Ova257 264 peptide

Cells were stained with fluorescein-conjugated mAbs to mouse CD3α (145-2C11), CD4 (RM4–5), CD8α (53–6.7), CD11c (HL3), CD40 (3/23), CD44 (IM7), CD45.1 (A20), CD62L (MEL-14), CD80 (16-10A1), CD86 (GL1), CD154 (MR1), I-A/I-E (M5/114.15.2), B220 (RA3-6B2), Vα2 TCR (B20.1), γδTCR (GL3), IFN-γ (XMG1.2), IL-17A (TC11-18H10), isotype-matched control mAb (BD Biosciences), CD209b (22D1), CD253/TRAIL (N2B2) (eBioscience), CD11b (M1/70), CD178/FasL (MFL3), F4/80 (BM8), Ly6C (HK1.4), Ly6G (1A8), γδTCR (GL3) (Biolegend), mPDCA-1/BST2 (JF05-1C2.4.1) (Miltenyi Biotec), H-2K^b OVA tetramer (MBL) and Siglec-H (440c; Hycult Biotechnology). For the intracellular expression of cytokines, cells were incubated for 4 hrs with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml; Sigma-Aldrich) plus ionomycin (500 ng/ml; Sigma-Aldrich) or OVA_257–264 peptide (SIINFEKL; 10 μM) plus GolgiPlug (BD Biosciences) during the final 2 hrs. Subsequently, the cells were resuspended in Fixation-Permeabilization solution (BD Cytofix/Cytoperm kit; BD Biosciences) and intracellular cytokine staining was carried out according to the manufacturer’s directions. Fluorescence staining was analyzed with a FACSCalibur flow cytometer and CELLQuest software (both from BD Biosciences).

Flat-bottom microtiter wells in 24-well plates were coated with Dimer X-2Kb:Ig fusion protein loaded with OVA257–264 peptide (BD Pharmingen, San Jose, CA) and recombinant B7-1/Fc chimeric protein (R&D Systems, Minneapolis, MN) as previously described (42 (link)). For two signal (2SI) stimulation, purified naive OT-I CD8+ T cells were plated at a density of 3 × 10⁵ cells in 1.5 mL Allos medium in each well of a coated plate with 2.5 U/mL recombinant human IL-2 (R&D Systems, Minneapolis, MN). For three signal (3SI) stimulation, naive OT-I CD8 T cells were plated under 2SI conditions as described above and additionally supplemented with 2 U/mL of murine rIL-12 (R&D Systems, Minneapolis, MN). The cells were incubated for three days before further analysis.