Biotinylated mAbs were detected with PerCP streptavidin (BD Pharmingen). Labeled cells were analyzed on an FACSAria (BD Biosciences) For generation of protein-specific memory T cells, C57BL/6 mice (5/group) were immunized by two sc injections of Ag85B (10 μg/mouse), Ag85A (10 μg/mouse), or PstS1 (10 μg/mouse) proteins at 2-week interval. BALB/c mice were immunized by four intranasal administrations of TT (1 μg) with the cholera toxin adjuvant (0.5 μg) at 1-week interval.

Four weeks after the last injection, spleen cells were harvested and used for immunological assays in vitro or in vivo. Experiments performed with unfractionated Ag85B-specific splenocytes were referred to as Ag85B-specific memory CD4+ T cells since all the specific responses triggered by Ag85B restimulation were mainly CD4+ T cell mediated (Supporting Information Fig. 4). For in vivo studies, 1.2 × 107 spleen cells from Ag85B immunized or naïve mice were iv inoculated into buy C59 wnt naïve mice. One day later, recipients were injected

sc with 10 μg of Ag85B, 50 μg PstS1, or combined proteins. Six days after protein injection, splenocytes were harvested and T-cell responses were assayed. Splenic DCs were isolated as described previously [55]. Briefly, spleen cells were centrifuged in Nycodenz density gradient (1.077 g/mL, Nycomed Pharma) at 1700 × g for 20 min at 4°C. The low-density fraction was collected and subjected Carfilzomib cost to magnetic cell sorting using anti-CD11c-Microbeads (Miltenyi Biotec). Purity routinely ranged between 96 and 98% CD11c+ cells. In some experiments, cells were further incubated with PE-anti-CD8α and then sorted into CD8α+ and CD8α− subpopulations using an FACSAria cell sorter. SPTLC1 Where indicated, DCs were cultured for 18 h in complete Iscove’s modified Dulbecco Medium, with or without Ag85B (10 μg/mL) or PstS1 (10 μg/mL). Where indicated, DCs were preincubated with piceatannol for 30’ at 37°C, washed, and then plated with the stimuli. In some experiments, neutralizing Abs to IL-6, neutralizing Ab to IL-1β, or their isotype controls

were added to the cultures. Culture supernatants were assayed for cytokine release by specific quantitative sandwich ELISA kits for levels of IL-6, IL-23 (eBioscience), and IL-1β (R&D Systems). In some experiments, DCs were assayed in a mixed leukocyte reaction using allogeneic spleen cells as responders. For in vivo stimulation of DCs, mice (5/group) were inoculated iv with Ag85B (10 μg/mouse), PstS1 (50 μg/mouse) protein, or PBS. Spleens were harvested 3 h later and the DCs were purified. Unfractionated spleen cells from Ag85B- or PstS1-immunized mice were cultured in round-bottomed 96-well plates (3.5 × 105 cells/well) in complete RPMI-1640 in the presence or absence of 5 μg/mL Ag85B, PstS1, or combination of proteins. Alternatively, splenocytes were co-cultured with 105 DCs pulsed overnight with the same proteins.