Transfection of BM DCs

BM cells from femur and tibia were cultured in 20 ng ml⁻¹ GMCSF (Peprotech) for 6 days. For confocal microscopy, BM DCs were incubated with 0.5 mg ml⁻¹ soluble ovalbumin-pH rodo for 1 h and analyzed by confocal microscopy after staining with anti-CD11c. For re-expression of WASp, WASp KO BM DCs were transfected with eGFP-WASpWT or eGFP-WASpΔVCA constructs⁶⁰ using Amaxa transfection (Primary cell 4D nucleofector kit, Lonza). After 6 h, GFP⁺ and GFP⁻ cells were FACS sorted using FACS Jazz. To assess acidification capacity, BM DCs after Amaxa transfection were incubated with ovalbumin-pH rodo beads for 30 min and pH rodo fluorescence determined in cells that had taken up one bead. To determine proliferation of OT-I CD8⁺ T cells, BM DCs after Amaxa transfection were incubated with 0.5 mg ml⁻¹ ovalbumin and LPS overnight. BM DCs were co-cultured with OT-I CD8⁺ T cells at 1:10 DC:T-cell ratio and analyzed by FACS after 48 h.