Targeting antigens to dendritic cell (DC)-specific receptors, such as DC-SIGN, induces potent T cell-mediated immune responses. DC-SIGN is a transmembrane C-type lectin receptor with a long extracellular neck region and a carbohydrate recognition domain (CRD). Thus far, only antibodies binding the CRD have been used to target antigens to DC-SIGN. We evaluated the endocytic pathway triggered by antineck antibodies as well as their intracellular routing and ability to induce CD8 ؉ T-cell activation. In contrast to anti-CRD antibodies, antineck antibodies induced a clathrin-independent mode of DC-SIGN internalization, as demonstrated by the lack of colocalization with clathrin and the observation that silencing clathrin did not affect antibody internalization in human DCs. Interestingly, we observed that anti-neck and anti-CRD antibodies were differentially routed within DCs. Whereas anti-CRD antibodies were mainly routed to late endosomal compartments, anti-neck antibodies remained associated with early endosomal compartments positive for EEA-1 and MHC class I for up to 2 hours after internalization. Finally, cross-presentation of protein antigen conjugated to antineck antibodies was approximately 1000-fold more effective than nonconjugated antigen. Our studies demonstrate that anti-neck antibodies trigger a distinct mode of DC-SIGN internalization that shows potential for targeted vaccination strategies. IntroductionDendritic cells (DCs) play a key role in initiating adaptive immune responses by capturing antigens and presenting them to T cells. The discovery of receptors that are mainly expressed by DCs, such as several members of the C-type lectin receptor (CLR) family, allows for vaccination strategies that target antigens directly to DCs in vivo. 1 Targeted delivery of antigens through CLRs stimulates antigen presentation and results in immunity when DC maturation stimuli are coadministered. 2 Although most of the vaccines currently on the market mainly induce humoral responses, many DC-targeted vaccines also induce strong cytotoxic T cell (CTL) responses. 1 This is attributed to the ability of certain DC subsets to present exogenous antigens on MHC class I, a process called cross-presentation.CLRs bind carbohydrate structures in a Ca 2ϩ -dependent manner via their carbohydrate recognition domain (CRD). The CRD binds specific mannose, galactose, or fucose structures present on self or non-self-proteins. 3 Human DC-SIGN represents a member of the CLR family that has been explored for DC vaccination strategies. The extracellular part of DC-SIGN is composed of a C-terminal CRD and a neck region consisting of 7 complete, and 1 incomplete, 23-residue tandem repeat regions. DC-SIGN has been demonstrated to recognize many pathogens, including viruses, bacteria, fungi, and parasites. 4 After binding, these pathogens are internalized and pathogen-derived antigens are presented via MHC class I and II molecules to CD8 ϩ and CD4 ϩ T cells, respectively. 5,6 By analogy, vaccination strategies targeting antigens to the CR...

Probiotics can modulate the immune system, conferring beneficial effects on the host. Understanding how these microorganisms contribute to improve the health status is still a challenge. Previously, we have demonstrated that Enterococcus faecalis CECT7121 implants itself and persists in the murine gastrointestinal tract, and enhances and skews the profile of cytokines towards the Th1 phenotype in several biological models. Given the importance of dendritic cells (DCs) in the orchestration of immunity, the aim of this work was to elucidate the influence of E. faecalis CECT7121 on DCs and the outcome of the immune responses. In this work we show that E. faecalis CECT7121 induces a strong dose-dependent activation of DCs and secretion of high levels of IL-12, IL-6, TNFα, and IL-10. This stimulation is dependent on TLR signaling, and skews the activation of T cells towards the production of IFNγ. The influence of this activation in the establishment of Th responses in vivo shows the accumulation of specific IFNγ-producing cells. Our findings indicate that the activation exerted by E. faecalis CECT7121 on DCs and its consequence on the cellular adaptive immune response may have broad therapeutic implications in immunomodulation.

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8 + T-cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC-SIGN (DC-specific-ICAM3-grabbing-nonintegrin/CD209) is a C-type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC-SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC-SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti-DC-SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti-DC-SIGN antibodies conjugated to OVA induced strong and persistent antigen-specific CD4 + and CD8 + T-cell responses, which efficiently protected from infection with OVA-expressing Listeria monocytogenes. Thus, we propose DC targeting via DC-SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens. Keywords: Crosspresentation · DC-SIGN · Dendritic cells · Listeria · VaccineAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe development of effective vaccines against intracellular infectious agents is one of the biggest challenges in science and an urgent unmet medical need. In addition, other highly prevalent diseases like cancer strongly require new vaccination strategies Correspondence: Prof. Tim Sparwasser e-mail: tim.sparwasser@twincore.de that promote effective and long lasting cytotoxic CD8 + T-cell (CTL) responses.With the discovery of DCs in 1973 by the Nobel Prize laureate Ralph Steinman, a new window of opportunities opened in vaccine development. Consequently, for many years efforts have been made to develop vaccines based on ex vivo generated DCs * These authors equally contributed to this work. Eur. J. Immunol. 2013Immunol. . 43: 2543Immunol. -2553 carrying specific antigens from tumors or pathogens [1]. Although these studies showed promising results in mice and in a number of clinical studies, the production of antigen-bearing cellular DC vaccines is a labor intensive and costly procedure [1]. Therefore, it hardly represents an option to vaccinate against infectious diseases like tuberculosis, malaria, or HIV, which mainly affect developing countries. More recently, a new strategy has been explored to target defined antigens to DCs by using antibodies or glycosylated molecules that bind specific surface receptors directly in vivo [2][3][4]. Many of the receptors studied for DC targeting purposes belong to the family of C-type lec...