Antigen presentation by dendritic cells and immunomodulation by cholera toxin B subunit. Implications for vaccine development

Sammanfattning: Antigen presentation by dendritic cells is the first event that takes place upon initiation of an acquired immune response. The inductive events during antigen presentation will essentially govern the orientation and the magnitude of the subsequent immune responses. We have investigated different aspects of antigen presentation by dendritic cells, especially when antigen was coupled to the mucosal carrier protein cholera toxin B subunit (CTB). CTB linked to protein antigens functions as a mucosal carrier protein to induce local antibody production together with systemic T cell tolerance. The carrier properties of CTB is mainly attributed to its ability to bind GM1 ganglioside present on virtually all cells. Antigen presentation of CTB conjugated antigens by DC was evaluated in vitro. For this purpose peptides and proteins were chemically coupled or genetically fused to CTB. We discovered that coupling of antigen to CTB dramatically enhanced the antigen-presenting capacity of DC and other professional APC, defined as an increase in proliferation and IFN-g production by antigen-specific T cells. This effect was mainly due to the GM1 binding properties of CTB, since incubation of CTB-coupled antigens with its natural receptor GM1 blocked the enhancement of APC function. However, CTB-conjugation also increased the expression of the costimulatory molecules B7.2 and CD40 on APC, implying that CTB coupling also has an immunomodulatory effect. Differental positioning of a T cell epitope within the CTB molecule affected the subsequent production of IFN-g without affecting proliferation. This effect is proposed by us to be linked to the peptides 56-63 in the CTB molecule, since replacement of this region with a T cell epitope abrogated the IFN-g blocking effect of CTB. Although CTB is supposed to have a tolerogenic effect upon delivery through the muco-epithelial surfaces, epicutaneous application of antigens coupled to CTB was shown to induce mainly a Th1 biased response in vivo. Application of both the native toxin (CT) and its B subunit increased numbers of infiltrating Langerhans cells to the skin. Whereas CT induced a mixed Th1 and Th2 profile to admixed antigen applied to the skin, CTB induced a clear Th1 profile with IL-2 and IFN-g production upon in vitro challenge with protein, underscoring the importance of the microenvironment for the direction of the immune response by APC. That buccal exposure to antigen leads to uptake by Langerhans cells in the buccal epithelium and subsequent migration of Langerhans cells to the draining lymph nodes was demonstrated. We also showed that DC vaccination was able to induce mucosal antibody responses and to protect against a mucosal extracellular pathogen in a mouse model of Bordetella pertussis infection. Furthermore, both autologous and heterologous DC were able to protect against infection, an observation that could have future implications in cases where conventional vaccines are inefficient or non-existing. In summary we show that CTB conjugation enhances antigen presentation by DC through increased antigen uptake and that apart from its GM1 binding properties it also posesses immunomodulatory functions.