No specific immune response was detected with SE used to formulate the GLA in our studies. Oil-in-water emulsion is considered an adjuvant by itself (e.g. MF59) and is believed to form a depot at the injection sites protecting the antigen

from clearance, allowing its slow long-term release into the surrounding tissues and prolonging the duration of the interaction between antigen and the responding cell 59, 60. Formulations are also believed to enhance solubility and stability of adjuvants. For example, unformulated MPLA is insoluble and forms aggregates 61. We could not detect any difference in cell recruitment and lymph node inflammation between buy EMD 1214063 MPLA and GLA-SE supporting the second notion. Under this context, it is possible that formulation of MPLA with SE may increase T-cell responses. However, our paper focuses on the immune response induced by GLA-SE, a clinical feasible adjuvant, and its capacity to render DC maturation in vivo. In addition to showing the capacity of a vaccine adjuvant to render DCs immunogenic in vivo, our results provide ways to help identify those Epacadostat innate stimuli and their combinations that can provide the link between innate and the desired adaptive immunity. C57BL/6, B6.TLR4−/−, and CD11c-DTR

mice were purchased from Jackson Laboratory. Mice in specific pathogen-free conditions were studied at 6–10 weeks according to institutional guidelines and approval of the Rockefeller University institutional animal care and use committee (IACUC). Mice were injected s.c. with 20 μg of GLA-SE or as control, oil-in-water SE (Immune Design, Seattle, WA). Spleens and lymph nodes were collected 6 or 18 h later and treated with collagenase D (400 U/mL) for 20 min at 37°C. DC maturation C-X-C chemokine receptor type 7 (CXCR-7) was analyzed by increased expression of CD80, CD86, and CD40 after gating on CD11c+ MHCII+ DCs. For cytokine production, spleens

were harvested 4 h after in vivo stimulation. CD11c+ MHCII+ DCs were purified by cell sorting (FACSAria; BD Biosciences) and plated at 5×104 cells/well in a 96-well plate for 18 h prior to assay of cytokines in the supernatants by multiplex ELISA (Meso Scale Discovery, Gaithersburg, MD). To test allostimulatory capacity, spleen and node CD11c+ MHCII+ DCs were cell-sorted 12 h after GLA-SE or SE injection. C57BL/6 DCs were fixed with 1% PFA (paraformaldehyde) for 10 min at 4°C and added in graded numbers to 2×105 carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled (Molecular Probes, Eugene, OR) Balb/C T cells. After 5 days, cell proliferation was analyzed by CFSE dilution in CD3+CD4+ cells. For DC antigen presentation in vivo, WT and MHCII−/− mice were injected with 5 μg of gag-p24 together with 20 μg of GLA-SE or control adjuvant SE. After 4 h, splenic CD11c−/− DCs were purified and adoptively transferred into naïve mice (i.v). Antigen-specific responses were evaluated by intracellular IFN-γ after prime-boost.