DNA activates human immune cells through a CpG sequence-dependent manner.

While bacterial DNA and cytosine-guanosine-dinucleotide-containing oligonucleotides (CpG ODN) are well described activators of murine immune cells, their effect on human cells is inconclusive. We investigated their properties on human peripheral blood mononuclear cells (PBMC) and subsets thereof, such as purified monocytes, T and B cells. Here we demonstrate that bacterial DNA and CpG ODN induce proliferation of B cells, while other subpopulations, such as monocytes and T cells, did not proliferate. PBMC mixed cell cultures, as well as purified monocytes, produced interleukin-6 (IL-6), IL-12 and tumour necrosis factor-alpha upon stimulation with bacterial DNA; however, only IL-6 and IL-12 secretion became induced upon CpG ODN stimulation. We conclude that monocytes, but not B or T cells, represent the prime source of cytokines. Monocytes up-regulated expression of antigen-presenting, major histocompatibility complex class I and class II molecules in response to CpG DNA. In addition, both monocytes and B cells up-regulate costimulatory CD86 and CD40 molecules. The activation by CpG ODN depended on sequence motifs containing the core dinucleotide CG since destruction of the motif strongly reduced immunostimulatory potential.     

Redundant and unique regulation of activated mouse B lymphocytes by IL-4 and IL-21

IL-21 distinctively regulates B cell growth and death, and it redundantly functions with IL-4 for IgG production. B cells likely encounter IL-4 and IL-21 in vivo, as both are secreted by activated T cells. Therefore, the action of both these cytokines was investigated during activation of B cells. IL-21 or the combination of IL-4 and IL-21 inhibited proliferation by purified mouse B cells to LPS or CpG DNA, whereas these cytokines enhanced proliferation after engaging the BCR or CD40. Although B cell subsets expressed somewhat varied levels of the IL-21 receptor, LPS-stimulated follicular and marginal B cell subsets were also dominantly susceptible to IL-21-induced growth arrest and cell death. After activation of B cells with CD40 and LPS, IL-4 and IL-21 distinctively regulated the expression of CD23, CD44, and CD138, and they cooperatively promoted IgG1 class-switching and synthesis. These findings support a model in which the presence of IL-4 and IL-21 inhibits B cells activated by polyclonal innate signals, and they promote B cell expansion and differentiation during T cell-dependent antibody responses, although the individual responses to IL-4 and IL-21 do not always overlap.     

Adjuvant effects of salidroside from Rhodiola rosea L. on the immune responses to ovalbumin in mice

Salidroside, a major component of Rhodiola rosea L., was evaluated for its adjuvant effects on the immune responses in mice by ovalbumin (OVA) stimulation. BALB/c mice were immunized subcutaneously with OVA 100 μg or OVA 100 μg dissolved in saline containing alum (100 μg) or salidroside (12.5, 25, or 50 μg) on Days 1 and 15. Two weeks later (Day 28), blood samples were collected to analyze OVA-specific IgG, IgG1, and IgG2b antibodies. Meanwhile, splenocytes were harvested to assess lymphocyte proliferation, cytokines (IL-2, IL-4, and IFN-γ) production, and CD4(+), CD8(+) lymphocyte subsets. The results indicated that co-administration of salidroside with OVA significantly enhanced the ConA-, LPS-, and OVA-induced splenocyte proliferation, produced more IL-2, IL-4, IFN-γ, and IgG, IgG1, and IgG2b antibody levels, and increased the percentage of CD4(+), CD8(+) lymphocyte subsets than OVA alone. Thus, salidroside possess immunological adjuvant activity by regulating humoral and cellular immune responses in mice.     

Anti-proliferative effects of phosphodiester oligodeoxynucleotides

The immunostimulatory effects of cytosine-phosphate-guanosine (CpG)-containing oligodeoxynucleotides (ODNs) have been extensively documented. In this paper, we describe the inhibitory effects of ODNs that contain natural phosphodiester backbones (O-ODNs) on the immunostimulation caused by CpG-containing phosphorothioated ODNs (CpG-S). CpG-S stimulation of mouse splenocyte proliferation was reduced by the addition of O-ODNs that contained or lacked the CpG-motif (CpG-containing phosphodiester oligodeoxynucleotide, CpG-O or GpC-O). The total number of cultured splenocytes was up-regulated by CpG-S, whereas repetitive addition of O-ODNs to the cell cultures inhibited this effect. The frequency of T2-like B cells was found to be increased by CpG-S. The culture supernatants of CpG-S-treated splenocytes contained elevated levels of IL-10 and IL-6. However, IL-10 and IL-6 production was down-regulated significantly by the combination of CpG-S and either CpG-O or GpC-O. The O-ODN mediated inhibition of proliferation was less pronounced in IL-10-/- mice. Thus, the O-ODNs, irrespective of CpG content, exerted inhibitory activities on the proliferation of B cells. These anti-proliferative effects appear to be mediated both by the down-regulation of IL-10 production and increased apoptosis.     

Early in vitro priming of distinct T(h) cell subsets determines polarized growth of visceralizing Leishmania in macrophages

An in vitro priming system of murine naive splenocytes was established to investigate early immune responses to LEISHMANIA: chagasi, the agent of visceral leishmaniasis in the New World. Priming of splenocytes from resistant C3H and CBA or susceptible BALB and B10 mice with L. chagasi resulted in blast transformation and in proliferating parasite-specific CD4(+) T cells secreting a differential complement of cytokines (IFN-gamma and low IL-10 levels for resistant T cells; IFN-gamma, IL-4 and high IL-10 levels for susceptible T cells). After priming, intracellular parasite load was much higher in susceptible than in resistant-type splenocyte cultures. On the other hand, infection of purified splenic macrophages from either resistant or susceptible mice with live L. chagasi promastigotes, resulted in comparable parasite loads. Moreover, when early CD4(+) T cell priming in splenocyte cultures was disrupted with anti-CD4 mAb, polarized parasite growth was abolished, becoming comparable in resistant and susceptible cultures. Neutralizing IL-4 activity during splenocyte priming did not affect the final parasite load in susceptible cultures. However, neutralizing IL-10 activity markedly decreased parasite load in susceptible, but not in resistant splenic macrophages. These results suggest that IL-10 plays an important role in L. chagasi infection in susceptible hosts. The results also indicate that innate control of growth of a visceralizing LEISHMANIA: in splenic macrophages results from the ability to activate different CD4(+) T cell subsets.     

Ethanol affects the generation, cosignaling molecule expression, and function of plasmacytoid and myeloid dendritic cell subsets in vitro and in vivo

The influence of ethanol (EtOH) on multiple dendritic cell (DC) subsets, in the steady state or following their mobilization in vivo, has not been characterized. Herein, generation of mouse bone marrow-derived DC (BMDC) in response to fms-like tyrosine kinase 3 ligand was inhibited by physiologically relevant concentrations of EtOH with selective suppression of plasmacytoid (p)DC. EtOH reduced surface expression of costimulatory molecules (CD40, CD80, CD86) but not that of coinhibitory CD274 (B7-H1) on resting or CpG-stimulated DC subsets. Interleukin (IL)-12p70 production by activated DC was impaired. Consistent with these findings, EtOH-exposed BMDC exhibited a reduced capacity to induce na?ve, allogeneic T cell proliferation and impaired ability to prime T cells in vivo. DC subsets freshly isolated from EtOH-fed mice were also examined. Liver DC, inherently immature and resistant to maturation, exhibited little change in their low surface cosignaling molecule expression, whereas splenic DC showed reduced expression of surface costimulatory molecules in response to CpG stimulation in vivo. These splenic DC elicited reduced na?ve, allogeneic T cell proliferation in vitro, and the stimulatory capacity of resting but not CpG-activated liver DC was reduced by chronic EtOH administration. T cells from animals primed with EtOH-exposed DC produced elevated levels of IL-10 following ex vivo challenge with donor alloantigen. Thus, EtOH impairs cytokine-driven differentiation and function of myeloid DC and pDC in vitro. Hepatic DC from chronic EtOH-fed mice are less affected than splenic DC, which exhibit impaired functional maturation following CpG stimulation. These results indicate a potential mechanism by which alcohol consumption is associated with immunosuppression.     

Specific siRNA downregulated TLR9 and altered cytokine expression pattern in macrophage after CpG DNA stimulation

Bacterial CpG DNA or synthetic oligonucleotides(ODNs)that contain unmethylated CpG motifs(CpG ODN)candirectly activate antigen-presenting cells(APCs)to secrete various cytokines through the intraceilular receptorTLR9.Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial tothe subsequent immune responses.To date,cytokine profiles in APCs upon CpG ODN stimulation in vitro are notfully investigated.In the present study,vector-based siRNA was used to downregulate TLR9 expression.Cytokineprofiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid uponCpG ODN stimulation.We found that not all the cytokine expressions by the macrophage were decreased whileTLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased,but IL-6,IFN-β and IL-10 expressions were not affected.Interestingly,the level of IFN-α was even increased.This alterationof cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role ofCpG DNA is more complicated in the pathogenesis and prevention of diseases.Cellular & Molecular Immunology.2005;2(2):130-135.     

Bacterial DNA and CpG-containing oligodeoxynucleotides activate cutaneous dendritic cells and induce IL-12 production: implications for the augmentation of Th1 responses

BACKGROUND: Unmethylated CpG sequences in bacterial DNA act as adjuvants selectively inducing Th1 predominant immune responses during genetic vaccination or when used in conjunction with protein Ag. The precise mechanism of this adjuvant effect is unknown. Because dendritic cells (DC) are thought to be crucially involved in T cell priming and Th1/Th2 education during vaccination via skin, we characterized the effects of bacterial DNA and CpG-containing oligodeoxynucleotides (CpG ODN) on cutaneous DC. METHODS AND RESULTS: Stimulation with CpG ODN 1826 (6 micrograms/ml) induced activation of immature Langerhans cell (LC)-like DC as determined by an increased expression of MHC class II and costimulatory molecules, loss of E-cadherin-mediated adhesion and increased ability to stimulate allogeneic T cells. Composition-matched control ODN 1911 lacking CpG sequences at equal concentrations was without effect. In comparison to LPS and ODN 1911, CpG ODN 1826 selectively stimulated DC to release large amounts of IL-12 (p40) and little IL-6 or TNF-alpha within 18 h and detectable levels of IL-12 p70 within 72 h. Stimulation with Escherichia coli DNA, but not calf thymus DNA, similarly induced DC maturation and IL-12 p40 production. Injection of CpG ODN into murine dermis induced enhanced expression of MHC class II and CD86 by LC in the overlying epidermis and intracytoplasmic IL-12 p40 accumulation in a subpopulation of activated LC. CONCLUSION: Bacterial DNA and CpG ODN stimulate DC in vitro and in vivo and may preferentially elicit Th1-predominant immune responses because they can activate and mobilize DC, inducing them to produce IL-12.     

The role of CD11c(+) hepatic dendritic cells in the induction of innate immune responses

The role of the liver in the initiation and maintenance of tolerance is a critical immune function that involves multiple lineages of immune cells. Included within these populations are liver dendritic cells (DCs). Although there has been significant work on the phenotypic and functional roles of splenic and bone marrow dendritic cells, as well as their subsets, comparable studies in liver have often been difficult. To address this issue we have isolated, from C57BL/6 mice, relatively pure populations of DCs and compared phenotype and function to the data from spleen using flow cytometry, cell sorter assisted purification and culture, morphology by cytospin and May-Giemsa staining, cell cycle progression, antigen uptake, cytokine production and allo-activation potential. natural killer (NK)1.1(-)CD11c(+) liver DC subsets (conventional DCs, T cell receptor (TcR)beta(-)NK1.1(-)CD11c(+)B220(-) and plasmacytoid DCs, TcRbeta(-)NK1.1(-)CD11c(+)B220(+)) efficiently endocytose dextran and produce significant levels of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40 in response to Toll-like receptor (TLR) ligands, with responses higher than splenic DCs. There is also a differential capability of hepatic DCs to respond to innate signals. Indeed, CD11c(+) hepatic DCs have a greater capacity to respond to innate stimulation but are less capable of inducing CpG activated-allogeneic T cells. These data suggest that hepatic dendritic cells function as a critical bridge between innate and adaptive immunity and are capable of inducing stronger innate responses with a lower capacity for allo-stimulation than splenic dendritic cells. These properties of liver dendritic cells contribute to their unique role in the induction of tolerance.     

Interleukin 5 Regulation of Peritoneal B-Cell Proliferation and Antibody Secretion

The influence of recombinant interleukin 5 (rIL-5) on murine peritoneal B-cell proliferation and antibody secretion was examined. Larger, low buoyant density peritoneal B cells proliferated better with rIL-5 than the smaller resting B cells. this was also true for splenic B cells; however, comparison of the respective populations showed the large peritoneal B-cell responses to be superior. Limiting dilution analyses showed that from 25% to about 40% of large peritoneal B cells proliferated in response to rIL-5 when lipopolysaccharide (LPS) was present. No detectable difference in the fraction of proliferating splenic B cells was seen in the presence of rIL-5. These results are consistent with expression of IL-5 receptors on about 70% of low-density peritoneal B cells as determined by fluorescent staining with anti-Il-5 receptor monoclonal antibody (MoAb). IL-5 also enhanced spontaneous and mitogen-driven IgM secretion by both peritoneal and splenic B lymphocytes; the increases exhibited by peritoneal B cells, however, were at least twice those exhibited by splenic B cells. Spontaneous and mitogen-driven secretion of auto-antibodies to bromelain-treated mouse erythrocytes (BrMRBC) by peritoneal B cells were also increased by this interleukin. Furthermore, rIL-5 enhanced peritoneal B-cell plaque-forming cell (PFC) responses to TNP-LPS but not to TNP-Ficoll. Both an anti-IL-5R MoAb and an anti-IL-5 MoAb blocked the rIL-5-induced enhancement of proliferation and auto-antibody PFC responses. Hence, IL-5 appears to be important for the regulation of proliferation and antibody secretion by many murine peritoneal B cells.     

IL-18基因佐剂协同HSV-1 gB DNA疫苗免疫诱导的特异性免疫应答

目的研究IL-18 cDNA协同单纯疱疹病毒Ⅰ型（HSV-1）糖蛋白B（gB）核酸疫苗免疫对机体体液免疫和细胞免疫应答的影响。方法利用pcDNA3载体分别构建HSV-1gB和IL-18的真核表达质粒pgB和pIL-18,分pcDNA3、pgB和pgB＋pIL-18三组,肌肉注射免疫接种BALB/c小鼠3次,每次间隔2周,每次接种质粒100μg,第3次免疫后2周,用ELISA检测特异性抗体滴度;利用^3H-TdR掺入法进行T细胞特异性抗原刺激实验;耳廓肿胀实验检测迟发型超敏（DTH）反应。结果与pgB单独免疫组相比,pIL-18的协同免疫可以显著增强ELISA特异性抗体滴度、T细胞增殖反应和DTH反应。结论IL-18cD-NA的协同免疫可以显著提高pgBDNA疫苗诱导的体液免疫和细胞免疫水平,增强核酸疫苗对机体的免疫保护作用。     

Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides flanked by certain bases (CpG ODN) have been shown to activate murine and human B cells and to induce proinflammatory cytokines by monocytes/macrophages and dendritic cells (DC). However, the CpG ODN sequences optimal for mice and humans are different. In the current study, the effects of CpG ODN, which were defined to stimulate strong responses in either mouse or human leukocytes, were compared for stimulation of bovine B lymphocyte proliferation and macrophage cytokine mRNA expression. The optimal CpG ODN was then tested for induction of cytokines in peripheral blood mononuclear cells (PBMC) and purified B lymphocytes, monocytes, and macrophages. At a high ODN concentration (40 microM), all but two CpG ODN tested stimulated B cell proliferation, which was dependent on unmethylated CpG motifs. CpG ODN 2059 containing the GTCGTT motif shown to activate human leukocytes also promoted the highest level of bovine B cell proliferation at a lower concentration (10 microM) when compared with CpG ODN containing AACGTT or GACGTT motifs active for murine leukocytes. Furthermore, ODN 2059 induced interleukin-6 (IL-6) production by B lymphocytes and IL-6 and IL-12 production by PBMC, monocytes, and macrophages. In contrast, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) production was either very low or undetectable. Consistent with increased IL-12 production, ODN 2059 also stimulated interferon-gamma (IFN-gamma) production by PBMC. Importantly, the levels of cytokines induced by ODN 2059 were comparable to those generated in response to Escherichia coli DNA. The weak TNF-alpha response combined with the vigorous IL-6 and IL-12 response to ODN 2059 indicate the potential use of this CpG ODN as an adjuvant to enhance both antibody-mediated and IFN-gamma-mediated macrophage activation, which are important for protection against disease caused by intracellular pathogens of cattle.     

Adjuvant effects of salidroside from Rhodiola rosea L. on the immune responses to ovalbumin in mice

Salidroside, a major component of Rhodiola rosea L., was evaluated for its adjuvant effects on the immune responses in mice by ovalbumin (OVA) stimulation. BALB/c mice were immunized subcutaneously with OVA 100 μg or OVA 100 μg dissolved in saline containing alum (100 μg) or salidroside (12.5, 25, or 50 μg) on Days 1 and 15. Two weeks later (Day 28), blood samples were collected to analyze OVA-specific IgG, IgG1, and IgG2b antibodies. Meanwhile, splenocytes were harvested to assess lymphocyte proliferation, cytokines (IL-2, IL-4, and IFN-γ) production, and CD4⁺, CD8⁺ lymphocyte subsets. The results indicated that co-administration of salidroside with OVA significantly enhanced the ConA-, LPS-, and OVA-induced splenocyte proliferation, produced more IL-2, IL-4, IFN-γ, and IgG, IgG1, and IgG2b antibody levels, and increased the percentage of CD4⁺, CD8⁺ lymphocyte subsets than OVA alone. Thus, salidroside possess immunological adjuvant activity by regulating humoral and cellular immune responses in mice.     

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.     

Plasmacytoid dendritic cells: the key to CpG

The vertebrate immune system has established TLR9 to detect microbial DNA based on unmethylated CG dinucleotides within certain sequence contexts (CpG motifs). In humans, the expression of toll-like receptor 9 (TLR9) is restricted to B cells and plasmacytoid dendritic cells (PDC). The PDC is characterized by the ability to rapidly synthesize large amounts of type I IFN (IFN- and IFN-β) in response to viral infection. In contrast to other dendritic cell subsets which express a broad profile of TLRs, the TLR profile in PDC is restricted to TLR7 and TLR9. So far, CpG DNA is the only defined microbial molecule recognized by PDC. An intriguing feature of PDC is its ability to simultaneously produce the two major Th1-inducing cytokines in humans, IFN- and IL-12, both at high levels. The ratio of IFN- versus IL-12 and the quantity of these cytokines are regulated by T helper cell-mediated costimulation via CD40 ligation. The ratio also depends on the differentiation stage of the PDC at the time of stimulation and the type of CpG ODN used. We propose a model in which the establishment of Th1 responses in vivo is improved by appropriately stimulated PDC that otherwise – in the absence of CpG DNA – support Th2 or Th0 responses and thus have been called DC2.     

Adjuvants LT-K63 and CpG enhance the activation of dendritic cells in neonatal mice

Dendritic cells (DC) play a major role in the priming of T cells and initiating specific immune responses. We assessed the effects of the adjuvants LT-K63 and CpG on neonatal DC in vivo and in vitro . Cytokine levels (IL-10, IL-12p70 and IL-12p40/IL-23p40) were measured and the expression of the activation markers CD86, CD40 and MHCII on CD11c⁺ DC was analysed by using FACS. The proportion of MHCII^high CD11c⁺ DC was higher in neonatal mice immunized with a pneumococcal conjugate (PncTT) and LT-K63 or CpG compared with that when PncTT was alone. In vitro stimulation with LT-K63 enhanced the expression of CD86 more on CD11c⁺ DC from spleens of mice immunized as neonates than those immunized as adults, whereas in vitro stimulation with CpG enhanced the expression of CD86 and CD40 on CD11c⁺ DC similarly in both age groups. CpG stimulation in vitro enhanced IL-10 and IL-12(p70) production in mice immunized as neonates with PncTT and either adjuvant, but not PncTT alone. The adjuvants LT-K63 and CpG enhance the activation of CD11c⁺ DC in mice immunized as neonates and can thereby overcome one of the limiting factors in the initiation of the immune response to conjugate vaccines in early life. The fact that neonatal DC are more susceptible to stimulation with either adjuvant, LT-K63 or CpG, could imply that neonatal CD11c⁺ DC are more easily activated than adult CD11c⁺ DC, and/or be a consequence of the predominance of different DC subsets in neonatal and adult mice.     

CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: A new class of vaccine adjuvants

Foreign DNA has been shown to impinge on immune cell function by an as yet unidentified mechanism. We and others have demonstrated that single-stranded (ss) DNA containing the motif CpG flanked by two 5′ purines and two 3′ pyrimidines are mitogenic for B cells and activate macrophages to release tumor necrosis factor-α, interferon-γ, interleukin (IL)-6 or IL-12. Because of these proinflammatory responses we investigated if ssDNA would serve as a potential vaccine adjuvant. Here we show that CpG-containing oligonucleotides represent a powerful adjuvant for both humoral and cellular immune responses. When ssDNA was incorporated into inocula, specific antibody titers of the IgG2 isotype were enhanced by greater than 100-fold. Primary cytotoxic T lymphocyte responses generated to either unprocessed protein antigen or major histocompatibility complex class I-restricted peptide were exceedingly strong. Evidence is also provided that oligomers directly influenced T cell receptor-triggered T cell proliferation. Thus ssDNA oligomers may serve as inexpensive and safe vaccine adjuvants and, in addition, differential effects due to sequence may allow for directed responses.     

Effective collaboration between IL-4 and IL-21 on B cell activation

Although IL-4 and IL-21 synergistically promote proliferation and differentiation of activated B cells, the mutual role in their collaboration is not known. When splenic B cells were sequentially stimulated with anti-IgM Ab and anti-CD40 Ab plus IL-4 and then with IL-21 at a 2-day interval, proliferation, frequency of class switching to IgG1 and plasma cell differentiation were continuously enhanced until day 5 of culture. Amounts of AID and Blimp1 mRNA in sequentially activated B cells with IL-4 and IL-21 increased more than those in activated B cells without IL-21. However, sequential stimulation of B cells with anti-IgM Ab and anti-CD40 Ab plus IL-21 and then with IL-4 at more than 1-day interval did not display the synergistic effect. Furthermore, sequential stimulation of activated B cells with a low dose of IL-4, which did not induce Ig class switching, at the beginning of culture and with IL-21 or IL-4 on day 2 of culture induced proliferation and differentiation of CXCR4(-) or CXCR4(+) B cells, respectively. Thus, IL-21 effectively promotes proliferation and differentiation of CXCR4(-) B cells pre-activated with anti-IgM Ab and anti-CD40 Ab plus IL-4.     

Synergy between CpG- or non-CpG DNA and specific antigen for B cell activation

DNA or oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG DNA) activate antigen-presenting cells and switch on T(h)1 immunity to antigen. B cells are synergistically activated by CpG DNA in combination with non-physiologic B cell stimulators such as polyclonal mitogen and surface Ig cross-linkers. This study shows the unexpected finding that not only CpG ODN, but also non-CpG and methylated ODN synergize with specific antigen, hen egg lysozyme (HEL), in stimulating HEL-specific B cells to proliferate, to express the early activation marker CD69 and to activate the NF-kappaB pathway. In vivo, non-CpG and methylated CpG ODN also enhanced anti-HEL antibody production in HEL-immunized mice, with a bias towards the production of T(h)1-associated isotypes. The synergy with all ODN to enhance B cell immune function was epitope-specific since neither denatured HEL nor other antigens enhanced the ODN effect on HEL-specific B cells. Furthermore, the synergy was independent of whether the ODN backbone was phosphorothioate or phosphodiester, or whether natural vertebrate genomic DNA was used. In all functional analyses, non-CpG and methylated CpG ODN showed lower activity than CpG ODN. These studies demonstrate that the presence of specific physiologic antigen might broaden the spectrum of DNA/ODN that stimulate B cells, with potential implications for the initiation and regulation of normal and pathologic immune responses.