Use of CpG oligodeoxynucleotides as immune adjuvants

Summary: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs directly stimulate human B cells and plasmacytoid dendritic cells (pDCs), thereby promoting the production of T helper 1 (Th1) and pro‐inflammatory cytokines and the maturation/activation of professional antigen‐presenting cells. These activities enable CpG ODNs to act as immune adjuvants, accelerating and boosting antigen‐specific immune responses by 5–500‐fold. These effects are optimized by maintaining close physical contact between the CpG DNA and the immunogen. Animal challenge models establish that protective immunity can be accelerated and magnified by coadministering CpG DNA with vaccines. Ongoing clinical studies indicate that CpG ODNs are safe and well tolerated when administered as adjuvants to humans, and in some cases, they increase vaccine‐induced immune responses.     

Adjuvant Activity of CpG Oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs directly stimulate human B cells and plasmacytoid dendritic cells (pDCs), thereby promoting the production of Th1 and proinflammatory cytokines and the maturation/activation of professional antigen-presenting cells. These activities enable CpG ODNs to act as immune adjuvants, accelerating and boosting antigen-specific immune responses by 5- to 500-fold. The CpG motifs present in bacterial DNA plasmids may contribute to the immunogenicity of DNA vaccines. Ongoing clinical studies indicate that CpG ODNs are safe and well tolerated when administered as adjuvants to humans and can improve vaccine-induced immune responses.     

Adjuvant activity of CpG oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs directly stimulate human B cells and plasmacytoid dendritic cells (pDCs), thereby promoting the production of Th1 and proinflammatory cytokines and the maturation/activation of professional antigen-presenting cells. These activities enable CpG ODNs to act as immune adjuvants, accelerating and boosting antigen-specific immune responses by 5- to 500-fold. The CpG motifs present in bacterial DNA plasmids may contribute to the immunogenicity of DNA vaccines. Ongoing clinical studies indicate that CpG ODNs are safe and well tolerated when administered as adjuvants to humans and can improve vaccine-induced immune responses.     

Formulation with CpG oligodeoxynucleotides increases cellular immunity and protection induced by vaccination of calves with formalin-inactivated bovine respiratory syncytial virus

Vaccination of calves with formalin-inactivated bovine respiratory syncytial virus (FI-BRSV) induces low levels of cellular immunity that may not be protective. Since inactivated and subunit vaccines formulated with CpG oligodeoxynucleotides (ODNs) have been shown to induce cellular immune responses, we studied the ability of a FI-BRSV vaccine formulated with CpG ODN to elicit cellular immunity against BRSV. Neonatal calves were immunized with FI-BRSV, FI-BRSV formulated with CpG ODN or medium and challenged with BRSV after two immunizations. Calves vaccinated with FI-BRSV formulated with CpG ODN developed increased numbers of IFN-gamma secreting cells in the peripheral blood and broncho-tracheal lymph nodes and enhanced BRSV-specific serum IgG2 in comparison to FI-BRSV immunized animals. Calves that received the FI-BRSV vaccine formulated with CpG ODN also experienced a reduction in the amount of BRSV in the lung tissue. Based on these observations, CpG ODN appears to be a suitable candidate adjuvant for inactivated BRSV vaccines.     

Hierarchical recognition of CpG motifs expressed by immunostimulatory oligodeoxynucleotides

Synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs trigger human PBMC to proliferate and secrete Ig, cytokines and chemokines. CpG ODN have entered clinical trials, and show promise as vaccine adjuvants, antiallergens, and for the treatment of infectious diseases and cancer. ODNs under consideration for human use vary in the sequence, number and location of the CpG motifs they contain. Yet little is known of the magnitude of the immune response elicited by these diverse ODNs, or the rules governing their interaction with immune cells. This work compares the proliferative, IgM, IL-6 and IP-10 response of PBMC from normal donors to a diverse panel of CpG ODNs. Results indicate that ODNs expressing 3-4 different CpG motifs are strongly stimulatory. The location of these motifs is important, with those at the 5' end exerting the greatest influence on ODN activity. These findings provide a basis for the rational design of ODNs optimized for clinical use.     

Therapeutic Applications of Synthetic CpG Oligodeoxynucleotides as TLR9 Agonists for Immune Modulation

Vertebrate toll-like receptors (TLRs) sense invading pathogens by recognizing bacterial and viral structures and, as a result, activate innate and adaptive immune responses. Ten human functional TLRs have been reported so far; three of these (TLR7, 8, and 9) are expressed in intracellular compartments and respond to single-stranded nucleic acids as natural ligands. The pathogen structure selectively recognized by TLR9 in bacterial or viral DNA was identified to be CpG dinucleotides in specific sequence contexts (CpG motifs). Short phosphorothioate-stabilized oligodeoxynucleotides (ODNs) containing such motifs are used as synthetic TLR9 agonists, and different classes of ODN TLR9 agonists have been identified with distinct immune modulatory profiles. The TLR9-mediated activation of the vertebrate immune system suggests using such TLR9 agonists as effective vaccine adjuvants for infectious disease, and for the treatment of cancer and asthma/allergy. Immune activation by CpG ODNs has been demonstrated to be beneficial in animal models as a vaccine adjuvant and for the treatment of a variety of viral, bacterial, and parasitic diseases. Antitumor activity of CpG ODNs has also been established in numerous mouse models. In clinical vaccine trials in healthy human volunteers or in immunocompromised HIV-infected patients, CpG ODNs strongly enhanced vaccination efficiency. Most encouraging results in the treatment of cancers have come from human phase I and II clinical trials using CpG ODNs as a tumor vaccine adjuvant, monotherapy, or in combination with chemotherapy. Therefore, CpG ODNs represent targeted immune modulatory drugs with a broad range of potential applications.     

CpG oligodeoxynucleotides and plasmid DNA stimulate Atlantic salmon (Salmo salar L.) leucocytes to produce supernatants with antiviral activity

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report herein that plasmid DNA and synthetic oliogodeoxynucleotides (ODNs) containing unmethylated CpG induce production of antiviral cytokine activity in Atlantic salmon leucocytes, whereas ODNs with an inverted motif (GpC) or with methylated cytosines have nearly no stimulatory effect. The adherent cell population, representing mainly macrophages, is directly activated by CpG-ODN, while the effect on the non-adherent population is weak. Since the peak antiviral activity in ODN-stimulated leucocytes is seen after 48h, this might indicate that the unmethylated DNA stimulates the adherent cells to produce co-stimulatory molecules, which in turn stimulates production of antiviral cytokines in the non-adherent cell population. The potent immune activation by CpG ODNs points to possible new applications as adjuvant in fish vaccines.     

CpG Oligodeoxynucleotides Enhance Host Defense during Murine Tuberculosis

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate immune cells to produce cytokines. CpG ODNs protect mice against infections with intracellular bacteria by the induction of a T helper 1 (Th1) response. To determine the effect of CpG ODNs in pulmonary tuberculosis, mice were treated with CpG ODNs or control ODNs at the time of intranasal infection. CpG ODNs reduced mycobacterial outgrowth for up to 5 weeks after Mycobacterium tuberculosis infection and were associated with a decrease in inflammation in lung tissue. CpG treatment was also associated with elevated levels of gamma interferon (IFN-gamma) and decreased levels of interleukin 4 in the lungs and an increased capacity of splenocytes to secrete Th1-type cytokines. CpG ODNs given 2 weeks after infection were still able to reduce mycobacterial outgrowth and to enhance a Th1 response 5 weeks postinfection. Administration of CpG ODNs to IFN-gamma-gene-deficient mice failed to reduce mycobacterial outgrowth. These data suggest that CpG ODNs improve host defense during pulmonary tuberculosis by an IFN-gamma-dependent mechanism.     

Cationic liposomes containing soluble Leishmania antigens (SLA) plus CpG ODNs induce protection against murine model of leishmaniasis

Development of an effective vaccine against leishmaniasis is possible due to the fact that individuals cured from cutaneous leishmaniasis (CL) are protected from further infection. First generation Leishmania vaccines consisting of whole killed parasites reached to phase 3 clinical trials but failed to show enough efficacies mainly due to the lack of an appropriate adjuvant. In this study, an efficient liposomal protein-based vaccine against Leishmania major infection was developed using soluble Leishmania antigens (SLA) as a first generation vaccine and cytidine phosphate guanosine oligodeoxynucleotides (CpG ODNs) as an immunostimulatory adjuvant. 1, 2-Dioleoyl-3-trimethylammonium-propane was used as a cationic lipid to prepare the liposomes due to its intrinsic adjuvanticity. BALB/c mice were immunized subcutaneously (SC), three times in 2-week intervals, with Lip-SLA-CpG, Lip-SLA, SLA + CpG, SLA, or HEPES buffer. As criteria for protection, footpad swelling at the site of challenge and spleen parasite loads were assessed, and the immune responses were evaluated by determination of IFN-γ and IL-4 levels of cultured splenocytes, and IgG subtypes. The group of mice that received Lip-SLA-CpG showed a significantly smaller footpad swelling, lower spleen parasite burden, higher IgG2a antibody, and lower IL-4 level compared to the control groups. It is concluded that cationic liposomes containing SLA and CpG ODNs are appropriate to induce Th1 type of immune response and protection against leishmaniasis.     

新型CpG ODN增强乙肝疫苗诱导IgG2a类抗体产生的实验研究

目的：寻找能增强乙肝疫苗刺激IgG2a类抗体产生，使机体处于Th1样免疫环境的新型乙肝疫苗佐剂。方法：选用自行设计的A、B、C型CpGODN，并以发表的A、B型CpGODN作为阳性对照，与重组乙肝疫苗混合后于第0．4周免疫BALB／C小鼠，用ELISA法检测免疫小鼠血清中HBsAb水平及种类。结果：各型CpGODN都能增强乙肝疫苗刺激HBsAb的产生水平，CpGODN＋乙肝疫苗免疫的小鼠血清中HBsAb类型为IgG2a〉〉IgG1，而单独应用商品化重组乙型肝炎疫苗的小鼠血清中HBsAb类型为IgG1〉〉IgG2a。结论：各型CpGODN对重组乙型肝炎疫苗[含AI（OH）3佐剂]均具有增效作用，而且可以诱导机体产生倾向于Th1途径的免疫应答反应。     

Inhibition of the hepatitis B virus replication in vitro by an oligodeoxynucleotide containing cytidine-guanosine motifs

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides within specific sequence contexts (CpG motifs) are known as potent activators of the immune system and inducers of several Th1-associated immunomodulatory cytokines. CpG ODNs show promise as vaccine adjuvants and immunoprotective agents in animals. Here, we investigated the inhibitory effects of D type CpG ODN on hepatitis B virus (HBV) replication in vitro. The experiments were performed in HepG2 2.2.15 cells, which contain an integrated tandem dimer of the HBV genome and are routinely used for anti-HBV study. HepG2 2.2.15 cells co-cultured with peripheral blood mononuclear cells (PBMCs) plus CpG ODN for 3 days, remarkably reduced the secretion of HBsAg and HBeAg, when compared to cells treated with PBMCs plus non-CpG ODN. The levels of intracellular HBV DNA and HBV mRNA were also decreased. Treatment of HepG2 2.2.15 cells with the culture supernatants of PBMCs activated by CpG ODN can remarkably suppress the secretion of HBsAg and HBeAg as compared with that of PBMCs without CpG ODN activation under the same conditions. There were no inhibitory effects on the replication of HBV to be found for CpG ODN treatment alone. These results suggest that CpG ODN can inhibit indirectly HBV replication in vitro via activating the immune cells, and could contribute to the development of an immunoregulator against HBV infection.     

Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous Leishmaniasis but maintains its potency and durability

The inoculation of live, nonattenuated Leishmania major to produce a lesion in a selected site that heals, referred to as leishmanization, is to date the only vaccine against leishmaniasis that has proven to be effective in humans. Its use has been restricted or abandoned entirely, however, due to safety concerns. In an attempt to develop a leishmanization protocol that minimizes pathology while maintaining long-term protection, live parasites were coinjected with CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs) alone or in combination with whole-cell lysates of heat-killed L. major promastigotes bound to alum (ALM). C57BL/6 mice infected intradermally by using L. major plus CpG ODN with or without ALM developed few or no dermal lesions and showed an early containment of parasite growth, while mice infected with L. major with or without ALM developed sizable dermal lesions that required up to 10 weeks to heal. The CpG ODNs provoked a transient inflammation that included an early recruitment and accumulation of gamma interferon-producing CD4(+) lymphocytes in the site. Attenuation of the live vaccine did not compromise its ability to confer long-term immunity, as mice receiving L. major and CpG ODN plus ALM were totally protected against reinfection with L. major for up to 6 months. By comparison, the immunity elicited by two efficient nonlive vaccines began to wane by 6 months. Our results suggest that immune modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.     

The enhanced effect of a hexameric deoxyriboguanosine run conjugation to CpG oligodeoxynucleotides on protection against allergic asthma

BACKGROUND: Oligodeoxynucleotides containing a CpG motif (CpG ODNs), as potent inducers of T(H)1 immunity, are considered promising candidates for immune modulation in asthma. We have previously demonstrated that conjugation of a hexameric deoxyriboguanosine run to the 3' terminus (3' dG(6)-run) of phosphodiester (PE) CpG ODNs enhanced their immuno-stimulatory activities in vitro. OBJECTIVE: This study aimed to evaluate the effect of a 3' dG(6)-run conjugation to PE or phosphorothioate (PS) CpG ODNs on protection against murine allergic asthma in vivo. METHODS: Balb/c mice were sensitized to ovalbumin by intraperitoneal injection with or without CpG ODNs (PS CpG ODNs, PE CpG ODNs, and those with 3' dG(6)-run) and subsequently challenged with ovalbumin. We evaluated airway hyperresponsiveness, eosinophil proportion in bronchoalveolar lavage fluid, airway inflammation, and ovalbumin-specific antibody responses. RESULTS: The conjugation of a 3' dG(6)-run to PE CpG ODNs enhanced the production of IFN-gamma from ovalbumin-specific T(H) cells and prevented the development of asthma in terms of airway hyperresponsiveness, airway eosinophilia, and ovalbumin-specific IgE responses; these effects were comparable to those of PS CpG ODNs. Enhanced effects of the 3' dG(6)-run were also observed in PS CpG ODNs, though they were lower than those in PE CpG ODNs. CONCLUSION: This study suggests that conjugation of a 3' dG(6)-run to CpG ODNs might provide an effective method for immune modulation of allergic asthma.     

Differential signaling by CpG DNA in DCs and B cells: not just TLR9

CpG-containing oligodeoxynucleotides (CpG ODNs) act on Toll-like receptor 9 (TLR9) that is expressed on B cells and plasmacytoid dendritic cells (pDCs) to stimulate the innate immune system, however, different types of CpG ODNs induce distinct responses. Recent papers suggest some CpG ODNs could require a second receptor or cofactor to signal. The different signaling complexes assembled might impact on the affinity with which CpG ODNs signal to TLR9 or activate additional pathways that lead to distinct immune responses.     

Interleukin-12- and gamma interferon-dependent protection against malaria conferred by CpG oligodeoxynucleotide in mice

Unmethylated CpG dinucleotides in bacterial DNA or synthetic oligodeoxynucleotides (ODNs) cause B-cell proliferation and immunoglobulin secretion, monocyte cytokine secretion, and activation of natural killer (NK) cell lytic activity and gamma interferon (IFN-gamma) secretion in vivo and in vitro. The potent Th1-like immune activation by CpG ODNs suggests a possible utility for enhancing innate immunity against infectious pathogens. We therefore investigated whether the innate immune response could protect against malaria. Treatment of mice with CpG ODN 1826 (TCCATGACGTTCCTGACGTT, with the CpG dinucleotides underlined) or 1585 (ggGGTCAACGTTGAgggggG, with g representing diester linkages and phosphorothioate linkages being to the right of lowercase letters) in the absence of antigen 1 to 2 days prior to challenge with Plasmodium yoelii sporozoites conferred sterile protection against infection. A higher level of protection was consistently induced by CpG ODN 1826 compared with CpG ODN 1585. The protective effects of both CpG ODNs were dependent on interleukin-12, as well as IFN-gamma. Moreover, CD8+ T cells (but not CD4+ T cells), NK cells, and nitric oxide were implicated in the CpG ODN 1585-induced protection. These data establish that the protective mechanism induced by administration of CpG ODN 1585 in the absence of parasite antigen is similar in nature to the mechanism induced by immunization with radiation-attenuated P. yoelii sporozoites or with plasmid DNA encoding preerythrocytic-stage P. yoelii antigens. We were unable to confirm whether CD8+ T cells, NK cells, or nitric oxide were required for the CpG ODN 1826-induced protection, but this may reflect differences in the potency of the ODNs rather than a real difference in the mechanism of action of the two ODNs. This is the first report that stimulation of the innate immune system by CpG immunostimulatory motifs can confer sterile protection against malaria.     

Immunomodulatory effects of CpG oligodeoxynucleotides on established th2 responses

CpG oligodeoxynucleotides (CpG ODNs) are known to induce type 1 T-helper-cell (Th1) responses. We have previously demonstrated that CpG ODNs administered during sensitization prevent Th2-mediated eosinophilic airway inflammation in vivo. We also reported that key Th1 cytokines, gamma interferon (IFN-gamma) and interleukin 12 (IL-12), are not necessary for this protection. Recent in vivo data suggest that CpG ODNs might also reverse established pulmonary eosinophilia. In order to clarify how CpG ODNs can inhibit established Th2 responses, we evaluated the cytokine production from splenocytes from antigen- and alum-immunized mice. Restimulation with antigen induced IL-5, which was clearly inhibited by coculture with CpG ODNs in a concentration-dependent manner. CpG ODNs also induced IFN-gamma, but in a concentration-independent manner. The inhibition of IL-5 production was not mediated through natural killer cells or via CD8(+) T lymphocytes. Although IFN-gamma plays an important role in inhibition of antigen-induced IL-5 production by CpG ODNs, IFN-gamma was not the sole factor in IL-5 inhibition. CpG ODNs also induced IL-10, and this induction correlated well with IL-5 inhibition. Elimination of IL-10 reduced the anti-IL-5 effect of CpG ODNs, although incompletely. This may be because IFN-gamma, induced by CpG ODNs, is also inhibited by IL-10, serving as a homeostatic mechanism for the Th1-Th2 balance. Overproduction of IFN-gamma was downregulated by CpG ODN-induced IL-10 via modulation of IL-12 production. These data suggest that CpG ODNs may inhibit established Th2 immune responses through IFN-gamma and IL-10 production, the latter serving to regulate excessive Th1 bias. These properties of CpG ODNs might be a useful feature in the development of immunotherapy adjuvants against allergic diseases such as asthma.     

CpG Oligodeoxynucleotides as TLR9 Agonists

Toll-like receptors (TLRs) are part of the innate immune system, and they belong to the pattern recognition receptors (PRR) family. The PRR family is designed to recognize and bind conserved pathogen-associated molecular patterns, which are not generated by the host and are restricted and essential to microorganisms. TLR9, which recognizes unmethylated CpG (cytosine guanosine dinucleotide), is a very promising target for therapeutic activation. Stimulation of TLR9 activates human plasmacytoid dendritic cells and B cells, and results in potent T helper-1 (T_h1)-type immune responses and antitumor responses in mouse tumor models and in patients. Several pharmaceutical companies, such as Pfizer, Idera, and Dynavax, are developing CpG oligodeoxynucleotides (ODNs) for the treatment of cancer, along with other conditions, such as infections and allergy. CpG ODNs have shown promising results as vaccine adjuvants and in combination with cancer immunotherapy. Several TLR9 agonists are being developed and have entered clinical trials to evaluate their safety and efficacy for the treatment of several hematopoietic and solid tumors. In this review, we discuss the use of CpG ODNs in several phase I and II clinical trials for the treatment of NHL, renal cell carcinoma, melanoma, and non-small cell lung cancer, either alone or in combination with other agents.     

Prophylactic vaccines mimic synthetic CpG oligonucleotides in their ability to modulate immune responses

Synthetic oligonucleotide ligands that bind to toll-like receptors are known to modulate the immune response via the activation of antigen presenting cells, and were therefore proposed as a novel form of vaccine adjuvant. Clinical-grade they are, however, not readily available. Here, we show that commonly used prophylactic vaccines for infectious diseases like measles, mumps and tuberculosis exhibit the same immune modulating behavior as synthetic CpG oligonucleotides in terms of their ability to stimulate IFN-α production and plasmacytoid dendritic cell maturation. Featuring the additional advantages of low-cost and proven safety, these vaccines could therefore be attractive alternatives to CpG oligonucleotides as adjuvants for immunotherapy. This previously undiscovered characteristic of prophylactic vaccines also sheds new light on the mechanisms by which they operate and is extremely interesting for vaccine development. Moreover, the finding that prophylactic vaccines trigger TLRs like synthetic oligonucleotides opens the possibility to predict the immune response of new vaccines.     

Respiratory syncytial virus(RSV)-induced allergy may be controlled by IL-4 and CX3C fractalkine antagonists and CpG ODN as adjuvant: hypothesis and implications for treatment

Based on the hypothesis that respiratory syncytial virus (RSV) sG protein causes allergy in patients, it is suggested that treatment of RSV patients with antagonists of IL-4 and FKN early in infection will prevent the increased level of IL-4 in the serum. Together with CpG ODNs that induce Toll-like receptor 9⁺ (TLR9⁺) plasmacytoid dendritic cells to release type I IFN-α and -β will reactivate the inhibited Th1 cells and the antiviral cytotoxic T leukocytes. In addition, binding of CpG ODNs to TLR9⁺ B cells will stop IgE synthesis and antiviral IgG and IgA will continue. Together, the IL-4 and FKN antagonists and CpG ODNs reactivate the adaptive immune response to clear the virus and protect the patient from a second RSV infection. It is also suggested that the less-pathogenic RSV strain Long may be a candidate for vaccine development after deletion of the FKN and superantigen domains from the G gene.     

Multiple effects of immunostimulatory DNA on T cells and the role of type I interferons

In addition to stimulating antigen-specific immune responses, infectious agents cause nonspecific activation of the innate immune system, notably up-regulation of costimulatory/adhesion molecules on APCs and cytokine production. In recent years it has become apparent that stimulation of the immune system by microorganisms is a property of a number of different cellular components, including DNA. As discussed earlier and elsewhere in this volume, the DNA of infectious agents--and indeed of all non-vertebrates tested--differs from mammalian DNA in being enriched for unmethylated CpG motifs. With appropriate flanking sequences, CpG DNA and synthetic CpG ODNs cause strong activation of APCs and other cells. In this article we have focussed on the capacity of CpG DNA/ODNs to alter T cell function. Whether these compounds act directly on T cells or function indirectly by activating other cells, especially APCs, is controversial [7, 8, 13, 14]. In contrast to other workers [8], we have yet to find definitive evidence that CpG DNA/ODNs can provide a co-stimulatory signal for purified T cells subjected to TCR ligation ([14] and unpublished data of authors). For this reason we lean to the notion that CpG DNA/ODNs modulate T cell function by inducing activation of APC rather than by acting directly on T cells. When injected in vivo in the absence of specific antigen, CpG DNA/ODNs have two striking effects on T cells, namely (1) induction of overt activation (proliferation) of memory-phenotype CD8+ cells, and (2) partial activation of all T cells, including na?ve-phenotype T cells. Both actions of CpG DNA/ODNs are heavily dependent on the production of IFN-I by APC. For memory-phenotype (CD44hi) CD8+ cells, neither CpG DNA nor IFN-I can cause proliferation of purified APC-depleted T cells in vitro. Hence, under in vivo conditions, CpG DNA-induced proliferation of CD44hi CD8+ cells is probably mediated through the production of a secondary cytokine, i.e., by a cytokine that is directly stimulatory for CD44hi CD8+ cells. Based on the available evidence, it is highly likely that the effector cytokine is IL-15. With this assumption, our current model is that proliferation of CD44hi CD8+ cells induced by injection of CpG DNA/ODNs reflects production of IFN-I which, in turn, leads to synthesis of IL-15. Which particular cell types produce these two cytokines is unclear, although APCs are probably of prime importance. In addition to inducing proliferation of memory-phenotype CD8+ cells via IL-15, the IFN-I induced by CpG DNA/ODNs can also induce partial activation of naive T cells. This form of activation leads to up-regulation of CD69 and other molecules but does not cause entry into cell cycle. It is of interest that the partial activation of naive T cells induced by IFN-I is associated with decreased T proliferative responses. Thus, proliferation of purified na?ve T cells elicited by combined TCR/CD28 ligation in vitro is greatly reduced by addition of IFN-I. This inhibitory effect of IFN-I does not influence cytokine production and probably reflects production of cell cycle inhibitors. Surprisingly, except at high doses, IFN-I fails to exert an anti-proliferative effect when T proliferative responses are driven by viable APCs. Indeed, in this situation, IFN-I enhances antigen-specific T proliferative responses, both in vivo and in vitro. This adjuvant effect of IFN-I is presumably a reflection of APC activation, but direct evidence on this issue is still lacking. In this article we have emphasized that contact with CpG DNA/ODNs has multiple effects on T cell function in vivo. Many of these effects seem to be related to the production of certain cytokines by APCs, notably IFN-I and IL-15. It should be stressed, however, that CpG DNA/ODNs probably lead to the production of many other cytokines. Hence, our current models of how CpG DNA/ODNs influence T cell function are undoubtedly oversimplified.