Effect of dendritic cells treated with CpG ODN on atopic dermatitis of Nc/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease and the pathogenesis of AD is associated with the release of various cytokines/chemokines due to activated Th(2) immune responses. Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotide in the context of particular base sequence (CpG motifs) are known to have the immunostimulatory activities in mice and to convert from Th(2) to Th(1) immune responses in AD. We aimed to investigate that CpG ODN, especially phosphodiester form, can stimulate the protective immunity in NC/Nga mice with AD. We isolated BMDCs from NC/Nga mice and then, cultured with GM-CSF and IL-4 for 6 days, and treated for 2 days by either phosphorothioate ODN or phosphodiester ODN. CpG ODN-treated DCs resulted in more production of IL-12. When CpG ODN-treated DCs were intravenously injected into the NC/Nga mice, the NC/Nga mice with CpG ODN-treated DCs showed significant improvement of AD symptoms and decrease of IgE level. Histopathologically, the NC/Nga mice skin with CpG ODN-treated DCs showed the decreased IL-4 and TARC expression comparing with non-injected mice. These results may suggest that phosphodiester CpG ODN-treated DCs might function as a potent adjuvant for AD in a mouse model.     

A protective role of locally administered immunostimulatory CpG oligodeoxynucleotide in a mouse model of genital herpes infection

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. We therefore investigated whether such a CpG-containing ODN (CpG ODN) given mucosally in the female genital tract could enhance innate immunity and protect against genital herpes infection. Groups of C57BL/6 mice were treated intravaginally with either CpG ODN or a non-CpG ODN control in the absence of any antigen either 2 days before or 4 h after an intravaginal challenge with a normally lethal dose of herpes simplex virus type 2 (HSV-2). Mice treated with CpG ODN exhibited significantly decreased titers of HSV-2 in their vaginal fluids compared with non-CpG ODN-treated mice. Furthermore, CpG ODN pretreatment significantly protected against development of disease and death compared to non-CpG ODN pretreatment. Most strikingly, CpG ODN conferred protection against disease and death even when given after the viral challenge. The CpG ODN-induced protection was associated with a rapid production of gamma interferon (IFN-gamma), interleukin-12 (IL-12), IL-18, and RANTES in the genital tract mucosa following CpG ODN treatment. The observed protection appeared to be dependent on IFN-gamma, IL-12, IL-18, and T cells, as CpG ODN pretreatment did not confer any significant protection in mice deficient in IFN-gamma, IL-12, IL-18, or T cells. Further, a complete protective immunity to reinfection was elicited in CpG ODN-treated, HSV-2-challenged mice, suggesting a role for mucosally administered CpG ODN in inducing the development of an acquired immune response in addition to its potent stimulation of innate immunity.     

CpG oligodeoxynucleotides enhance neonatal resistance to Listeria infection

Infection by Listeria monocytogenes causes serious morbidity and mortality during the neonatal period. Previous studies established that immunostimulatory CpG oligodeoxynucleotides (ODN) can increased the resistance of adult mice to many infectious pathogens, including Listeria. This work examines the capacity of CpG ODN to stimulate a protective immune response in newborns. Results indicate that dendritic cells, macrophages, and B cells from 3-day-old mice respond to CpG stimulation by secreting IFN-gamma, IL-12, and/or TNF-alpha. Spleen cells from CpG-treated neonates produce large amounts of cytokine and NO when exposed to bacteria in vitro. Newborns treated with CpG ODN are protected from lethal Listeria challenge and generate Ag-specific CD4 and CD8 T cells that afford long-term protection against subsequent infection. These results demonstrate that cellular elements of the neonatal immune system respond to stimulation by CpG ODN, thereby reducing host susceptibility to infectious pathogens.     

Stimulation of innate immune responses by CpG oligodeoxynucleotide in newborn lambs can reduce bovine herpesvirus-1 shedding

Stimulation of the innate immune system is potentially very important in neonates who have an immature adaptive immune system and vaccination cannot be used to reduce the risk of infection. CpG oligodeoxynucleotide (ODN) can stimulate innate immune responses in newborn chickens and mice, but similar studies are lacking in other mammalian species. We have shown previously that CpG ODN can both stimulate an acute-phase immune response and induce the antiviral effector molecule, 2'5'-A synthetase, in adult sheep. Therefore, the immunostimulatory activity of A class and B class CpG ODN was evaluated in newborn lambs, and the capacity of CpG ODN-induced responses to reduce viral shedding was evaluated following aerosol challenge with the respiratory pathogen, bovine herpesvirus-1 (BHV-1). In vitro CpG ODN stimulation of peripheral blood mononuclear cells (PBMC) isolated from newborn lambs (3-5 days old) and adult sheep induced equivalent CpG-specific proliferative responses and interferon-alpha (IFN-alpha) secretion. CpG ODN-induced IFN-alpha secretion by neonatal PBMCs was, however, significantly (p < 0.01) enhanced 6 days after subcutaneous (s.c.) injection of 100 microg/kg CpG ODN 2007. Newborn lambs injected s.c. with B class CpG ODN 2007 or the inverted GpC control ODN formulated in 30% Emulsigen (MVP Laboratories, Ralston, NE) displayed CpG ODN-specific increases in body temperature (p < 0.0001), serum 2'5'-A synthetase activity (p = 0.0015), and serum haptoglobin (p = 0.07). CpG ODN-treated lambs also displayed a transient reduction in viral shedding on day 2 postinfection (p < 0.05), which correlated (p < 0.03) with serum 2'5'-A synthetase levels on the day of viral challenge. These observations confirmed that CpG ODNs effectively activate innate immune responses in newborn lambs and CpG ODN-induced antiviral responses correlated with a reduction in viral shedding.     

A mouse model for immunization with ex vivo virus-infected dendritic cells

Dendritic cells (DCs) have been demonstrated to be an important if not essential inducer of cellular immune responses. The ability to grow these cells in vitro may open up new avenues for protective immunizations. In this study we have analyzed the virus-specific memory response generated following immunization with ex vivo-infected bone marrow-derived dendritic cells. We demonstrate that mouse DCs are efficiently infected with influenza virus but do not release infectious progeny virus. Ex vivo-infected DCs secrete interleukin-12 (IL-12) and induce a potent T helper (Th)1-like immune response when injected into mice. This was demonstrated by the generation of cytotoxic T lymphocytes, the production of high levels of gamma-interferon, and undetectable levels of IL-4 upon in vitro restimulation of splenocytes from immunized animals. In addition, the virus-specific antibody response is primarily of the IgG2a isotype, consistent with the expansion of Th1 cells. Animals immunized with DCs infected with X-31 influenza virus and challenged with PR8 influenza virus cleared the infection faster than animals not vaccinated. Thus, infected DCs efficiently activate the cellular immune response and induce heterosubtypic immunity in mice.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

Formulation with CpG oligodeoxynucleotides prevents induction of pulmonary immunopathology following priming with formalin-inactivated or commercial killed bovine respiratory syncytial virus vaccine

Commercial killed bovine respiratory syncytial virus (K-BRSV) and formalin-inactivated BRSV (FI-BRSV) tend to induce Th2-type immune responses, which may not be protective and may even be detrimental during subsequent exposure to the virus. In this study we assessed the ability of CpG oligodeoxynucleotides (ODNs) to aid in the generation of effective and protective BRSV-specific immune responses. Mice were immunized subcutaneously with FI-BRSV formulated with CpG ODN, Emulsigen (Em), CpG ODN and Em, or non-CpG ODN and Em. Two additional groups were immunized with K-BRSV or K-BRSV and CpG ODN. After two vaccinations, the mice were challenged with BRSV. FI-BRSV induced Th2-biased immune responses characterized by production of serum immunoglobulin G1 (IgG1) and IgE, as well as interleukin-4 (IL-4), by in vitro-restimulated splenocytes. Formulation of FI-BRSV with CpG ODN, but not with non-CpG ODN, enhanced serum IgG2a and IFN-gamma production by splenocytes, whereas serum IgE was reduced. Although the immune response induced by K-BRSV was not as strongly Th2 biased, the addition of CpG ODN to this commercial vaccine also resulted in a more Th1-type response. Furthermore, the addition of CpG ODN to the BRSV vaccine formulations resulted in enhanced neutralizing antibody responses. Significant production of IL-5, eotaxin, and eosinophilia was observed in the lungs of FI-BRSV- and K-BRSV-immunized mice. However, IL-5 and eotaxin levels, as well as the number of eosinophils, were decreased in the mice vaccinated with the CpG ODN-formulated vaccines. Finally, when formulated with CpG ODN, both FI-BRSV and K-BRSV significantly reduced virus production after challenge with BRSV.     

TLR-activated dendritic cells enhance the response of aged naive CD4 T cells via an IL-6-dependent mechanism

The most effective immunological adjuvants contain microbial products, such as TLR agonists, which bind to conserved pathogen recognition receptors. These activate dendritic cells (DCs) to become highly effective APCs. We assessed whether TLR ligand-treated DCs can enhance the otherwise defective response of aged naive CD4 T cells. In vivo administration of CpG, polyinosinic-polycytidylic acid, and Pam(3)CSK(4) in combination with Ag resulted in the increased expression of costimulatory molecules and MHC class II by DCs, increased serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that, in vitro, preactivation of DCs by TLR ligands makes them more efficient APCs for aged naive CD4 T cells. After T-DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR preactivation of both bone marrow-derived and ex vivo DCs improved responses. IL-6 produced by the activated DCs during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APCs by TLR ligands.     

Synergism between CpG-containing oligodeoxynucleotides and IL-2 causes dramatic enhancement of vaccine-elicited CD8+ T cell responses

Novel anticancer vaccination regimens that can elicit large numbers of Ag-specific T cells are needed. When we administered therapeutic vaccines containing the MHC class I-presented self-peptide tyrosinase-related protein (TRP)-2(180-188) and CpG-containing oligodeoxynucleotides (CpG ODN) to mice, growth of the TRP-2-expressing B16F1 melanoma was not inhibited compared with growth in mice that received control vaccinations. When we added systemic IL-2 to the TRP-2(180-188) plus CpG ODN vaccines, growth of B16F1 was inhibited in a CD8-dependent, epitope-specific manner. Vaccines containing TRP-2(180-188) without CpG ODN did not cause epitope-specific tumor growth inhibition when administered with IL-2. The antitumor efficacy of the different regimens correlated with their ability to elicit TRP-2(180-188)-specific CD8+ T cell responses. When we administered TRP-2(180-188) plus CpG ODN-containing vaccines with systemic IL-2, 18.2% of CD8+ T cells were specific for TRP-2(180-188). Identical TRP-2(180-188) plus CpG ODN vaccines given without IL-2 elicited a TRP-2(180-188)-specific CD8+ T cell response of only 1.1% of CD8+ T cells. Vaccines containing TRP-2(180-188) without CpG ODN elicited TRP-2(180-188)-specific responses of 2.8% of CD8+ T cells when administered with IL-2. There was up to a 221-fold increase in the absolute number of TRP-2(180-188)-specific CD8+ T cells when IL-2 was added to TRP-2(180-188) plus CpG ODN-containing vaccines. Peptide plus CpG ODN vaccines administered with IL-2 generated epitope-specific CD8+ T cells by a mechanism that depended on endogenous IL-6. This is the first report of synergism between CpG ODN and IL-2. This synergism caused a striking increase in vaccine-elicited CD8+ T cells and led to epitope-specific antitumor immunity.     

Cutting edge: Expression of functional CD137 receptor by dendritic cells

Interaction between dendritic cells (DCs) and T cells is a prerequisite for the initiation of a T cell response. The molecular nature of this interaction remains to be fully characterized. We report in this work that freshly isolated mouse splenic DCs and bone marrow-derived DCs express CD137 on the cell surface and in soluble form. Triggering CD137 increased the secretion of IL-6 and IL-12 from DCs. More importantly, infusion of an agonistic mAb to CD137 into naive mice enhanced the ability of DCs to stimulate T cell proliferation in response to both alloantigens and a nominal Ag in vitro. This enhancement of DC function is not mediated through activation of T cells, because the effect was also observed in RAG-1 knockout mice that lack T cells. Our findings implicate CD137 as an important receptor involved in the modulation of DC function.     

IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation

Cytokines that are induced by infection may contribute to the initiation of immune responses through their ability to stimulate dendritic cells (DCs). In this paper, we have addressed the role of IL-15 in DC activation, investigating its expression by DCs in response to three different signals of infection and examining its ability to stimulate DCs. We report that the expression of both IL-15 and the IL-15 receptor alpha-chain are increased in splenic DCs from mice inoculated with dsRNA (poly(I:C)), LPS, or IFN-alphabeta, and in purified murine splenic DCs treated with IFN-alphabeta in vitro. Furthermore, IL-15 itself was able to activate DCs, as in vivo or in vitro exposure of splenic DCs to IL-15 resulted in an up-regulation of costimulatory molecules, markedly increased production of IFN-gamma by DC and an enhanced ability of DCs to stimulate Ag-specific CD8(+) T cell proliferation. The magnitude of all of the IL-15-induced changes in DCs was reduced in mice deficient for the IFN-alphabeta receptor, suggesting a role for IFN-alphabeta in the stimulation of DCs by IL-15. These results identify IL-15 as a stimulatory cytokine for DCs with the potential for autocrine activity and link its effects to expression of IFN-alphabeta.     

Cytokine induction by a bacterial DNA-specific modified base

Unmethylated CpG dinucleotides in DNA contribute to a rapid inflammatory response in mammals. Here we show that N(6)-methyladenine (N(6)-MeA), a bacterium-specific modified base, also causes cytokine production. An oligodeoxyribonucleotide (ODN) containing N(6)-MeA induced cytokines when injected into mice. Co-injection of N(6)-MeA and CpG ODNs enhanced cytokines 2- to 3-fold, as compared with the injection of a CpG ODN alone. Plasmid DNA containing N(6)-MeA, complexed with cationic lipids, induced IL-12. These results indicate that the bacterium-specific base, in addition to the unmethylated CpG motif, triggers the mammalian immune response, and suggest that N(6)-MeA-containing DNA could be useful for cellular immunotherapy and DNA vaccine.     

A C-type CpG ODN accelerates wound healing via regulating fibroblasts and immune response

Abolished or delayed wound healing is a serious problem in clinical surgery, therefore, the new therapy for wound healing is needed. Synthetic oligodeoxynucleotides containing one or more CpG motifs (CpG ODN) has been reported to activate the immune system and improves skin wound healing. The aim of the present study was to evaluate the role of a new C-type CpG ODN in wound healing. We found that the CpG ODN promoted cell proliferation and collagen I production in human skin fibroblasts cells. Besides, we also investigated the effect of CpG ODN on the activation of immune cells. The macrophages and plasmacytoid dendritic cells (pDCs) were incubated with CpG ODN. CpG ODN activated macrophage and pDCs via regulating TLR9/MyD88/NF-κB pathway and TLR9/MyD88/IRF7 pathway, respectively. To further evaluate the effect of CpG ODN on wound healing in vivo a wound healing model was established in mice. The results showed that CpG ODN treatment accelerated wound healing in mice. CpG ODN increased cytokines secretion in wound skin and elevated the ratio of CD4 ⁺ and CD8 ⁺ T cells in the spleen. Our results showed that CpG ODN accelerated wound healing, which was partly due to the regulation of fibroblasts and immune response. The findings suggested that the CpG ODN might be a proper medicament for the treatment of wound healing.     

Therapeutic effect of CpG motifs on the development of chronic graft-versus-host disease in mice

Transferring DBA/2 spleen cells into (C57BL/10xDBA/2) F1 (referred to as BDF1) mice induces a chronic graft-versus-host disease (GVHD), characterized by the production of Th(2) cytokines, hypergammaglobulinemia, and immune complex-mediated glomerulonephritis that resembles systemic lupus erythematosus. DNA motif consisting of an unmethylated CpG dinucleotide flanked by two 5' purines and two 3' pyrimidines (CpG ODN) induces Th(1) cytokine production in mice. This study examines the effect of administering CpG ODN to mice undergoing chronic GVHD, based on the premise that altering Th(1)/Th(2) activity might beneficially impact on disease progression.GVHD BDF1 mice injected with DBA/2 spleen cells were treated with weekly intraperitoneal injection of 50 microg CpG ODN. This treatment significantly suppressed the production of IgG anti-DNA autoantibody and reduced the development of glomerulonephritis. Serum IgG2a titers were higher in the CpG ODN than in non-CpG control group, whereas IgG1 titers were unchanged. As predicted, IFN-gamma levels were significantly higher in the CpG ODN-treated group, while IL-4 levels were lower, resulting in a shift in the Th(1)/Th(2) cytokine ratio. Results suggest that CpG ODN administration may be of therapeutic benefit in chronic GVHD.     

Ox40 costimulation enhances the development of T cell responses induced by dendritic cells in vivo.

Dendritic cells (DCs) are bone marrow-derived APCs that display unique properties aimed at stimulating naive T cells. Several members of the TNF/TNFR families have been implicated in T cell functions. In this study, we examined the role that Ox40 costimulation might play on the ability of DCs to regulate CD4(+) and CD8(+) T cell responses in vivo. Administration of anti-mouse Ox40 mAb enhanced the Th response induced by immunization with Ag-pulsed DCs, and introduced a bias toward a Th1 immune response. However, anti-Ox40 treatment enhanced the production of Th2 cytokines in IFN-gamma(-/-) mice after immunization with Ag-pulsed DCs, suggesting that the production of IFN-gamma during the immune response could interfere with the development of Th2 lymphocytes induced by DCs. Coadministration of anti-Ox40 with DCs during Ag rechallenge enhanced both Th1 and Th2 responses induced during a primary immunization with DCs, and did not reverse an existing Th2 response. This suggests that Ox40 costimulation amplifies an ongoing immune response, regardless of Th differentiation potential. In an OVA-TCR class II-restricted adoptive transfer system, anti-Ox40 treatment greatly enhanced the level of cytokine secretion per Ag-specific CD4(+) T cell induced by immunization with DCs. In an OVA-TCR class I-restricted adoptive transfer system, administration of anti-Ox40 strongly enhanced expansion, IFN-gamma secretion, and cytotoxic activity of Ag-specific CD8(+) T cells induced by immunization with DCs. Thus, by enhancing immune responses induced by DCs in vivo, the Ox40 pathway might be a target for immune intervention in therapeutic settings that use DCs as Ag-delivery vehicles.     

A dendritic cell population generated by a fusion of GM-CSF and IL-21 induces tumor-antigen-specific immunity

We have previously shown that the fusion of GM-CSF and IL-21 (GIFT-21) possesses a potent immune stimulatory effect on myeloid cells. In this study, we define the effect of GIFT-21 on naive murine monocytes (GIFT-21 dendritic cells [DCs]), which express increased levels of Gr-1, CD45R, MHC class I, CD80, CD86, and CXCR4 and suppress CD11c and MHC class II. Compared with conventional dendritic cells, GIFT-21 DCs produced substantially more CCL2, IL-6, TNF-α, and IFN-α and induced significantly greater production of IFN-γ by CD8(+) T cells in MHC class I-restricted Ag presentation assays. B16 melanoma and D2F2 Neu breast cancer growth was inhibited in mice treated with Ag-naive GIFT-21 DCs. This effect was lost in CD8(-/-) and CCR2(-/-) mice and when mice were treated with β(2)-microglobulin-deficient GIFT-21 DCs, indicating that GIFT-21 DCs migrated to and sampled from the tumors to present tumor Ags to CCL2 recruited CD8(+) T cells via MHC class I. We propose that autologous GIFT-21 DCs may serve as a cell therapy platform for the treatment of cancer.     

Immunization with murine breast cancer cells treated with antisense oligodeoxynucleotides to type I insulin-like growth factor receptor induced an antitumoral effect mediated by a CD8+ response involving Fas/Fas ligand cytotoxic pathway

We have demonstrated that in vivo administration of phosphorothioate antisense oligodeoxynucleotides (AS[S]ODNs) to type I insulin-like growth factor receptor (IGF-IR) mRNA resulted in inhibition of C4HD breast cancer growth in BALB/c mice. The present study focused on whether in vivo administration of C4HD tumor cells pretreated with IGF-IR AS[S]ODN and irradiated could provide protection against C4HD wild-type tumor challenge and also on elucidating the mechanism mediating this effect. Our results showed that mice immunized with IGF-IR AS[S]ODN-treated C4HD cells experienced a growth inhibition of 53.4%, 61.6%, and 60.2% when compared with PBS-treated mice, wild-type C4HD cell-injected mice, or phosphorothioate sense oligodeoxynucleotide-treated C4HD cell-injected mice, respectively. The protective effect was C4HD-specific, because no cross-protection was observed against other syngeneic mammary tumor lines. The lack of protection against tumor formation in nude mice indicated that T cells were involved in the antitumoral response. Furthermore, cytotoxicity and splenocyte proliferation assays demonstrated that a cellular CD8(+)-dependent immune response, acting through the Fas/Fas ligand death pathway, could be mediating the antitumor effect induced by immunization with AS[S]ODN-treated cells. Immunization also induced splenocytes to produce Ag-dependent IFN-gamma, indicating the presence of a type 1 response. We demonstrated for the first time that IGF-IR AS[S]ODN treatment of breast cancer cells induced expression of CD86 and heat shock protein 70 molecules, both involved in the induction of the immunogenic phenotype. Immunization with these tumor immunogens imparted protection against parental tumor growth through activation of a specific immune response.     

IL-12p70-dependent Th1 induction by human B cells requires combined activation with CD40 ligand and CpG DNA

The detection of microbial molecules via Toll-like receptors (TLR) in B cells is not well characterized. In this study, we found that both naive and memory B cells lack TLR4 (receptor for LPS) but express TLR9 (receptor for CpG motifs) and produce IL-6, TNF-alpha, and IL-10 upon stimulation with CpG oligonucleotides (ODN), synthetic mimics of microbial DNA. Consistent with the lack of TLR4, purified B cells failed to respond to LPS. Similar to CpG ODN, CD40 ligand (CD40L) alone induced IL-6, TNF-alpha, and IL-10. Production of these cytokines as well as IgM synthesis was synergistically increased when both CpG ODN and CD40L were combined. Unlike IL-6, TNF-alpha, and IL-10, the Th1 cytokine IL-12p70 was detected only when both CpG ODN and CD40L were present, and its induction was independent of B cell receptor cross-linking. CpG ODN did not increase the capacity of CD40L-activated B cells to induce proliferation of naive T cells. However, B cells activated with CpG ODN and CD40L strongly enhanced IFN-gamma production in developing CD4 T cells via IL-12. Together, these results demonstrate that IL-12p70 production in human B cells is under the dual control of microbial stimulation and T cell help. Our findings provide a molecular basis for the potent adjuvant activity of CpG ODN to support humoral immune responses observed in vivo, and for the limited value of LPS.     

Intratumoral delivery of CpG-conjugated anti-MUC1 antibody enhances NK cell anti-tumor activity

Monoclonal antibodies (mAbs) against tumor-associated antigens are useful anticancer agents. Antibody-dependent cellular cytotoxicity (ADCC) is one of the major mechanisms responsible for initiating natural killer cell (NK)-mediated killing of tumors. However, the regulation of ADCC via NK cells is poorly understood. We have investigated the cytolytic activity of NK cells against pancreatic cancer cells that were coated with an antibody directed against the human tumor antigen, Mucin-1 designated HMFG-2, either alone or conjugated to CpG oligodeoxynucleotide (CpG ODN). Conjugated antibodies were tested for their ability to elicit ADCC in vitro and in vivo against pancreatic cancer cells. NK cells cultured in the presence of immobilized CpG ODN, HMFG-2 Ab, or CpG ODN-conjugated HMFG-2 Ab were able to up-regulate perforin similarly. Interestingly, a significant higher ADCC was observed when CpG ODN-conjugated HMFG-2-coated tumor cells were co-cultured with NK cells compared to unconjugated HMFG-2 Ab or CpG ODN alone. Moreover, MyD88-deficient NK cells can perform ADCC in vitro. Furthermore, intratumoral injections of CpG ODN-conjugated HMFG-2 induced a significant reduction in tumor burden in vivo in an established model of pancreatic tumor in nude mice compared to CpG ODN or the HMFG-2 alone. Depletion of macrophages or NK cells before treatment confirmed that both cells were required for the anti-tumor response in vivo. Results also suggest that CpG ODN and HMFG-2 Ab could be sensed by NK cells on the mAb-coated tumor cells triggering enhanced ADCC in vitro and in vivo.     

CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.