DC express, among other, the CD205 (Ly75, DEC205) molecule that functions as an endocytic receptor involved in the uptake of extracellular antigens. chickens. The expression value after challenge was normalized to the basic value of expression in the day of challenge. 13567_2017_423_MOESM5_ESM.pdf (2.9M) GUID:?DBF99490-B6D5-4794-ADCD-D5EDDE5E2B3B Abstract Systems of antigen delivery into antigen-presenting cells represent an important novel strategy in chicken vaccine development. In this study, we verified the ability of Rous Clarithromycin sarcoma virus (RSV) antigens fused with streptavidin to be targeted by Rabbit Polyclonal to T3JAM specific biotinylated monoclonal antibody (anti-CD205) into dendritic cells and induce virus-specific protective immunity. The method was tested in four congenic lines of chickens that are either resistant or susceptible to the progressive growth of RSV-induced tumors. Our analyses confirmed that the biot-anti-CD205-SA-FITC complex was internalized by chicken splenocytes. In the cytokine expression profile, several significant differences were evident between RSV-challenged progressor and regressor chicken lines. A significant up-regulation of IL-2, IL-12, IL-15, and IL-18 expression was detected in immunized chickens of both regressor and progressor groups. Of these cytokines, IL-2 and IL-12 were most up-regulated 14?days post-challenge (dpc), while IL-15 and IL-18 were most up-regulated at 28?dpc. On the contrary, IL-10 expression was significantly down-regulated in all immunized groups of progressor chickens at 14?dpc. We detected significant up-regulation of IL-17 in the group of immunized progressors. LITAF down-regulation with iNOS up-regulation was especially observed in the progressor group of immunized chickens that developed large tumors. Based on the increased expression of cytokines specific for activated dendritic cells, we conclude that our system is able to induce partial stimulation of specific cell types involved in cell-mediated immunity. Electronic supplementary material The online version of this article (doi:10.1186/s13567-017-0423-8) contains supplementary material, which is available to authorized users. Introduction Generation of de novo adaptive responses, including responses to vaccines, is primarily elicited by dendritic cells (DC), specialized leukocytes adapted Clarithromycin for antigen capture, processing and presentation to T lymphocytes. Knowledge of these cells in a target species is therefore crucial in finding the most effective means of vaccination. The key role of T cell-mediated responses to cancer has been established in several models [1]. The antitumor immune response relies on DC, which act as professional antigen-presenting cells (APC). Altered DC function is common in tumors producing soluble factorscytokineswith immunosuppressive activity [2, 3]. DC express, among other, the CD205 (Ly75, DEC205) molecule that functions as an endocytic receptor involved in the uptake of extracellular antigens. In the chicken, the presence of this molecule was also confirmed, along with its endocytic properties [4]. Importantly, DC could be targeted with antigen-conjugated monoclonal antibodies specific for CD205, which are then efficiently internalized, processed in the endosomal compartment, and presented to both major histocompatibility complex I (MHC I) and MHC II molecules [5]. In this study, we used a monoclonal antibody (anti-CD205) for direct antigen delivery. This strategy of activating different DC populations by direct in vivo targeting of their surface receptors has been pioneered by Steinman and Nussenzweig, who used antigen coupling to antibodies to target receptors on DC surfaces [6C8]. We used genetic fusion of the antigen with streptavidin (SA), which in its tetrameric form binds a biotinylated antibody targeting a surface receptor on the APC. Thus prepared complexes can deliver immunogens into DC through endocytosis with the selected surface receptor, enabling antigen processing and presentation, and leading to induction of adaptive immunity [9, 10]. We applied this novel vaccination approach to our previously described model system of inbred lines resistant (CB, CB.RI; regressors) or susceptible (CC, CC.RI; progressors) to progressive growth of Rous sarcoma virus (RSV)-induced tumors [2, 3]. RSV harbors the oncogene v-test. Data of gene expression were prepared in Genex 5.3.7 software (GenEx). The following analysis was done in SAS 9.4 software. Groups were compared by repeated three-way ANOVA. Contrasts were used for detailed comparison. Linear discriminant function analysis based on all analyzed genes was used to show separation of different groups. The cytokine expression profiles of RSV-challenged chicken groups were classified using methods of principal component analysis (PCA) [33] and linear discrimination analysis Clarithromycin in XLSTAT software (StatSoft, Czech Republic). Results SA-RSV fusion proteins The complete sequence of Clarithromycin the RSV antigens v-src, env, pol and gag were fused to the N- and C-terminus of the tetramerization core of streptavidin. The SA fusion with whole antigens v-src and env did not form stable tetramers due to their size. These two antigens were therefore split into two overlapping parts. In env, the signal, transmembrane and intracellular domain were also excluded (Figure?1A). All fusion proteins were produced in and purified close to homogeneity (Figure?1B). The fusion proteins with antigens fused to the C-.