Dectin-1 a C-type lectin recognizing fungal and mycobacterial pathogens can deliver

Dectin-1 a C-type lectin recognizing fungal and mycobacterial pathogens can deliver intracellular signals that activate dendritic cells (DCs) resulting in initiation of SMI-4a immune responses and growth of Th17 CD4+ T cell responses. to CD1c+CD1a+ dermal DCs but not to epidermal Langerhans cells. Anti-hDectin-1-mediated DC activation resulted in upregulation of costimulatory molecules and secretion of multiple cytokines and chemokines in a Syk-dependent manner. DCs activated with the anti-hDectin-1 mAb could significantly enhance both neo and foreign antigen-specific CD8+ T cell responses by promoting both the expansion of SMI-4a CD8+ T cell and their functional activities. We further exhibited that delivering antigens to DCs via hDectin-1 using anti-hDectin-1-antigen conjugates resulted in potent antigen-specific CD8+ T cell responses. Thus hDectin-1 expressed on DCs can contribute to the induction and activation of cellular immunity against intracellular pathogens such as mycobacteria that are recognized by DCs via Dectin-1. Vaccines based on delivering antigens to DCs Rabbit Polyclonal to TRIM24. with an agonistic anti-hDectin-1 mAb could elicit CD8+ T cell-mediated immunity. are predominantly Th17 (13 14 and to a far lesser extent Th1 (23 24 Soluble factors including IL-6 and IL-1β secreted by antigen-presenting cells (APCs) promote the growth of Th17 responses (13 14 25 which are crucial for mounting protective immunity to intracellular bacterial pathogens such as mycobacteria (26) and (27). Dectin-1 contains a putative internalization signal sequence for the lysosomal endosome (28 29 and thus can contribute to pathogen-specific T cell responses. Mouse DCs that take up antigens via Dectin-1 can present antigenic peptides to both CD4+ and CD8+ T cells (21 22 One recent study showed that ovalbumin (OVA)-transgenic mice immunized with conjugates of anti-Dectin-1 and OVA generated strong CD4+ T cell responses but weak CD8+ T cell responses (22). However a more recent study (30) showed that mouse DCs activated with β-glucans could primary cytotoxic CD8+ T cell responses. These studies (21 22 suggested that antigens delivered to DCs via Dectin-1 could result in potent antigen-specific CD8+ T cell responses when DCs were activated by signaling via Dectin-1. Thus we hypothesized that antigens delivered to DCs SMI-4a via hDectin-1 with a concomitant activation of the DCs via the same hDectin-1 might result in potent antigen-specific CD8+ T cell responses. This hypothesis was tested using an agonistic anti-hDectin-1 mAb and anti-hDectin-1 mAb-antigen (neo and foreign antigens) conjugates. Our data showed that DCs activated with anti-hDectin-1 mAb resulted in enhanced CD8+ T cell responses. We further exhibited that anti-hDectin-1 mAb and its conjugates with both neo and non-self antigens could take action via DCs to elicit potent antigen-specific CD8+ T cell responses. Materials and Methods Antibodies and other reagents Anti-CD1a (BD Biosciences CA) and anti-CD1c (Biolegend CA) and anti-CD207 (BIIR clone 15B10) antibodies were used in immunofluorescence. SMI-4a Anti-human IgG (Fab) conjugated with alkaline phosphatase (AP) and all other antibodies SMI-4a used for staining cells were purchased from Southern Biotech (CA) and BD Biosciences respectively. IFNα IL-4 and GM-CSF were purchased from the pharmacy at Baylor University Medical Center (TX). IL-2 and CFSE were purchased from Peprotech (NJ) and Molecular Probes (CA) respectively. Piceatannol curdlan and laminarin were from Sigma (MO). lipopolysaccharide (LPS) was purchased from Invivogen (CA). HLA-A*0201 tetramers of influenza viral (Flu) M158-66 and MART-126-35 (27L) were purchased from Beckman Coulter (CA). Flu M158-66 MART-126-35 and MART-126-35 (27L) peptides were synthesized by Biosynthesis (TX). Anti-hDectin-1 monoclonal antibody (mAb) Receptor ectodomain.hIgG (human IgG1Fc) and AP (human placental alkaline phosphatase) fusion proteins were produced for immunizing mice and screening mAbs respectively. A mammalian vector for human Fc and AP fusion proteins was designed as previously described (31). Fusion proteins were produced using the FreeStyle? 293 Expression System (Invitrogen CA) according to the manufacturer’s protocol. Receptor ectodomain.hIgG was purified by 1 ml HiTrap protein A affinity chromatography (GE Healthcare CA). Six-week-old BALB/c mice.