Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsα in CD11b+ DCs

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40−/−, Batf3−/−, and IL-17A−/− mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3−/− mice, lacking CD8α⁺ DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b⁺ DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.     

Coupling of antigen to cholera toxin for dendritic cell vaccination promotes the induction of MHC class I-restricted cytotoxic T cells and the rejection of a cognate antigen-expressing model tumor

We previously demonstrated that cholera toxin (CT) is highly efficient as a combined carrier and adjuvant for dendritic cell (DC) vaccination, inducing strong Th1-dominated B cell and CD4(+) T cell responses. In this study we show that vaccination with DC pre-pulsed ex vivo with CT-conjugated OVA (OVA-CT) gives rise to OVA-specific CD8(+) T cells that produce IFN-gamma and are cytotoxic for OVA-expressing E.G7 tumor cells both in vitro and in vivo. The induction of specific CD8(+) CTL by OVA-CT-treated DC was associated with enhanced presentation of OVA peptide (SIINFEKL) on MHC class I in combination with an overall activation of the pulsed DC. Vaccination of mice with OVA-CT-pulsed DC resulted in rejection of already established MHC class I-positive, MHC class II-negative, OVA-expressing E.G7 tumors in an antigen-specific, CD8(+) T cell-dependent fashion and was associated with high numbers of tumor-infiltrating CD8(+) T cells. Conjugation of antigen to CT facilitated DC uptake of the linked antigen through the GM1 receptor-binding B subunit and induced strong activation-maturation signals through the biologically active A subunit. These results have interesting implications for DC vaccination aimed at inducing CTL immune responses.     

Mucosal immune network in the gut for the control of infectious diseases

The common mucosal immune system (CMIS) consists of an integrated cross-communication pathway of lymphoid tissues made up of inductive and effector sites for host protection against pathogenic microorganisms. Major effector molecules of the CMIS include IgA antibodies and cytokines, chemokines and their corresponding receptors. Secretory IgA (S-IgA), the major immunoglobulin, is induced by gut-associated lymphoreticular tissue (GALT)-derived B cells with the help of Th1- and Th2-type CD4(+) T lymphocytes. Cytotoxic T lymphocytes (CTLs) in the mucosal epithelium, a subpopulation of intraepithelial lymphocytes (IELs), also help maintain the mucosal barrier. The CMIS is unique in that it can provide both positive and negative signals for the induction and regulation of immune responses in both the mucosal and systemic compartments after oral or nasal antigen exposure. Prevention of infection through mucosal surfaces can be achieved by the CMIS through connections between inductive (e.g. GALT) and effector tissues. When vaccine antigens are enterically administered together with mucosal adjuvants [e.g. cholera toxin (CT), heat-labile toxin produced by Escherichia coli (LT) and IL-12], antigen-specific Th1/Th2 and IgA B cell responses are induced simultaneously in the mucosal effector compartment. Since these antigen-specific immune responses are not generated by oral vaccine without mucosal adjuvant, safe and effective adjuvants for the induction of antigen-specific S-IgA and CTL responses are essential for the development of mucosal vaccines for protection against infectious diseases. Finally, recent findings suggest the presence of a CMIS-independent IgA induction pathway, which also must be considered in the development of mucosal vaccines.     

Cholera toxin and its B subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development

Cholera toxin (CT) is a strong mucosal adjuvant for codelivered antigens, whereas its nontoxic B subunit (CTB) is an efficient mucosal carrier molecule for the generation of immune responses to linked antigens. We investigated the effects of CT and CTB on the immunogenicity of in vitro-treated antigen-pulsed dendritic cells (DC) following intravenous injection into mice. Prior to infusion, DC were pulsed for 90 min with either free ovalbumin (OVA), OVA mixed with CT or CTB, or chemical conjugates of OVA with CT and CTB (OVA-CT and OVA-CTB). DC pulsed with OVA or with OVA and CTB gave rise to modest antibody and T-cell responses. Conjugation of OVA with CTB enhanced both the subsequent B-cell and T-cell responses to OVA and preferentially induced Th2 responses. CT was shown to be a strong adjuvant when it was coadministered to DC with OVA and was even stronger when it was coadministered with OVA-CTB and primed for a mixed Th1-Th2 response. The antibody and T-cell responses were further enhanced if OVA was coupled to CT, implying that CT can utilize a combined carrier and adjuvant function vis-a-vis linked antigens for DC vaccination. The immunopotentiating capacity of CT- and CTB-linked antigen was associated with both upregulated secretion of interleukin-1beta by the pulsed DC and increased expression of CD80 and CD86 on the DC surface. These results imply that CT and CTB can be used to both markedly increase and partially direct the DC vaccine-induced immune response with respect to Th1 and Th2 responses, which has obvious implications for DC-based vaccine development.     

Effects of the adjuvant cholera toxin on dendritic cells: stimulatory and inhibitory signals that result in the amplification of immune responses

Cholera toxin (CT) is a potent mucosal adjuvant. When administered through the mucosal route CT amplifies B and T lymphocyte responses to co-administered antigens. Since the discovery of CT as a mucosal adjuvant, other bacterial enterotoxins have been found to have this property. These molecules or their detoxified derivatives are all important for the development of mucosal vaccines for human use, and it is thus necessary to understand their mechanism of action. CT has immunomodulatory effects on different cell types, however, the interaction of CT with dendritic cells (DCs), which have a primary role in the priming of immune responses, may be crucial for its adjuvant activity.     

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin.

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.     

Cholera toxin adjuvant greatly promotes antigen priming of T cells

Cholera toxin (CT) given perorally is a powerful mucosal immunogen and adjuvant. Information that explains the adjuvant effect of CT may be used for the development of more effective oral vaccines and might also contribute to our understanding of the mechanisms involved in regulating mucosal immunity. The present study was undertaken to investigate if CT administered together with keyhole limpet hemocyanin (KLH) would act to promote or inhibit priming of KLH-specific T cells and whether the adjuvant effect of CT is restricted to mucosal immune responses or is a generalized phenomenon due to direct immunomodulating effects of CT. We found that CT adjuvant greatly augmented the effectiveness of a single oral priming immunization with KLH: re-challenge with KLH in vitro 1 week following immunization gave several-fold stronger proliferation in KLH-specific spleen, mesenteric lymph node, Peyer's patch and gut lamina propria T cells from KLH + CT adjuvant as opposed to KLH only-treated mice. Moreover, several-fold stronger cytokine production, i.e. interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10 and interferon-Y accompanied the enhanced proliferative response of T cells from CT adjuvant-treated mice. The adjuvant effect of CT was not restricted to mucosal immune responses but was evident also following a single parenteral immunization with KLH + CT. Limiting dilution analysis revealed that CT adjuvant promoted a 20- to 40-fold increase in the frequency of primed KLH-specific T cells. Phenotypic and functional analyses clearly demonstrated that CT adjuvant primarily enhanced priming of CD4⁺ rather than CD8⁺ T cells and the pattern of lymphokine secretion disclosed that CT most probably promoted antigen priming of both T_hl and T_h2 type of CD4⁺ T precursor cells.     

Genetically manipulated bacterial toxin as a new generation mucosal adjuvant

Cholera toxin (CT) and heat-labile toxin (LT) of Escherichia coli act as adjuvants for the enhancement of mucosal and serum antibody (Ab) responses to mucosally co-administered protein antigen (Ag). Both LT and CT induce B7-2 expression on antigen-presenting cells (APCs) for subsequent co-stimulatory signalling to CD4+ T cells. CT directly affects CD4+ T cells activated via the TCR-CD3 complex with selective inhibition of Th1 responses whereas LT maintains Th1 cytokine responses with inhibition of interleukin (IL)-4 production. Interestingly, while CT failed to induce mucosal adjuvant activity in the absence of IL-4, LT did so. Nontoxic mutant (m)CTs (S61F and E112K) retain adjuvant properties by inducing CD4+ Th2 cells, which provided effective help for the Ag-specific mucosal immunoglobulin (Ig)A, as well as serum IgG1, IgE and IgA Ab responses. The mCT E112K has been shown to exhibit two distinct mechanisms for its adjuvanticity. Firstly, mCT enhanced the B7-2 expression of APCs. Secondly, this nontoxic CT derivative directly affected CD4+ T cells and selectively inhibited Th1 cytokine responses. Thus, several lines of evidence indicate that enzyme activity can be separated from adjuvant properties of CT and this offers promise for the development of safe delivery of vaccines for mucosal IgA responses.     

CD8alpha(-) and CD8alpha(+) subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of na?ve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8alpha expression. We have studied the phenotype and maturation state of mouse splenic CD8alpha(-) and CD8alpha(+) DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8alpha(-) and CD8alpha(+) DCs into potent and equally efficient activators of na?ve CD4(+) and CD8(+) T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.     

Dendritic cells in colonic patches and iliac lymph nodes are essential in mucosal IgA induction following intrarectal administration via CCR7 interaction

This study examined dendritic cells (DC) following intrarectal (IR) vaccination with the mucosal adjuvant cholera toxin (CT). Three rounds of IR vaccination with ovalbumin (OVA) and CT resulted in brisk levels of systemic and mucosal Ig responses. Immunohistochemical studies revealed that CD11c+ MHC class II+ cells accumulated primarily in the colonic patches (CP) and lamina propria of the large intestine (LI-LP), iliac LN (ILN) and MLN following IR vaccination with CT. Adoptively transferred CFSE-labeled OVA-specific CD4+ T cells proliferated significantly, secreting predominantly Th1-type cytokines in the CP (48 h after IR vaccination with CT) and Th2-type cytokines in the ILN (96 h after IR vaccination with CT). Following three IR vaccinations, CP-null mice that were generated by in utero treatment with anti-IL-7R Ab showed reduced levels of serum IgG and fecal IgA antibodies, suggesting a crucial role for CP in the initiation of systemic and mucosal immune responses. Of most interest, IR vaccination reduced IgA levels in fecal extracts significantly more in the CCR7-/- mice than in the wild-type mice. These results indicate that IR vaccination primarily mobilizes CD11c+ cells in the CP and ILN to induce optimal mucosal immune responses by CCR7 interaction.     

Human CD8⁺ T cells drive Th1 responses through the differentiation of TNF/iNOS-producing dendritic cells

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.     

Indoleamine 2,3-dioxygenase expression and functional activity in dendritic cells exposed to cholera toxin

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-metabolizing enzyme expressed by dendritic cells (DC), has the potential to inhibit T cell responses and to promote tolerance. In contrast, cholera toxin (CT), the enterotoxin produced by Vibrio cholerae, promotes T cell responses, partly through its ability to induce DC maturation and promote antigen presentation. We hypothesized that the adjuvant activity of CT is associated with a lack of induction of IDO in DC. To test this hypothesis, monocyte-derived DC were pulsed with CT, and the IDO mRNA expression, IDO functional activity and cytokine production were measured as well as the ability of DC to induce T cell responses in vitro. Cholera toxin exposure induced enhanced levels of IDO mRNA in DC but no functional IDO protein activity. Cholera toxin pulsing however primed DC for CD40L-induced IDO protein activity. CD40L stimulation of CT-pulsed DC induced a modest IL-12p40 production, but not IL-12p70 or IL-23 secretion. Furthermore, CT-pulsed DC induced strong allogeneic and autologous T cell responses in vitro, which were not affected by the IDO-specific inhibitor 1-methyl tryptophan. Our results show that CT per se does not induce the expression of functional IDO protein, although it primes DC for CD40L-mediated IDO production and IL-12p40 secretion. Furthermore, CT-treated DC were equally powerful in their T cell stimulatory capacity as cytokine-matured DC.     

Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-kappaB translocation

Cholera toxin (CT) is a potent adjuvant; however, the mechanism for its ability to enhance mucosal immunity has not been fully elucidated. We report here that CT exerts its adjuvant properties by signaling through the GM1 ganglioside receptor. When ganglioside-defective mice were given the antigen (Ag) ovalbumin (OVA) with CT by the oral route, CT failed to support either OVA-specific antibody or CD4+ T cell responses. In vitro treatment of murine bone marrow-derived dendritic cells (DC) with CT induced full maturation as evidenced by up-regulation of the costimulatory molecules, as well as by an enhanced ability to effectively present OVA for Ag-specific T cell responses. On the other hand, ganglioside-defective DC failed to differentiate to full function as Ag-presenting cells in response to CT. Since ganglioside-defective DC showed a mature phenotype after stimulation with lipopolysaccharide (LPS), the effects of CT on DC was independent of signal transduction through adjuvant receptor for LPS, the Toll-like receptor 4. Furthermore, CT also induced nuclear translocation of nuclear factor (NF)-kappaB in DC in a GM1-dependent fashion. These results highlight gangliosides expressed by DC for recognition of the non-self protein bacterial enterotoxin, which employ a unique signaling pathway to induce both innate and adaptive immunity.     

Human dendritic cells transfected with allergen-DNA stimulate specific immunoglobulin G4 but not specific immunoglobulin E production of autologous B cells from atopic individuals in vitro

Atopic/allergic diseases are characterized by T helper 2 (Th2)-dominated immune responses resulting in immunoglobulin E (IgE) production. DNA-based immunotherapies have been shown to shift the immune response towards Th1 in animal models. In further studies we showed that human dendritic cells (DC) transfected with allergen-DNA are able to stimulate autologous CD4(+) T cells from atopic individuals to produce Th1 instead of Th2 cytokines and to activate interferon-gamma (IFN-gamma)-producing CD8(+) T cells. The aim of this study was to analyse whether DC transfected with allergen-DNA are also able to influence immunoglobulin production of B cells from atopic donors. For this purpose, human monocyte-derived DC from grass-pollen allergic donors were transfected with an adenovirus encoding the allergen Phleum pratense 1 and cocultured with B cells, autologous CD4(+) T cells, and CD40 ligand-transfected L-cells. B cells receiving help from CD4(+) T cells stimulated with allergen-transfected dendritic cells produced more allergen-specific IgG4 compared to stimulation with allergen protein pulsed DC or medium, while total IgG4 production was not affected. In contrast, specific IgE production was not enhanced by stimulation with allergen-DNA transfected DC compared to medium and inhibited compared to allergen protein-pulsed DC with similar effects on total IgE production in vitro. Allergen-DNA transfected dendritic cells are able to direct the human allergic immune response from Th2-dominance towards Th1 and Tc1 also resulting in decreased IgE and increased IgG4 production.     

CD137 ligand reverse signaling has multiple functions in human dendritic cells during an adaptive immune response

T cell activation via dendritic cells (DC) is an important step in the adaptive immune response, which requires DC maturation, migration to lymph nodes and presentation of antigen to T cells. CD137 receptor expressed on activated T cells is a potent costimulatory molecule. Here, we investigated the functions of CD137 ligand (CD137L) in human monocyte-derived DC during an immune response. Cross-linking of CD137L on DC leads to cell maturation in an autocrine fashion, mostly via release of TNF-alpha. Reverse signaling of CD137L also mediates migration of DC via up-regulation of the CCR7 chemokine receptor, demonstrated by an in vivo MIP-3beta-dependent SCID mouse migration model. Finally, CD137L-activated DC induce differentiation of human T cells into potent Th1 effectors. Cocultivation of autologous T cells and CD137L-activated DC in an antigen-specific reaction leads to T cell proliferation and the release of IL-12p70 and IFN-gamma. These findings deliver new insights into the multiple effects of reverse signaling of CD137L in human DC during the initiation of an adaptive immune response, including the key features of DC maturation, migration and, ultimately, antigen-specific T cell differentiation.     

Strong adjuvant properties of cholera toxin on gut mucosal immune responses to orally presented antigens.

There is a great need for substances that can act as adjuvants on local mucosal immune responses to perorally (p.o.) administered immunogens and which could be included in future oral vaccines. In this study we show that in mice cholera toxin (CT) is a potent adjuvant on enteric mucosal immune responses to related (cholera B subunit) as well as unrelated (KLH) antigens presented by the p.o. route. The adjuvant action of CT was dose-dependent and was achieved only when CT was given p.o. and together with the antigen. Both priming (memory induction) and boosting of the gut mucosal immune system by the oral route were greatly potentiated by CT. High numbers of specific antibody-producing cells as well as substantial mucosal memory in the lamina propria were stimulated by p.o. priming immunizations if CT adjuvant was included. Anamnestic responses could be elicited by a single p.o. booster immunization for at least 10 weeks and probably much longer. The adjuvant action of CT is suggested to involve activation of adenylate cyclase and cyclic AMP-mediated signals with differential effects on B and regulatory T intestinal lymphocytes. The adjuvant-active dose of CT, 100-500 ng, was lower than the immunogenic dose (2 micrograms) and much below the p.o. dose needed for detectable net fluid secretion in mouse intestine (5-10 micrograms). Cholera B subunit (10 micrograms) administered p.o. together with 500 ng of CT was 50 times more effective in stimulating gut mucosal anti-toxin responses compared with B subunit vaccine alone. Our results suggest that CT or substances that use similar adjuvant mechanisms may substantially increase the mucosal immunogenicity and efficacy of non-replicating oral vaccines.     

Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent

Cholera toxin (CT) is a potent vaccine adjuvant when administered via parenteral, mucosal, or transcutaneous routes. It also inhibits innate inflammatory responses induced by pathogen-derived molecules, such as lipopolysaccharide (LPS). We demonstrated previously that CT promotes the induction of regulatory type 1 T cells (Tr1) as well as T helper type 2 cells (Th2). T cells from mice immunized with antigen in the presence of CT produced high levels of interleukin (IL)-10 and IL-5 and low levels of IL-4 and interferon-gamma (IFN-gamma). Here, we demonstrate that immunization with antigen in the presence of CT induced a population of antigen-specific CD4(+) T cells that produced IL-10 in the absence of IL-4, in addition to cells that coexpressed IL-4 and IL-10 or produced IL-4 only. CT-generated Tr1 cells inhibited antigen-specific proliferation as well as IFN-gamma production by Th1 cells, and this suppression was cell contact-independent. It is interesting that coincubation with Th1 cells significantly enhanced IL-10 production by the Tr1 cells. As IL-10 can promote the differentiation of Tr1 cells, we investigated cytokine production by dendritic cells (DC) following exposure to CT. Previous data showed that CT can modulate the expression of costimulatory molecules and inhibit the production of chemokines and cytokines, including IL-12 and tumor necrosis factor alpha and enhance IL-10 production. Here, we show that CT synergizes with LPS to induce IL-6 and IL-1beta in addition to IL-10 production by immature DC. Therefore, CT may promote the induction of Th2 and Tr1 cells in part via selective modulation of DC cytokine production and costimulatory molecule expression.     

Impairment of dendritic cell function by excretory-secretory products: a potential mechanism for nematode-induced immunosuppression

To determine whether helminth-derived products modulate dendritic cell (DC) function, we investigated the effects of excretory-secretory products (ES) and adult worm homogenate (AWH) derived from the gastrointestinal nematode Heligmosomoides polygyrus (Hp) on murine bone marrow-derived DC (BMDC). Compared to the TLR9 ligand CpG, Hp-derived products alone failed to induce DC activation. ES, but not AWH, inhibited BMDC cytokine and chemokine production and co-stimulatory molecule expression (CD40, CD86 and MHC class II) induced by TLR ligation. TLR ligand-independent, PMA-induced DC activation was unaffected by ES. Recipients of ES-treated BMDC pulsed with OVA had suppressed Ab responses in vivo, irrespective of the Th1 or Th2 isotype affiliation, compared to recipients of control OVA-pulsed BMDC. Importantly, suppression occurred even in the presence of the potent type 1 adjuvant CpG. In contrast to untreated OVA-pulsed BMDC, ES-treated BMDC pulsed with OVA had reduced co-stimulatory molecule and cytokine expression. CD4(+)CD25(+)Foxp3(-) T cells, which secreted high IL-10 levels, were generated in co-cultures of OT-II OVA-specific TCR-transgenic CD4(+) T cells and ES-treated BMDC. These IL-10-secreting T cells suppressed effector CD4(+) T cell proliferation and IFN-gamma production, the latter effect mediated by an IL-10-dependent mechanism. Together, these results demonstrate that nematode ES impaired DC function and suppressed both Th1 and Th2 adaptive immune responses possibly by inducing regulatory T cells.     

Cell-surface bound pertussis toxin induces polyclonal T cell responses with high levels of interferon-gamma in the absence of interleukin-12

Pertussis toxin (PTx), an exotoxin produced by Bordetella pertussis, has long been used as a mucosal adjuvant. We examined the T cell stimulatory properties of PTx in order to dissect its mechanisms of adjuvanticity. PTx or the B-oligomer of PTx (PTxB) failed to activate purified murine CD4+ or CD8+ T cells, as measured by a lack of proliferation or expression of early T cell activation markers. However, these T cells proliferated extensively in response to the toxin in the presence of syngeneic DC, and proliferation was accompanied by a high level of IFN-gamma production in the absence of IL-12. Interestingly, such responses were independent of signals mediated by MHC-TCR interaction. Both PTx and PTxB were found to bind stably to the surface of DC, and increased the adherence of DC to surrounding cells. These data suggest that polyclonal T cell responses mediated by the toxin are likely to be caused by the toxin bound on the surface of APC, either cross-linking cell surface molecules on T cells, or directly stimulating T cells together with the co-stimulatory molecules expressed on APC. B. pertussis may use this toxin as a mechanism to evade a specific immune response.     

Antigen processing of vesicular stomatitis virus in situ. Interdigitating dendritic cells present viral antigens independent of marginal dendritic cells but fail to prime CD4(+) and CD8(+) T cells

Acute macrophage (M phi) depletion, using a liposome-mediated 'suicide technique', markedly suppressed priming of splenic CD4(+) and CD8(+) T-cell responses to vesicular stomatitis virus (VSV). However, phagocytic marginal dendritic cells (MDC), but not interdigitating dendritic cells (IDC), are now known to be also depleted by this technique. To clarify the role splenic dendritic cell (DC) subsets and M phi play in priming for a virus-specific T-cell-mediated immune response, DC and M phi were purified from VSV-infected mice and assayed for the presence of epitopes recognized by VSV helper T (Th) cells and cytotoxic T lymphocytes (CTL). Antigen pulse experiments performed in situ demonstrated that VSV Th cell and CTL epitopes became transiently associated only with DC, but not M phi or B cells, indicating that DC represent the critical antigen-presenting cell (APC) population in vivo for this virus. The failure of MDC/M phi-deficient mice to become primed was not due to the complete elimination of antigen-presenting DC because VSV peptide/class I and II complexes were detected on IDC following lipsome-mediated elimination of phagocytic cells. However, the VSV-induced chemokine response was dramatically suppressed in these mice. Thus, despite the expression of VSV peptide/class I and II complexes, IDC are not sufficient to prime VSV Th cells in the absence of MDC and/or splenic M phi.