After washing, plates were incubated with biotinylated anti-mouse IL-2 monoclonal antibody and then with streptavidin-horseradish peroxidase (all from BD Pharmingen, USA). the highest number of IL-2 producing cells detected in mice primed and boosted by the nasal route. This work shows the critical role of immunization routes in modulating quality and localization of immune responses in prime-boost vaccine strategies. antibody-mediated inhibition of T cell trafficking (Ciabattini et al., 2011). In the present work, the T cell clonal expansion following IN or subcutaneous (SC) priming and the secondary immune response after boosting by either the homologous or heterologous routes was studied in mice. Ovalbumin (OVA) mixed with the synthetic TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826, widely described as an effective adjuvant for both parenteral and mucosal immunization (Tengvall et GSK256066 2,2,2-trifluoroacetic acid al., 2006; Bode et al., 2011), was used as the model GSK256066 2,2,2-trifluoroacetic acid vaccine formulation. The antigen-specific CD4+ T cell priming after IN or SC immunization was studied employing the adoptive transfer model of OVA-specific transgenic CD4+ T cells (Kearney et al., 1994; Pettini et al., 2009). The local and systemic antibody responses, as well as the T cell response in the spleen and in draining lymph nodes, were characterized following homologous or heterologous combination of prime-boost routes. Materials and Methods Animals Eight-weeks old female OT-II TCR-transgenic (H-2b), C57BL/6J, and BALB/c mice, purchased from GSK256066 2,2,2-trifluoroacetic acid Charles River (Lecco, Italy), were maintained under specific pathogen-free conditions in the animal facilities at the University of Siena, and treated according to national guidelines (Decreto Legislativo January 27, 1992 n. 116, implementing 86/609/CEE Directive). All animal studies were approved by the Ethics Committee Comitato Etico Locale dellAzienda Ospedaliera Universitaria Senese and the Italian Ministry of Health (number 4/2011, July 20, 2011). Adoptive transfer of transgenic CD4+ T cells and priming studies Adoptive transfer experiments were performed as previously described (Pettini et al., 2009). Briefly, lymphocytes collected from OT-II transgenic mice were enriched for CD4+ T cells and stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE, 7.5?M, Invitrogen). An amount of 2.5??106 of CFSE-labeled T cells was injected into the tail vein of each recipient mouse. After 24?h, C57BL/6J mice were immunized with OVA grade V (Sigma-Aldrich; 25?g/mouse) and CpG ODN1826 (TCC ATG ACG TTC CTG ACG TT, hereafter CpG ODN; Eurofins MWG Operon, Ebersberg, Germany; 20?g/mouse) by the IN or SC routes. IN immunized mice were lightly anesthetized by intraperitoneal injection of tiletamine and zolazepam hydrochloride (Zoletil 20, Laboratoires Virbac, France, 6?mg/kg) and xylazine (Xilor 2%, Bio 98 Srl, Italy, 3?mg/kg), held in a vertical position and then inoculated with drops into a single nostril with a total volume of 20?l of Phosphate Buffered Saline (PBS) solution. SC immunization was performed dorsally in the region of the neck in a total volume of 100?l of PBS. Groups of three mice were sacrificed 0, 3, 5, and 7?days following immunization. Cervical (CLN), mediastinal (MedLN), axillary (AxLN), iliac (ILN), mesenteric (MLN) lymph nodes, and spleen (SPL) were harvested from each mouse and individually mashed onto a nylon screen. Cells were washed twice in Hanks Balanced Salt Solution (HBSS, Gibco), incubated with Fc-blocking solution [0.5?mg CD16/CD32 mAb (clone 93) (eBioscience, USA), 5% v/v mouse serum, 5% v/v rat serum, 0.2% w/v sodium azide (all from Sigma-Aldrich) in 100?ml of HBSS] for 30?min at 4 C, and stained with PerCP-conjugated anti-mouse CD4 (clone RM 4C5, BD Pharmingen) for 30?min at 4 C. Samples were analyzed by flow cytometry (FACScalibur, Becton Dickinson, San Diego, CA, USA). Data analysis was performed by using Flow Jo software (Tree Star, Ashland, MULK OR, USA). Immunization using prime-boost combinations and sample collection BALB/c mice (six per group) were primed by the IN or SC route with OVA grade V (100?g/mouse) mixed with the adjuvant CpG ODN (20?g/mouse) at days 0 and 10.
