-gamma, IL-12 p70, and IL-18 after subsequent LPS challenge. We conclude that CpG potentiates innate IFN-gamma production from NK cells by increasing IL-18 availability, but that the suppressive effects of LPS on innate cellular immunity dominate during combined LPS and CpG pretreatment. Multiple Toll-like receptor engagement in vivo during infection can result in functional polarization of innate immunity dominated by a specific Toll-like receptor response.

Adjuvants, Immunologic↗
PubMed1999

Differential regulation of the IL-12 p40 promoter and of p40 secretion by CpG DNA and lipopolysaccharide.

Challenge of macrophages with DNA containing an internal CpG motif results in IL-12 p40 secretion. In the presence of IFN-gamma, CpG DNA induces more p40 secretion than does LPS. In the RAW 264 macrophage cell line, both CpG DNA and LPS activate a p40 promoter-reporter construct, and the promoter response to either agent is augmented 2- to 5-fold by IFN-gamma. While either LPS or CpG DNA induces p40 promoter activity, only CpG DNA induces an increase in p40 mRNA or protein secretion. Even though IFN-gamma augmented LPS-driven p40 promoter activity in RAW 264 cells, the combination of IFN-gamma and LPS induced less p40 mRNA or protein secretion than the combination of IFN-gamma and CpG DNA. The ability of IFN-gamma to augment LPS or CpG DNA-induced p40 promoter activation was observed with truncation mutants of the IL-12 promoter containing as few as 250 bp 5' of the TATA box. Although LPS alone is a poor inducer of p40 transcription, both LPS and CpG DNA induce similar nuclear translocation of NF-kappaB. This binding is not augmented by costimulation with IFN-gamma. Thus, CpG DNA induces p40 transcription by a mechanism that includes NF-kappaB translocation; however, CpG DNA appears to induce other factor(s) necessary for p40 transcription. These results illustrate fundamental differences between CpG DNA and LPS with respect to activation of IL-12 p40 secretion.

Adjuvants, Immunologic↗
PubMed1999

Bacterial DNA or oligonucleotides containing unmethylated CpG motifs can minimize lipopolysaccharide-induced inflammation in the lower respiratory tract through an IL-12-dependent pathway.

To determine whether the systemic immune activation by CpG DNA could alter airway inflammation, we pretreated mice with either i.v. bacterial DNA (bDNA) or oligonucleotides with or without CpG motifs, exposed these mice to LPS by inhalation, and measured the inflammatory response systemically and in the lung immediately following LPS inhalation. Compared with non-CpG oligonucleotides, i. v. treatment with CpG oligonucleotides resulted in higher systemic concentrations of polymorphonuclear leukocytes, IL-10, and IL-12, but significantly reduced the concentration of total cells, polymorphonuclear leukocytes, TNF-alpha, and macrophage inflammatory protein-2 in the lavage fluid following LPS inhalation. The immunoprotective effect of CpG-containing oligonucleotides was dose-dependent and was most pronounced in mice pretreated between 2 and 4 h before the inhalation challenge, corresponding to the peak levels of serum cytokines. bDNA resulted in a similar immunoprotective effect, and methylation of the CpG motifs abolished the protective effect of CpG oligonucleotides. The protective effect of CpG oligonucleotides was observed in mice with either a disrupted IL-10 or IFN-gamma gene, but release of cytokines in the lung was increased, especially in the mice lacking IFN-gamma. In contrast, CpG DNA did not protect mice with a disrupted IL-12 gene against the LPS-induced cellular influx, even though CpG DNA reduced the release of TNF-alpha and macrophage inflammatory protein-2 in the lung. These findings indicate that CpG-containing oligonucleotides or bDNA are protected against LPS-induced cellular airway inflammation through an IL-12-dependent pathway, and that the pulmonary cytokine and cellular changes appear to be regulated independently.

Administration, Inhalation↗
PubMed2001

Antimetastatic effect of CpG DNA mediated by type I IFN.

The mechanisms involved in the antimetastatic effect of CpG-containing DNA were investigated in a mouse model of experimental metastasis. Tumor cell colony formation in lungs or livers of mice after i.v. inoculation with syngeneic fibrosarcoma or thymoma cells was determined. The i.v. injection of plasmid DNA or synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs before tumor cell application strongly inhibited metastasis. Because synthetic CpG-ODN was not directly tumor cytotoxic, the target cells for this CpG-ODN effect were determined. The cytotoxic activity on standard natural killer (NK) targets as well as on fibrosarcoma cells of splenic NK cells and NKT cell-containing liver mononuclear cells derived from CpG-ODN-treated mice was strongly enhanced. Participation of NK/NKT cells in the CpG-induced antimetastatic effect was demonstrated by reduction of the antimetastatic effect in mice depleted of NK/NKT cells and beta2-microglobulin-deficient mice. Neutralization of interleukin 12, interleukin 18, or IFN-gamma did not interfere with the CpG-induced antimetastatic effect. However, in sera of CpG-ODN-treated mice, high levels of IFN-alpha were detected, and in IFN-alpha/beta receptor-deficient mice, the CpG-ODN-induced antimetastatic effect was strongly reduced. These data indicate that CpG-ODNs activate NK/NKT cells for antimetastatic activity indirectly via IFN-alpha/beta receptor activation. The exploitation of the stimulatory activity of CpG-ODN for the innate immune system might be a useful strategy for antimetastatic therapy.

Animals↗
PubMed2005

Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs detected by Toll-like receptor 9 of dendritic cells and B cells have potent immunomodulatory effects. CpG oligodeoxynucleotides induce cytokines, activate natural killer cells, and elicit T-cell responses leading to antitumor effects, including improved efficacy of chemotherapeutic agents and, as we reported recently, synergy between CpG oligodeoxynucleotide 1826 and single-dose radiotherapy of an immunogenic mouse fibrosarcoma. The present study extends this finding to the fractionated radiotherapy of the fibrosarcoma tumor and assesses the ability of CpG oligodeoxynucleotide 1826 to increase the radioresponse of a tumor (nonimmunogenic fibrosarcoma). The experiments used a murine immunogenic fibrosarcoma tumor, fibrosarcoma growing in the leg of mice, and response to radiotherapy was assessed by tumor growth delay and tumor cure rate (TCD50, radiation dose yielding 50% tumor cure). Multiple s.c. peritumoral or i.t. administrations of CpG oligodeoxynucleotide 1826 at a dose of 100 microg per mouse were given when established tumors were 6 mm in diameter. Local tumor irradiation was initiated when tumors grew to 8 mm in diameter; radiation was delivered in 1 to 9 Gy fractions given twice daily separated by 6 to 7 hours for 5 consecutive days to achieve a total dose of 10 to 90 Gy. CpG oligodeoxynucleotide 1826, given as a single agent, had only a small antitumor effect, but it dramatically enhanced fibrosarcoma response to radiotherapy. Although 83.1 (79.2-90.0) Gy total dose were needed to achieve tumor cures in 50% of mice treated with radiotherapy alone, only 23.0 (11.5-32.7) Gy total dose were needed in mice treated with both CpG oligodeoxynucleotide 1826 and radiotherapy. The magnitude of potentiation of tumor radioresponse at the TCD50 level was by a factor of 3.61, a much higher value than that (a factor of 1.93) that we reported for single-dose radiotherapy. Mice cured of their tumors by combined CpG oligodeoxynucleotide 1826 plus radiotherapy were highly resistant to s.c. tumor take or development of tumor nodules in the lung from i.v. injected tumor cells when rechallenged with fibrosarcoma cells 100 to 120 days after the treatment, suggesting the development of a memory response. CpG oligodeoxynucleotide 1826 also increased radioresponse of the nonimmunogenic fibrosarcoma tumor by a factor of 1.41 and 1.73 when CpG oligodeoxynucleotide 1826 was given s.c. and i.t., respectively. These findings show that CpG oligodeoxynucleotides are highly potent enhancers of tumor response to both single-dose and fractionated radiation and as such have potential to improve clinical radiotherapy.

Animals↗
PubMed2001

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12.

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll-like receptor (TLR) expression. TLR1-TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDC but not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL-8, IP-10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN-alpha and IL-12 including bioactive IL-12 p70. Previous incubation of PDC with IL-3 decreased the amount of CpG-induced IFN-alpha and shifted the cytokine response in favor of IL-12. CpG ODN-activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN-stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL-12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activated T cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell-controlled restriction to foreign antigens is maintained.

Adjuvants, Immunologic↗
PubMed2005

Successful combination of local CpG-ODN and radiotherapy in malignant glioma.

Oligodeoxynucleotides containing CpG motifs (CpG-ODN) display broad immunostimulating activity and are currently under clinical trial in various malignancies, including recurrent glioblastomas. Combining CpG-ODN with another therapy that could induce antigen release might enhance tumor-specific immune response. We investigated whether radiotherapy (RT) could be associated advantageously to intratumoral injections of CpG-ODN. Fisher rats bearing 9L glioma were treated with various combinations of RT and CpG-28, an oligonucleotide with good immunostimulating activity. RT and CpG-28 induced complete tumor remission in one-third of the animals. When both treatments were combined, complete tumor remission was achieved in two-thirds of the animals (p < 0.001 when compared to non-treated rats, p < 0.03 when compared to CpG-28 alone). Such efficacy was not observed in nude mice, underlying the role of T cells in antitumor effects. The combination of both treatments appeared optimal when the delay between RT and CpG-28 administration was <3 days (from 100% survival for a 3 days delay, to 57% survival for a 21 days delay, p < 0.05). Tumor infiltration by immune cells and expression within tumors of the CpG receptor, TLR9, were not modified by irradiation. These results support an attractive strategy of sequential radiotherapy and immunotherapy by CpG-ODN and have potential implications for future clinical trials with CpG-ODN.

Animals↗
PubMed2005

CpG-oligodeoxynucleotides induce mobilization of hematopoietic progenitor cells into peripheral blood in association with mouse KC (IL-8) production.

The immune system of vertebrates detects bacterial DNA as a "danger signal" based on the presence of unmethylated CpG motifs. We examined whether oligodeoxynucleotides (ODNs) with CpG motifs (CpG-ODNs) also induce mobilization of hematopoietic progenitor cells (HPCs). Mice challenged with CpG-ODNs showed an increase in peripheral blood colony-forming units (CFU) with a peak at day 4 after treatment, associated with an increase, starting 30 min after CpG treatment, in serum levels of mouse keratinocyte-derived chemokine (mKC), a functional homolog of human interleukin (IL) 8; production of granulocyte-colony-stimulating factor (CSF) was also detected. Mobilization and mKC induction were sequence-specific and dose-dependent occurring even with low doses of CpG-ODNs. Interestingly, intestinal cells were involved in mKC production. HPC mobilization by CpG-ODNs was dependent on peripheral blood mononuclear cells since mobilization was reduced in neutrophil-depleted mice. Moreover, CpG-ODN treatment significantly increased G-CSF mobilizing capacity. Finally, pretreatment with an anti-mKC neutralizing antibody significantly reduced CpG-induced mobilization, further supporting a role for mKC. Thus, bacterial DNA is a "danger signal" not only for immune cells but also for hematopoietic cells, communicating the need for increased hematopoiesis during infections and for the renewal of the immune system. The HPC mobilization activity of CpG-ODNs will need to be considered in the design of treatment regimens for cancer clinical trials using CpG-ODNs in association with chemotherapy.

Animals↗