ITAM signaling is required for maintaining peptide-MHCII complexes on the surface of DCs. To assess the presence of pMHCII complexes on the surface of DCs, we used the B11 T-cell hybridoma specific for β-gal peptide presented by I-A^b. In these experiments, WT and DF DCs were pulsed with a saturating dose of peptide (10μM), washed, and immediately fixed (A) or chased for 24 hours before fixation (B). After fixation, DCs were cultured overnight with B11 cells, after which point, culture supernatants were analyzed for IL-2 by enzyme-linked immunosorbent assay. Similarly, WT and Vav^NULL DCs were pulsed with peptide and immediately fixed (C) or chased for 24 hours before fixation (D) and then used to stimulate B11 cells as described above. Data represent mean IL-2 production ± SD for triplicate samples and are representative of 5 independent experiments; *P < .05, **P < .01.