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We quantified immunoglobulin E (IgE) on peritoneal mast cells of interleukin-4 (IL-4)-gene knockout (-/-) mice and wild-type (+/+) controls using a cytofluorometric method, and examined the expression of IgE receptors, estimated by quantifying the total binding of IgE on the mast cells of IL-4 (-/-) mice. The mast cells of IL-4 (+/+) mice, identified and measured using microscope fluorometry, had a fluorescence intensity five to six times higher than that of non-mast cells, while the mast cells obtained from IL-4 (-/-) mice had fluorescence intensities within the range of those of non-mast cells. Two weeks after an infection with Nippostrongylus brasiliensis, the fluorescence intensity of the mast cells of IL-4 (+/+) mice increased to a level about twice as high as that before immunization. However, no significant increase after infection was observed in IL-4 (-/-) mice. Furthermore, the mast cells of IL-4 (-/-) mice did not bind IgE when incubated with IgE at concentrations that saturated IgE receptors on the mast cells of wild-type controls, thereby indicating that the expression of IgE receptors on mast cells was impaired in the IL-4-deficient mice. Using a reverse transcription-polymerase chain reaction, we found gene expression of all three subunits (alpha-, beta- and gamma-chains) of the IgE receptor in IL-4 (-/-) like that in IL-4 (+/+) mice. The results thus suggest that the binding of IgE may be essential to induce the translation of mRNA to IgE-receptor proteins. We also observed that there were about twice as many peritoneal mast cells in the IL-4 (-/-) mice as there were in the IL-4 (+/+) mice, in both immunized and non-immunized animals. This was unexpected in view of previous findings suggesting that IL-4, in concert with stem cell factor and IL-3, stimulates the proliferation and differentiation of mast cells in vitro.

Using IL-6-deficient (IL-6 -/-) or wild-type mice, we investigated whether IL-6 is involved in the intestinal adjuvant activity of cholera toxin (CT) and to what extent IL-6 is required for mucosal IgA responses against soluble protein Ags or live Helicobacter felis infection. In naive IL-6 -/- mice we found normal total IgA levels in serum, bronchial and intestinal lavage and unaltered frequencies of IgA plasma cells in intestinal lamina propria. In Peyer's patches (PP) and mesenteric lymph nodes (MLN) IgA-producing cells were as frequent in IL-6 -/- as in wild-type mice. Immunohistochemical analysis of PP revealed germinal centers that co-localized IgA+ cells, indicating B cell activation and isotype switching in situ in the intestinal immune inductive site. Phenotypic analysis of the distribution of conventional B-2 cells (B220+CD5-/Mac-1-) and B-1 cells (B220+, CD5+/Mac-1+) in intestine-associated tissues gave comparable results in IL-6 -/- and wild-type mice. The ability to respond with mucosal IgA following oral and intranasal immunization with specific Ag, KLH or OVA, in the presence of CT adjuvant or to live H. felis infection was similar in IL-6 -/- and wild-type mice. CT exerted strong and comparable adjuvant functions in IL-6 -/- and wild-type mice. Repeated oral immunizations with CT alone stimulated immune protection against CT-induced diarrhea in ligated loops that was of similar magnitude in IL-6 -/- and wild-type mice. We conclude that, although IL-6 has been ascribed a crucial role in terminal differentiation of IgA B cells in vitro, we found no evidence to support the notion that IL-6 is critically required for IgA B cell development or specific mucosal IgA responses in vivo.

The role of antibodies and antigen deposition for the development of immunological memory has been incompletely investigated. We addressed whether long-term protection and T-cell memory can be stimulated against a genital tract infection with human Chlamydia trachomatis serovar D in B-cell-deficient (muMT) mice. At 6 months following a primary infection with C. trachomatis, both muMT and wild-type (WT) mice exhibited strong and comparable protection against reinfection. Evidence of long-lasting CD4+ T-cell memory was found in both muMT and WT mice, typified by comparable delayed-type hypersensitivity (DTH) reactions against chlamydial antigens. No bacterial or chlamydial DNA was found in the genital tract of muMT memory mice, suggesting that immunological memory was maintained in the absence of antigen. Whereas few T cells were present in the genital tract of memory mice, rapid recruitment of CD4+, and some CD8+, T cells into the genital tract tissue was observed after challenge with live bacteria. Accumulation of T cells in the genital tract was preceded by a short transient infection of similar magnitude in both muMT and WT memory mice, arguing against a long-term protective role of local antibodies. The rapid recruitment of CD4+ T cells into the genital tract was associated with a transient detection of interferon-gamma (IFN-gamma) mRNA in the genital tract in chlamydia-immune memory mice, which was not found in naïve, challenged mice. Thus, long-term protection in the genital tract against C. trachomatis infection is conveyed by IFN-gamma-producing CD4+ memory T cells, which appear to be maintained in the absence of antibodies and local antigen deposition.

In recent studies we have demonstrated that immunological B- and T-cell memory may be stimulated effectively by oral immunization, simply by admixing protein antigens with cholera toxin (CT) adjuvant. Here we extend information by employing a hapten-carrier system allowing us to separate B- and T-cell memory and to evaluate the requirement of memory T cells for effective reactivation of mucosal memory B cells. We found that 2 weeks following oral priming immunizations with dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) plus CT adjuvant, significant serum anti-DNP antibodies of IgG, IgA and IgM immunoglobulin classes were demonstrated. However, after 2 years only IgM anti-DNP antibodies could still be detected in serum. When memory lymphocytes were isolated from these mice, from both systemic and gut-associated lymphoid tissues, and challenged with antigen in vitro, vigorous IgM, but no IgG or IgA, anti-DNP production was observed. By contrast, when the DNP-KHL-primed memory mice were challenged in vivo by an oral booster immunization with DNP-KLH plus CT adjuvant, strong systemic IgG and local mucosal IgA anti-DNP responses were recorded, while IgM anti-DNP production was poor. Moreover, the mucosal memory B cells from DNP-KHL-immunized mice were more responsive in vivo to an oral booster immunization with the carrier-specific antigen, DNP-KLH, compared to that provided by an unrelated carrier, DNP-human serum albumin (HSA), which gave only poor mucosal and systemic anti-DNP B-cell responses. Taken together our data suggest that mucosal memory B cells are recirculating cells that have retained their ability to produce IgM antibodies and, therefore, have not undergone switch differentiation involving gene rearrangements with constant mu-chain deletions. Furthermore, mucosal B-cell memory and CD4+ T-cell memory are closely interconnected phenomena, requiring both components for effective expression and probably also for maintenance of immunological memory in the mucosal immune system.

This study was conducted to investigate whether cholera toxin (CT), used as a mucosal adjuvant, would promote the development in mice of immunological memory to unrelated antigens administered by the oral route. We found that oral priming immunizations with keyhole limpet haemocyanin (KLH) in combination with CT adjuvant induced long-term, at least 22 months and perhaps lifelong, immunological memory in the intestinal lamina propria (LP). In contrast, oral priming immunizations with KLH alone failed to stimulate immunological memory. Moreover, memory responses in the KLH plus CT-immunized mice were elicited by antigen alone, i.e. without CT adjuvant, suggesting that once immunological memory is established in the intestinal mucosa, e.g. by oral vaccination, elicitation of secondary-type responses does not require the presence of CT and thus could result from re-encounter with specific bacterial or viral antigens in the intestine. We also found that a single priming-dose of KLH plus CT adjuvant was sufficient to stimulate long-term, antigen-specific memory in the intestinal mucosa. Finally, the ability of CT to induce immunological memory in the gut mucosa required the whole toxin and could not be achieved by using the toxoid, the cholera toxin B subunit (CTB), which lacks the adenylate cyclase/cAMP-activating property of the whole molecule. The results support the view that mucosal adjuvants, incorporated into oral vaccines, might be an effective means to achieve long-term immunological memory and protection against pathogenic micro-organisms at mucosal surfaces.

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.

The development of subunit vaccines requires the use of adjuvants that act by stimulating components of the innate immune response. Immune-stimulating complexes (ISCOMS) containing the saponin adjuvant Quil A are potential vaccine vectors that induce a wide range of Ag-specific responses in vivo encompassing both humoral and CD4 and CD8 cell-mediated immune responses. ISCOMS are active by both parenteral and mucosal routes, but the basis for their adjuvant properties is unknown. Here we have investigated the ability of ISCOMS to recruit and activate innate immune responses as measured in peritoneal exudate cells. The i.p. injection of ISCOMS induced intense local inflammation, with early recruitment of neutrophils and mast cells followed by macrophages, dendritic cells, and lymphocytes. Many of the recruited cells had phenotypic evidence of activation and secreted a number of inflammatory mediators, including nitric oxide, reactive oxygen intermediates, IL-1, IL-6, IL-12, and IFN-γ. Of the factors that we investigated further only IL-12 appeared to be essential for the immunogenicity of ISCOMS, as IL-6- and inducible nitric oxide synthase knockout (KO) mice developed normal immune responses to OVA in ISCOMS, whereas these responses were markedly reduced in IL-12KO mice. The recruitment of peritoneal exudate cells following an injection of ISCOMS was impaired in IL-12KO mice, indicating a role for IL-12 in establishing the proinflammatory cascade. Thus, ISCOMS prime Ag-specific immune responses at least in part by activating IL-12-dependent aspects of the innate immune system.