ITAM signaling is required for antigen presentation by DCs. WT and Vav^NULL DCs (50 000) were cultured with the indicated doses of OVA-coated latex beads and purified OT2 T cells. T cells were analyzed for expression of the activation markers CD69 and CD25 by FACS after 18 hours of stimulation (A), or analyzed for proliferation by CFSE dye dilution after 72 hours of stimulation (B). (C) WT and DF DCs (50 000) were cultured with the indicated doses of OVA peptide (continuous peptide) and purified CFSE-labeled OT2 T cells. Proliferation was determined by FACS for CFSE dye dilution on day 3 of culture. (D) Alternatively, WT and DF DCs were pulsed with a saturating dose of OVA peptide (100nM) for 2 hours and washed thoroughly (pulsed peptide). Subsequently, the indicated numbers of pulsed DCs were cultured with OT2 T cells for analysis of T-cell proliferation. Similarly, WT and Vav^NULL DCs were cultured with continuous peptide (E) or pulsed peptide (F) and analyzed for their ability to induce OT2 T-cell proliferation as described above. Data shown are representative of at least 5 independent experiments.