Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.     

CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

Unmethylated cytosine-phosphate-guanine (CpG) dinucleotides flanked by specific bases in bacterial DNA induce a favorable immune response by acting as danger signals to the host. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) also act like the unmethylated CpG oligonucleotides in bacterial DNA. In the present study, we investigated the effects of synthetic CpG-ODN on the protection of olive flounder (Paralichthys olivaceus) against infection by Philasterides dicentrarchi, a pathogen of scuticociliatosis, through two consecutive experiments (trial I and II). Fish were intraperitoneally (i.p.) injected with CpG-ODN 1668 or GpC-ODN 1720 at different doses (3 microg in trial I and 10 microg in trial II), and after one week the fish were i.p. challenged with P. dicentrarchi. In both trial I and II, fish injected with CpG-ODN 1668 showed significantly higher serum scuticocidal activity than fish injected with PBS alone, while the scuticocidal activity disappeared by heat-inactivation. This result suggests that CpG-ODN might activate an alternative pathway of complement of olive flounder, and complement-mediated killing might be an important innate immune factor in the resistance against P. dicentrarchi infection. Although the cumulative mortality was largely different between trials I and II, the relative survival rate of fish injected with a high dose of CpG-ODN 1668 was considerably higher than that of fish injected with a low dose of this ODN, while the relative survival rate was not different between fish injected with the high dose and low dose of GpC-ODN 1720. The results of the present study suggest that CpG-ODNs may be used as potential immunostimulants to lessen cultured fish loss caused by scuticociliates.     

Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs.

Immune stimulatory oligodeoxynucleotides (ODN) with unmethylated CpG motifs are potent inducers of both innate and adaptive immunity. It initially appeared that a single type of optimal CpG motif would work in all applications. We now report that specific motifs of CpG ODN can vary dramatically in their ability to induce individual immune effects and that these differences impact on their antitumor activity in different tumor models. In particular, a distinct type of CpG motif, which has a chimeric backbone in combination with poly(G) tails, is a potent inducer of NK lytic activity but has little effect on cytokine secretion or B cell proliferation. One such NK-optimized CpG ODN (1585) can induce regression of established melanomas in mice. Surprisingly, no such therapeutic effects were seen with CpG ODN optimized for activation of B cells and Th1-like cytokine expression (ODN 1826). The therapeutic effects of CpG 1585 in melanoma required the presence of NK but not T or B cells and were not associated with the induction of a tumor-specific memory response. In contrast, CpG 1826, but not CpG 1585, was effective at inducing regression of the EL4 murine lymphoma; this rejection was associated with the induction of a memory response and although NK cells were necessary, they were not sufficient. These results demonstrate that selection of optimal CpG ODN for cancer immunotherapy depends upon a careful analysis of the cellular specificities of various CpG motifs and an understanding of the cellular mechanisms responsible for the antitumor activity in a particular tumor.     

NK- and CD8(+) T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides.

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8(+) T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8(+) effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

Intratracheal synthetic CpG oligodeoxynucleotide causes acute lung injury with systemic inflammatory response

Bacterial genome is characterized by frequent unmethylated cytosine-phosphate-guanine (CpG) motifs. Deleterious effects can occur when synthetic oligodeoxynucleotides (ODN) with unmethylated CpG dinucleotides (CpG-ODN) are administered in a systemic fashion. We aimed to evaluate the effect of intratracheal CpG-ODN on lung inflammation and systemic inflammatory response. C57BL/6J mice received intratracheal administration of CpG-ODN (0.01, 0.1, 1.0, 10, or 100 μM) or control ODN without CpG motif. Bronchoalveolar lavage (BAL) fluid was obtained 3 or 6 h or 1, 2, 7, or 14 days after the instillation and subjected to a differential cell count and cytokine measurement. Lung permeability was evaluated as the BAL fluid-to-plasma ratio of the concentration of human serum albumin that was injected 1 h before euthanasia. Nuclear factor (NF)-κB DNA binding activity was also evaluated in lung homogenates. Intratracheal administration of 10 μM or higher concentration of CpG-ODN induced significant inflammatory cell accumulation into the airspace. The peak accumulation of neutrophils and lymphocytes occurred 1 and 2 days after the CpG-ODN administration, respectively. Lung permeability was increased 1 day after the 10 μM CpG-ODN challenge. CpG-ODN also induced nuclear translocation of NF-κB and upregulation of various inflammatory cytokines in BAL fluid and plasma. Histopathology of the lungs and liver revealed acute lung injury and liver damage with necrosis, respectively. Control ODN without CpG motif did not induce any inflammatory change. Since intratracheal CpG-ODN induced acute lung injury as well as systemic inflammatory response, therapeutic strategies to neutralize bacterial DNA that is released after administration of bactericidal agents should be considered.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-kappaB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-kappaB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-alpha expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Lipoplexes prepared from cationic liposomes and mammalian DNA induce CpG-independent, direct cytotoxic effects in cell cultures and in mice

BACKGROUND: Recent studies demonstrated the cytotoxic activity of bacterial DNA (pDNA) complexed with cationic lipids. This cytotoxicity is related to the ability of pDNA to induce potently the immune system, which is associated with release of inflammatory cytokines. Both activities seem to be related to the nonmethylated CpG sequences present in the pDNA. Here we study the cytotoxic activity of nonbacterial DNA complexed with cationic lipids against various tumor cell lines. METHODS: Various nucleic acids complexed with cationic liposomes were prepared and their cytotoxic activity was studied in cell cultures and in tumor-bearing mice. Cell uptake of lipoplexes was evaluated, and mechanism of DNA cytotoxic activity was studied. RESULTS: We found that nonbacterial (vertebrate) genomic DNA when complexed with cationic lipids is highly cytotoxic against C-26 and M-109 tumor cells. Cationic lipids alone were not toxic to these cells. The cytotoxic activity does not result from nonspecific acidification of the intracellular milieu, as substitution of DNA by poly-L-glutamate did not result in cytotoxicity, although the level of uptake of anionic charges per cell was similar to that of the nucleic acids, suggesting that this cytotoxic effect is specific to nucleic acids. By studying the nucleic acid fate using confocal microscopy, we found that cytotoxicity correlated with the release of DNA into the cytoplasm following uptake of lipoplexes. Injection of calf thymus DNA-based lipoplexes to mice with peritoneal C-26 metastases resulted in doubling of median survival time and long-term survival in 20% of the tumor-bearing mice. Judging by low levels of IFN-gamma, TNF-alpha and IL-6 in the treated mice, this effect cannot be ascribed to Th-1 inflammation, but rather to a direct cytotoxic effect on the tumor cells. CONCLUSIONS: The above data provide a new insight into the mechanisms of lipoplex-mediated antitumor effects in vitro and in vivo and new perspectives in cancer therapy.     

Phosphorothioate oligodeoxynucleotides promote the in vitro development of human allergen-specific CD4+ T cells into Th1 effectors

DNA vaccination is an effective approach in inducing the switch of murine immune responses from a Th2 to a Th1 profile of cytokine production that has been related to the activity of unmethylated CpG motifs present in bacterial, but not mammalian, DNA. We report here that some synthetic phosphorothioate, but not phosphodiester, oligodeoxynucleotides (ODNs) were able to induce B cell proliferation and to shift the in vitro differentiation of Dermatophagoides pteronyssinus group 1-specific human CD4+ T cells from atopic donors into Th cell effectors showing a prevalent Th1, instead of Th2, cytokine profile. This latter effect was completely blocked by the neutralization of IL-12 and IFN (alpha and gamma) in bulk culture, suggesting that the Th1-inducing activity of phosphorothioate ODNs was mediated by their ability to stimulate the production of these cytokines by monocytes, dendritic, and NK cells. Cytosine methylation abolished the Th1-inducing activity of ODNs; however, CpG dinucleotide-containing ODNs exhibited the Th1-shifting effect independently of the presence or the absence of CpG motifs (5'-pur-pur-CpG-pyr-pyr-3'). Moreover, the inversion of CpG to GpC resulted only in a partial reduction of this activity, suggesting that the motif responsible for the Th1-skewing effect in humans is at least partially different from that previously defined in mice. These results support the concept that the injection of allergens mixed to, or conjugated with, appropriate ODNs may provide a novel allergen-specific immunotherapeutic regimen for the treatment of allergic disorders.     

Signaling through NK cell-associated CD137 promotes both helper function for CD8+ cytolytic T cells and responsiveness to IL-2 but not cytolytic activity

NK cells possess both effector and regulatory activities that may be important during the antitumor immune response. In fact, the generation of antitumor immunity by the administration of an agonistic mAb against CD137 is NK cell-dependent. In this study, we report that NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-gamma secretion, but not their cytolytic activity. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or "helper" activity for CD8(+)CTL. Furthermore, tumor-specific CTL activity against P815 tumor Ags was abrogated following anti-CD137 treatment in NK-depleted mice. We further demonstrate that CD137-stimulated helper NK cells expressed the high-affinity IL-2R and were hyperresponsive to IL-2. Taken together with previous findings that CD137 is a critical receptor for costimulation of T cells, our findings suggest that CD137 is a stimulatory receptor for NK cells involved in the crosstalk between innate and adaptive immunity.     

Treatment of infectious diseases with immunostimulatory oligodeoxynucleotides containing CpG motifs

Bacterial DNA with CpG motifs can efficiently stimulate the vertebrate immune system. Thus, synthetic oligodeoxynucleotides that contain such CpG motifs (CpG-ODN) are currently used in preclinical and clinical studies to develop new allergy or cancer therapies and vaccine adjuvants. Recent animal studies indicate that CpG-ODN therapies can also be used for successful treatment of infections caused by bacteria, parasites or viruses. In these experiments, innate and adaptive immune responses against pathogens were augmented by CpG-ODN and subsequently induced resistance against infectious diseases. The stimulation of dendritic cells played a central role for the therapeutic effect of CpG-ODN. However, CpG-ODN can also have negative side effects, which accelerate disease progression in some viral infections. Clinical studies with CpG-ODN will determine their potential for the therapy of infectious diseases in humans.     

CpG寡脱氧核苷酸触发中华绒螯蟹酚氧化酶原系统信号传导途径的研究

为探讨CpG寡脱氧核苷酸(CpG-oligodeoxynucleotides,CpG-ODN)激活中华绒螯蟹血细胞酚氧化酶原系统(prophenoloxidase system,proPO系统)的信号传导途径,使用一定剂量的CpGODN-1670、ODN-R以及几种细胞信号传导的激活剂或抑制剂体外处理中华绒螯蟹血细胞,通过检测胞内外酚氧化酶(PO)活性的变化,对CpG ODN触发proPO激活系统的信号传导途径进行评价。结果显示,ODN-1670与ODN-R均可触发蟹血细胞proPO激活系统,试验剂量的ODN-1670可促进血细胞内外已有的proPO转化为PO,而对proPO颗粒的释放具有一定的抑制作用;ODN-R则不仅可使proPO转化为PO,还可促进血细胞脱颗粒。两者的信号传导途径相似,可能都包含了G-蛋白介导的蛋白激酶C(PKC)途径,酪氨酸蛋白激酶(RTK)途径对ODN-1670的触发proPO激活系统的活化过程进行负调控。       

Reduction of 1-methyl-1-nitrosourea-induced mammary gland carcinoma by in vivo application of immunostimulatory CpG motifs in Sprague-Dawley rats

In the present work the role of 13-cis retinoic acid and CpG oligodeoxynucleotides (CpG-ODN) in a 1-methyl-1-nitrosourea (MNU)-induced mammary gland carcinoma animal model was investigated. Treatment with both components, applied either alone or in combination, induced a significant decrease of the tumour burden and the volume of tumours only in rats that received CpG-ODN (p = 0.046, compared to the MNU control group). The data indicate that the Th-1 biased immunostimulatory capacities of CpG motifs may play a significant role in induction of protective immune responses against mammary gland tumours in Sprague-Dawley rats.     

CpG-containing oligodeoxynucleotides act through TLR9 to enhance the NK cell cytokine response to antibody-coated tumor cells

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcgammaRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-gamma (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1alpha was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 +/- 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-gamma compared with mice receiving either agent alone. TLR9-/- animals reconstituted with TLR9+/+ NK cells secreted IFN-gamma in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.     

CpG-Oligodeoxynucleotides activate tyrosinase-related protein 2–specific T lymphocytes but do not lead to a protective tumor-specific memory response

Purpose: Peritumoral CpG-oligodeoxynucleotide (ODN) treatment has been successful in tumor mouse models expressing strong antigens to induce activation of tumor-specific CD8⁺ T lymphocytes which contribute to the control of tumor growth. To get near to clinical reality, the tumor-specific CD8⁺ response was investigated in mice bearing the weakly immunogenic B16 melanoma tumor and using the melanocyte differentiation tyrosinase-related protein 2 (TRP-2) as a tracking antigen. Methods: The expansion and activation of TRP-2–specific T lymphocytes by CpG-ODNs was analyzed by tetramer staining and IFN- production assays, while the activity of these cells in both memory and primary response was evaluated in vivo. Results: After CpG-ODN treatment, the number of TRP-2 tetramer-stained CD8⁺ T lymphocytes was not significantly modified, but these cells produced higher levels of interferon (IFN-) in response to the antigen than those from untreated mice. Mice possessing these activated T lymphocytes, when evaluated for their antitumor memory response, showed marginal protection against intravenous (i.v.) and subcutaneous (s.c.) tumor rechallenge. These cells were not crucial for the control of primary tumor growth since strong reduction of subcutaneous tumor was observed after CpG-ODN treatment in both CD8⁺ T cell depleted or nondepleted mice. On the contrary, NK cell depletion markedly reduced CpG-ODN-induced tumor growth inhibition. Conclusions: Altogether, these data indicate the CpG treatment activates tumor-reactive effector CD8⁺ T lymphocytes, but, paralleling recent clinical observations, our model indicates that the mere activation of antitumor T cells is insufficient to result in a clinical response.Abbreviations CpG unmethylated CpG dinucleotides - ODNs oligodeoxynucleotides - TLR9 toll-like receptor 9 - TRP-2 tyrosinase-related protein 2     

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo

Background

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo.

Methods

Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured.

Results

In WT mice, CpG-ODN induced a strong activation of pulmonary NFκB as well as a significant increase in pulmonary TNF-α and IL-1β mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice.

Conclusion

This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.     

Immunostimulation of dendritic cells by cationic liposomes

A nano-aggregate liposome-polycation-DNA (LPD), composed of a cationic lipid, protamine and plasmid DNA was found to effectively deliver a human papillomavirus (HPV)-E7 epitope antigen to the antigen presenting cells of the immune system, eliciting enhanced anti-tumor immune responses in mouse models of cervical carcinoma. Both the cationic liposome and plasmid DNA were essential for the full immunostimulation activity of LPD. Interestingly, cationic liposomes alone could stimulate the antigen presenting dendritic cells (DC) leading to the expression of co-stimulatory molecules, CD80 and CD86. However, cationic lipids could not stimulate DC for the expression of pro-inflammatory cytokines. Moreover, they were unable to enhance the expression of NF-κB, suggesting that dendritic cells stimulation by cationic lipids is signaled through an NF-κB independent mechanism. DC stimulation was specific to cationic lipids, the zwitterionic and anionic lipids showed little or no activity. The ability of different cationic lipids to stimulate the expression of co-stimulatory molecules on DC varied significantly. In general, the cationic lipids bearing ethyl phosphocholine head groups were better stimulants than their trimethylammonium counterparts. In case of the cationic lipids bearing trimethyl ammonium head groups, the ones bearing unsaturated or shorter saturated hydrophobic chains exhibited enhanced immunostimulatory activity. The LPS-induced TNF-α expression by dendritic cells was inhibited by active cationic lipids but not the inactive ones, suggesting the possible involvement of lipopolysaccharide binding protein (LBP) in cationic lipid mediated DC stimulation. Based on the structure-specific activation of dendritic cells by cationic lipids, a model for the immunostimulation of DC by such lipids is proposed.     

Cancer immunotherapy with CpG-ODN

Bacterial DNA and synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) are the ligands for the Toll-like receptor 9 (TLR9), which is expressed by B-lymphocytes and a subset of dendritic cells. CpG-ODN are strong activators of both innate and specific immunity, and drive the immune response towards the Th1 phenotype. Given the promising results obtained in several experimental models of allergies or infections, CpG-ODN are now entering clinical trials for these diseases. In cancer, promising approaches combined CpG-ODN with tumor antigens, monoclonal antibodies or dendritic cells. When no relevant tumor antigen is known, CpG-ODN can be used alone to activate locally the innate immunity and trigger a tumor-specific immune response, overcoming the need for the identification of a tumoral antigen. Preclinical models have shown impressive results and several clinical trials are on-going worldwide in melanoma, lymphoma, renal carcinoma, breast cancer and glioblastoma.     

Identification of immunostimulatory oligodeoxynucleotide from Escherichia coli genomic DNA

Bacterial DNA containing immunostimulatory CpG motifs can stimulate antigen-presenting cells to express costimulatory molecules and to produce various cytokines in vivo and in vitro. In this study, we fragmented macromolecular E.coli genomic DNA with DNase I, and analyzed the ability of the resulting DNA fragments to induce the NF-kappaB activation and humoral immune response. Furthermore, using computational analysis and luciferase assay for synthetic ODNs based on the sequence of the immunostimulatory DNA fragments (DF-ODNs), an active component of DF-ODNs sequences was investigated. Experimental results demonstrated that DF-ODN is optimal for the NF-kappaB-responsive promoter activation in the mouse macrophage cell line and the humoral immune response in vivo. In agreement with the activity of the DFODNs processed by DNase I, a synthetic ODN based on the DF-ODN sequences is potent at inducing IL-12 mRNA expression in primary dendritic cells. These results suggest that the discovery and characterization of a highly active natural CpG-ODN may be achieved by the analyses of bacterial DNA fragments generated by a nuclease activity.