Spinophilin plays a functional role in antigen presentation in vitro. (a) DCs isolated from spinophilin KO (dotted lines) and WT mice (solid lines) were stimulated in vitro and the expression of cell surface markers was analyzed by flow cytometry. WT (black) and KO (red) solid lines indicate unstimulated cells, whereas dotted lines are cells stimulated (matured) overnight at day five in culture by 30 ng/ml LPS. Expression of cell surface markers was comparable between the two cell types. Data are representative of three independent experiments. (b, left) DCs were isolated from spinophilin WT (black) or KO (red) mice and cocultured with OT-II CD4+ T cells in the presence of an antigen (ovalbumin). After 18 h, supernatants were isolated and assayed for IL-2 by ELISA. Values shown are the mean of triplicate measurements ± SD. (c) DCs were isolated from spinophilin WT (black) and KO (red) mice and cultured for 5 d. Ovalbumin-647 was incubated with DCs for 10 min at 37°C and chased for 1 h. A representative of three experiments is shown (n = 8 animals total from three independent experiments; cells are gated on CD11c+). Uptake of FITC-RNAase and FITC-dextran yielded similar results (not depicted). (d) DCs from −/− or +/+ mice were cultured for 5–6 d, matured with 30 ng/ml LPS, and pulsed with an antigen (10 μg/ml ovalbumin aa 323–339 peptide). DCs were then cultured with OT-II CD4+ T cells for 20–60 min, fixed, and labeled for immunofluorescence microscopy. The number of fixed conjugates was counted for both KO and WT cells and found to be comparable (P = 0.15; n = 4 animals per group; n = 562 and 625 cells, respectively). Error bars indicate the SEM.