In parallel studies, 0·3 µM [3H]-thymidine was added after 60 h of culture, and incorporation was determined 12 h later. Cytokine production in the supernatant was determined by standard sandwich enzyme-linked immunosorbent assay (ELISA) for IL-2, IL-4, TNF-α and IFN-γ (Biolegend, San Diego, CA, USA). For in

vivo priming, B6 mice received intravenous (i.v.) 4 × 105 purified DC that were incubated with irradiated ActmOVA-Kbm1 T cells, as described above. Apoptotic cells were removed from the DC populations using the apoptotic cell removal kit (Miltenyi Biotec, Auburn, CA, USA). CD8+ T cell responses were analysed in spleens 7 days after DC transfer using intracellular cytokine staining to IFN-γ and TNF-α upon incubation with OVA257–264 (5 µg/ml) or control peptide

selleck chemicals llc TRP-2180–188 (5 µg/ml) for 5 h in the presence of brefeldin A. Surface staining for CD8 and CD44 and intracellular cytokine staining for IFN-γ was performed using a Cytofix/Cytoperm kit (BD Pharmingen, La Jolla, CA, USA), according to the manufacturer’s instructions [12,41]. For memory CD8+ T cell assessment, an in vivo cytotoxicity assay was performed 28 days after DC treatment. Briefly, mice received CFSEhigh-labelled splenocyte pulsed with OVA257–264 BMN 673 cell line (target cells) mixed with an equal number of CFSEmedium-labelled control cells. Twenty-four h later the ratio of CFSElow/CFSEhigh cells was determined by flow cytometry [42]. OVA-specific CD4+ T helper type 1 (Th1) and Th2 cells were enumerated by enzyme-linked immunospot assay (ELISPOT) 10 days after DC transfer after a 48-h in vitro stimulation with OVA323–339 Tobramycin (10 µg/ml), control peptide GP61–80 (10 µg/ml) or concanavalin A (ConA) (2 µg/ml; positive control), as described previously [43]. Challenge model.  Mice received i.v. 5 × 105 purified DC that were incubated with irradiated ActmOVA-Kbm1 T cells. Seven days later,

mice were challenged by subcutaneous (s.c.) injection of 2 × 106 EL-4-mOVA cells in the left flank and 2 × 106 EL-4 cells in the right flank. Tumour growth was measured every second day with vernier calipers. Tumour size was calculated as the product of bisecting tumour diameters. Therapeutic model.  In the therapeutic approach, mice were inoculated with 2 × 106 live EL-4-mOVA cells on the left flank and 2 × 106 EL-4 as control on the right flank. As soon as palpable tumours had formed, mice received 1 × 106 purified DC that had been exposed to irradiated ActmOVA cells, and tumour growth was monitored daily with a vernier caliper. In parallel studies mice received only EL-4-mOVA cells in the left flank to determine long-term survival, reoccurrence of tumours and possible loss of OVA-tumour antigen. Unless stated otherwise, the data are expressed as means [standard error of the mean (s.e.m.)]. Survival responses were analysed by Kaplan–Meyer using a log-rank test.