Cholera toxin B subunit (CTB) is an efficient mucosal carrier molecule for the generation of mucosal antibody responses and/or induction of systemic T-cell tolerance to linked antigens. CTB binds with high affinity to GM1 ganglioside cell surface receptors. In this study, we evaluated how conjugation of a peptide or protein antigen to CTB by chemical coupling or genetic fusion influences the T-cell-activating capacity of different antigen-presenting cell (APC) subsets. Using an in vitro system in which antigen-pulsed APCs were incubated with antigen-specific, T-cell receptor-transgenic T cells, we found that the dose of antigen required for T-cell activation could be decreased >10,000-fold using CTB-conjugated compared to free antigen. In contrast, no beneficial effects were observed when CTB was simply admixed with antigen. CTB conjugation enhanced the antigen-presenting capacity not only of dendritic cells and B cells but also of macrophages, which expressed low levels of cell surface major histocompatibility complex (MHC) class II and were normally poor activators of naive T cells. Enhanced antigen-presenting activity by CTB-linked antigen resulted in both increased T-cell proliferation and increased interleukin-12 and gamma interferon secretion and was associated with up-regulation of CD40 and CD86 on the APC surface. These results imply that conjugation to CTB dramatically lowers the threshold concentration of antigen required for immune cell activation and also permits low-MHC II-expressing APCs to prime for a specific immune response.Cholera toxin B subunit (CTB) and the closely related Escherichia coli heat-labile enterotoxin B subunit (LTB) are highly efficient carrier molecules for the induction of mucosal antibody responses (10, 17, 27, 31) and oral tolerance (2, 17, 42). The therapeutic applications of CTB-mediated oral tolerance, as demonstrated in animal models, include the prevention and treatment of T-cell-mediated autoimmune diseases (7,44,49), immunoglobulin E (IgE)-mediated allergic reactions (37,47,55), and infection-induced pathological inflammatory conditions (32, 43). The mechanism behind CTB's or LTB's efficacy as a mucosal carrier molecule has not been fully defined but is believed to be associated with the strong binding of CTB or LTB to the GM1 receptor present on most cells in the body, including epithelial cells and leukocytes. Efficient binding to GM1 could potentially increase the uptake of antigen across the mucosa and lead to an enhanced presentation of the conjugated molecule to the immune system (17, 34). Another possibility is that CTB per se has immunomodulating properties. Indeed, it has been shown that CTB induces major histocompatibility complex (MHC) class II expression on B cells (14), enhances antigen presentation by macrophages (M) in the absence of enhanced MHC II expression (29), and blocks the development of diabetes in NOD mice through the development of regulatory cells (40).Mucosal antigen-specific antibody formation and tolerance induction share the requirement ...

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Dendritic cells (DC) express a functional NADPH oxidase and produce reactive oxygen species (ROS) upon interaction with microbes and T cells. Exposure to ROS leads to DC activation and maturation, as evidenced by phenotypic and functional changes. We have evaluated how endogenous ROS production affects the cytokine secretion pattern and T cell-activating capacity of bone marrow-derived murine DC. DC treated with ROS scavengers, as well as DC from mice that lack a functional NADPH oxidase (and thereby inherently deficient in ROS production) produced significantly increased levels of IL-1b, IL-6, TNF-a and TGF-b in response to microbial activation. DC deficient in ROS production induced high levels of IFN-c and IL-17 in responding T cells after Ag-specific or superantigen-induced activation. Finally, we show that ROS deficiency affected the induction of a T cell-dependent inflammatory condition, collagen-induced arthritis (CIA). C57BL/6 mice that lack a functional NADPH oxidase developed a severe and erosive CD4-dependent CIA, whereas the majority of the congenic wild-type animals remained healthy. These data suggest that ROS act as immunomodulators in DC-driven T cell activation and perhaps also in T cell-dependent immunopathology.Key words: CD4 + T cells Á Cytokines Á Dendritic cells Á Immune regulation Á Rheumatology IntroductionImmature myeloid DC reside in non-lymphoid organs and sense the environment through the expression of a panel of patternrecognition receptors. In the immature stage, DC have a high capacity to ingest foreign Ag but they express low levels of molecules that are involved in T cell activation. Once activated through one or several pattern recognition receptors, the DC undergo phenotypic and functional changes that involve migration to a lymphoid organ, reduced ability to sample Ag, and enhanced expression and secretion of molecules that attract and activate naive T cells. Thus, both the localization of DC and the capacity of DC to activate T cells are maturation dependent. Depending on the nature of the activating stimuli and the signaling properties of the pattern-recognition receptors involved, the DC acquire certain traits that affect the quality of the ensuing T cell response. The best known example is the development of Th1 and Th2 responses, which depend to a large extent on the cytokine secretion profile induced in the DC (reviewed in [1]). Consequently, factors that affect the maturation of DC influence the subsequent T cell profile.ROS represent endogenous factors that may influence the function of DC. ROS induce both DC maturation [2, 3] and the synthesis of pro-inflammatory cytokines [4]. Conversely, antioxidants block DC maturation [5]. DC have all the components of a functional NADPH oxidase within their plasma membranes [6], and produce ROS in response to both cognate T cell interactions [7] and microbial components [8,9]. Thus, ROS could serve as endocrine regulators of DC function and thereby influence the nature of the ensuing immune response. There is evidence that ROS c...

Topical application of cholera toxin (CT) onto mouse skin can induce a humoral immune response to CT as well as to coadministered Ags. In this study, we examined the nontoxic cell-binding B subunit of CT (CTB) as a potential adjuvant for cutaneous immune responses when coadministered with the prototype protein Ag, OVA. CTB applied onto skin induced serum Ab responses to itself with magnitudes comparable to those evoked by CT but was poorly efficient at promoting systemic Ab responses to coadministered OVA. However, transcutaneous immunization (TCI) with either CT or CTB and OVA led to vigorous OVA-specific T cell proliferative responses. Furthermore, CTB potentiated Th1-driven responses (IFN-γ production) whereas CT induced both Th1 and Th2 cytokine production. Coadministration of the toxic subunit CTA, together with CTB and OVA Ag, led to enhanced Th1 and Th2 responses. Moreover, whereas TCI with CT enhanced serum IgE responses to coadministered OVA, CTB suppressed these responses. TCI with either CT or CTB led to an increased accumulation of dendritic cells in the exposed epidermis and the underlying dermis. Thus, in contrast to CT, CTB appears to behave very differently when given by the transcutaneous as opposed to a mucosal route and the results suggest that the adjuvanticity of CT on Th1- and Th2-dependent responses induced by TCI involves two distinct moieties, the B and the A subunits, respectively.

Introduction The tumour necrosis factor (TNF) family ligands BAFF (B-cell activating factor of TNF family) and APRIL (a proliferation-inducing ligand) are essential for B-cell survival and function. Elevated serum levels of BAFF and APRIL have been reported earlier in patients with systemic lupus erythematosus (SLE). Since autoantibody formation in the central nervous system (CNS) is a distinct feature of neuropsychiatric SLE (NPSLE), we have investigated whether NPSLE is associated with an enhanced intrathecal production of APRIL and BAFF.