DC Culture and Peptide Pulsing

Bone marrow derived dendritic cells (BM-derived DCs) culture was described before (28). Briefly, BM cell suspensions from WT, ICAM-1^−/−, ICAM-2^−/−, and ICAM-1/-2^−/− C57BL/6J mice were obtained by centrifugation (4,000 rpm, 4 min) of femurs and tibiae. BM cells were incubated, in 20 ml cultures containing 18 ml restimulation medium containing RPMI-1640 supplemented with 10% FBS (Thermo Fisher Scientific), 10 U/ml penicillin-streptomycin, 2 mM L-glutamine, 1% (v/v) non-essential amino acids, 1 mM sodium pyruvate, and 0.05 mM β-mercaptoethanol (Grogg Chemie AG) and 2 ml Flt-3L-containing supernatant [produced from SP2/0 transfected cell line secreting murine recombinant Flt-3L (29, 30)], for 6–9 days until activation with 1 μg/ml lipopolysaccharide (LPS from Salmonella; Sigma-Aldrich) for 24 h and pulsing with MOG_aa35−55 peptide for 1 h at 37°C.