OVA257–264 (SIINFEKL), tyrosine-related protein-2 tyrosinase-related protein (TRP)-2180–188 (SVDYDFFDWL), OVA323–339 (ISQAVHAAHAEINEAGR) and lymphocytic choriomeningitis virus–glycoprotein (LCMV GP)61–80 (GLKGPDIYKGVYQFKSVEFD) were obtained from A&A Laboratories (San Diego, CA, USA). DC were isolated from

spleens of naive mice or mice treated for 9 days with 10 µg human recombinant (hr)FLT3L as described previously [34]. hrFLT3L was a kind gift check details from Amgen (Thousand Oaks, CA, USA). DC were analysed for the expression of CD4, CD8α, CD11b, CD11c, CD40, CD54, CD80, CD86, Kb, Db and I-A/E by flow cytometric analysis (antibodies/isotype controls; eBioscience/Biolegend, San Diego, CA, USA; DCs were subsorted by flow cytometry based on their expression of CD11c, CD11b, CD8α or PDCA-1 by flow cytometry to purity of >95% and viability >95% (7-AAD staining). OT-1 and OT-2

T cells were isolated using CD8 or CD4 microbeads buy LDE225 (Miltenyi Biotec, Auburn, CA, USA) and labelled with 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE) (Molecular Probes, Eugene, OR, USA) as described previously [38]. Purity of sorted cells was >98% and viability was >97% as determined by CD4/CD8/Vα2/Vβ5 expression and 7-AAD staining. Purified DCs (1 × 105) were cultured with irradiated splenocytes in a 1:3 ratio in 96-well U-bottomed plates. After 3, 6 and 16 h supernatant was analysed for type I IFN by reporter assay [39] and IL-10, tumour necrosis factor (TNF)-α and TGF-β by quantitative polymerase chain reaction (PCR) using SybrGreen and the following primers: ml32 forward 5-GAAACTGGCGGAAACCCA-3, ml32 reverse 5-GGATCTGGCCCTTGAACCTT-3, TNF-α forward 5-GTACTGGCATGTGTATGTCA-3, Exoribonuclease TNF-α reverse 5-TGGTTGAGGGAATCATT-3, IL-10 forward 5-GGTTGCCAAGCCTTATCGGA-3, IL-10 reverse 5-ACCTGCTCCACTGCCTTGCT-3, TGF-β forward 5-GACCGCAACAACGCCATCTA-3, TGF-β reverse 5-GGCGTATCAGTGGGGGTCAG-3. The fold increase of specific RNA (mRNA after apoptotic cells exposure/mRNA before apoptotic cells) was

determined after normalization to L32 for each sample. Purified DCs (1 × 105) were cultured with irradiated purified ActmOVA-Kbm1 T cells in a 1:3 ratio in 96-well U-bottomed plates. After 24 h, 1 × 105 CFSE-labelled OT-1 or OT-2 T cells were added to the wells. This experimental set-up allows us to study exclusively cross-priming by the DC subsets because the mutated peptide binding groove of Kbm1 cannot bind the OVA257–264 peptide [40] and the lack of MHC class II on the T cells prevents direct activation of the OT-2 T cells [41]. As positive control, DCs were pulsed with OVA peptides for 10 min and washed thoroughly. OT-1 and OT-2 T cell proliferation and survival were determined after 70 h by analysis of CFSE dilution together with staining for Vα2, CD4/CD8 and 7-AAD.