The numbers of DCs secreting IL-12 were then assessed by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT). high antibody titres in experimental animals.14 == Determine 1. == A schematic representation of the synthetic lipid core peptide construct. An effective adjuvant should target the accompanying vaccine towards dendritic cells (DCs), to improve antigen presentation and activation of immune responses. DCs are leucocytes that excel in the uptake, processing and presentation of antigen to T cells. Appropriately activated DCs interact with CD4+T PF-06651600 lymphocytes through cell surface receptors such as major histocompatibility complex class II (MHCII), CD80 and/or CD86 and secrete cytokines such as interleukin (IL)-12 that induce activation of T cells. In mice, DCs are characterized by the expression of CD11c and can be divided into broad populations based on the expression of CD8 and CD4, accordingly termed CD8+, CD4+and the double-negative (DN) DC (CD8CD4).5,6In humans, CD4 and CD8 do not differentiate DC subpopulations. DCs also express pattern recognition molecules known as the Toll-like receptors (TLRs) which provide activation signals to DCs. The TLR family consists of 11 members (TLR111) that recognize different varieties of pathogen-associated molecular patterns,7generally leading to the maturation and migration of DCs to the lymph nodes to initiate an immune response. While mRNA for TLR2 and TLR4 has been found in all murine DC subsets, 8TLR9 protein expression and function have been shown in these cells.9,10 TLR2 is unique in that it recognizes a rather broad range of lipid ligands derived from different types of microbes (e.g. bacteria). Among these, TLR2 binds to lipoproteins/lipoteichoic acids derived from both natural (various pathogens) and synthetic sources, and also recognizes atypical types of lipopolysaccharide (LPS).11TLR2 heterodimerizes PF-06651600 with either TLR1 or TLR6 for the recognition tri- or di-acylated lipids, respectively,1219but this is not a strict requirement as synthetic lipopeptides such as macrophage-activating lipopeptide (MALP2)-SK4, Pam2C-SK4and Pam3C-SK4can signal in the absence of both receptors,20,21leading to the notion that TLR2 may KLF15 antibody form functional homodimers for signalling. In the present study, given that all adaptive immune responses are initiated by DCs, we investigated the PF-06651600 effect of LCP on DC phenotype and functionin vivoand whether the effects could be mediated by TLR2 signalling. == Materials and methods == == Lipid core peptide == The LCP construct was synthesized as previously described,1dissolved in sterile, endotoxin-free phosphate-buffered saline (PBS) and stored in aliquots at 80 until required. == Mice == Female C57BL/6 mice were purchased from the Central Animal Breeding House (Pinjarrah Hills, Qld, Australia) or from the Herston Medical Centre (Herston, Qld, Australia). Female OT-II mice were kindly provided by Dr Adriana Baz of the Queensland Institute of Medical Research (QIMR, Herston, Qld, Australia). == Immunizations and tissue collection == In these studies, we compared the effects of LCP alone and in conjunction with an immunogenic protein ovalbumin (OVA) on DC phenotype and function. Mice were injected intraperitoneally (i.p.) either with 50 g of LCP or 50 g of LCP + 50 g of OVA (Sigma-Aldrich, St Louis, MO) or with PBS alone. Blood was obtained via tail-snipping after 4 and 7 days, and the serum was collected and stored at 20 until it was used. Spleens were harvested at different time-points PF-06651600 post-injection as shown inFig. 2or after 7 days for functional assays. == Physique 2. == Flow-cytometry profiles of murine splenic dendritic cells (DCs) from lipid core peptide (LCP)-treated mice. (a) Multiple cohorts of mice were administered with either the synthetic compound LCP or phosphate-buffered saline (PBS) and CD11c+DC isolated after 3,.