Cholera toxin B subunit as a carrier molecule promotes antigen presentation and increases CD40 and CD86 expression on antigen-presenting cells.

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.

Vaccination with Bordetella pertussis-pulsed autologous or heterologous dendritic cells induces a mucosal antibody response in vivo and protects against infection.

This study demonstrates for the first time that vaccination with either autologous or heterologous dendritic cells (DC) pulsed with specific antigen induces protective immune responses against noninvasive bacteria, namely Bordetella pertussis. The DC-mediated protection is associated with strong B. pertussis-specific immunoglobulin G (IgG) and IgA responses in the lung.

Mucosal immunity and tolerance: relevance to vaccine development.

The mucosal immune system of mammals consists of an integrated network of lymphoid cells which work in concert with innate host factors to promote host defense. Major mucosal effector immune mechanisms include secretory antibodies, largely of immunoglobulin A (IgA) isotype, cytotoxic T cells, as well as cytokines, chemokines and their receptors. Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination or natural immunization by a mucosal route can effectively induce immune suppression. The diverse compartments located in the aerodigestive and genitourinary tracts and exocrine glands communicate via preferential homing of lymphocytes and antigen-presenting cells. Mucosal administration of antigens may result in the concomitant expression of secretory immunoglobulin A (S-IgA) antibody responses in various mucosal tissues and secretions, and under certain conditions, in the suppression of immune responses. Thus, developing formulations based on efficient delivery of selected antigens/tolerogens, cytokines and adjuvants may impact on the design of future vaccines and of specific immunotherapeutic approaches against diseases associated with untoward immune responses, such as autoimmune disorders, allergic reactions, and tissue-damaging inflammatory reactions triggered by persistent microorganisms.

Antigen presentation in the murine oral epithelium.

We have previously reported that the buccal mucosa can support delayed type hypersensitivity (DTH) reactions to contact sensitizers. In the present study, we show that cells isolated from the buccal epithelium are able to present soluble exogenous antigens to specific T cells. Single cell suspensions obtained by enzymatic dispersion of buccal epithelial sheets could present the native protein antigen hen-egg lysozyme (HEL) to the I-Ak-restricted CD4+ T-cell hybridoma specific for a.a 46-61 on HEL. T-cell activation resulted in interleukin-2 (IL-2) production which could be inhibited by anti-major histocompatibility complex (MHC) class-II antibodies of pertinent specificity. Immunohistochemical staining of whole buccal epithelial sheets revealed that all MHC II positive cells had a dendritic morphology and expressed ATPase activity, indicating that these cells represent a major antigen-presenting cell (APC) population in this tissue. Furthermore, single cell suspensions isolated from buccal epithelium (BEC) after local in vivo administration of either a native soluble protein, a synthetic dodecapeptide, or a contact sensitizer were able to activate antigen-specific T cells ex vivo. Kinetic analyses indicated that maximal APC activity in the oral epithelium occurred within 1 hr after local antigen administration, and had essentially vanished after 24 hr. Conversely, APC activity was undetectable in draining cervico-mandibular lymph node cell suspensions recovered 1 hr after local antigen injection but became manifest after 3-24 hr. These observations suggest that dendritic cells can acquire antigens in the buccal epithelium and migrate to draining lymph nodes where they present processed antigen to MHC class II-restricted T cells. This APC population may thus be a critical element in the initiation of Th1-driven DTH responses in the oral mucosa.

Differential in vivo effects of a superantigen and an antibody targeted to the same T cell receptor. Activation-induced cell death vs passive macrophage-dependent deletion.

Superantigens have multiple pleiotropic effects in vivo, causing the activation, proliferation, and deletion of specific T cells. In our study, we analyzed the effects of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) on peripheral T cells in vivo. As an internal control we took advantage of a IgG2a mAb, F23.1 (anti-V beta 8), that recognizes products from the same V beta gene family as that recognized by SEB. Suprisingly, not only SEB, but also F23.1 primes peripheral T cells to undergo oligonucleosomal DNA fragmentation typical for programmed cell death (PCD). Nonetheless the deletion and induction of PCD imposed by both agents obey rather different principles. First, SEB, not F23.1-induced PCD, concerns T cells that have passed through the S phase of the cell cycle, as demonstrated by experiments in which the thymidine analogue 5-bromo-2'desoxyuridine was detected in mono- and oligonucleosomal fragments of T cells undergoing PCD. Second, deletion of V beta 8+ T cells induced by SEB, not F23.1, can be blocked in vivo by high doses of retinol and, during the early phase, by glucocorticoid receptor blockade with RU-38486. Inasmuch as retinol fails to antagonize the glucocorticoid-induced PCD, at least two pathways are involved in early SEB-driven deletion, one that depends on the presence of endogenous glucocorticoid, and another that can be inhibited by retinol. Third, depletion of phagocytes in vivo by means of liposome-encapsulated dichloromethylene diphosphonate does not impede the activation and deletion of V beta 8+ cells by SEB, although it partially prevents the elimination of T cells binding F23.1 in vivo. Thus, macrophages are not rate-limiting for the action of SEB. In a further series of experiments, we demonstrate that SEB causes the secretion of a variety of cytokines (IL-1, -2, -4, -10, granulocyte-macrophage-CSF, IFN-gamma, and TNF) that may cause lethal septic shock. In contrast, F23.1 that efficiently induces all these mediators in vitro, fails to do so in vivo. In synthesis, the elimination of T cells induced by two different agents specific for V beta 8 obeys different principles: activation-induced cell death in the case of SEB and passive macrophage-mediated elimination in the case of F23.1.