Overexpression of fascin in LXR agonist–treated DCs leads to enhanced IS formation and further T-cell activation. Immature DCs were differentiated for 6 days and left untreated (untr) or treated from day 2 on with 2 μM LXR agonist T0901317 (T09). Mature DCs were obtained from iDCs by incubation with LPS for the last day. iDCs and mDCs were then transfected with pEGFP-C1-vector (vector) or pEGFP-C1-fascin (fascin) plasmid. (A) At 24 hours after transfection, DCs were pulsed with superantigen for 30 minutes and incubated with Jurkat T cells for an additional 30 minutes. CD3ϵ relocalization was visualized by indirect immunofluorescence. Typical examples of conjugates, negative (left) or positive (right) for relocalization of CD3ϵ, are shown. Bar represents 10 μm. The diagram shows the percentage of conjugates counted positive for CD3ϵ relocalization in means ± SEM of 4 independent experiments. Significance versus T09 + vector: *P ≤ .05. (B) At 24 hours after transfection, GFP-positive DCs were FACS sorted to a purity of 98%. FACS-sorted pEGFP-C1–positive DCs were pulsed with superantigen for 30 minutes and incubated with Jurkat T cells at a DC/T-cell ratio of 1:8 and 1:16 for 24 hours. Expression of CD25 and CD69 was analyzed by 2-color flow cytometry. Diagram shows geometric mean fluorescence intensities (MFIs) ± SEM related to those obtained from T09-treated vector–transfected DCs (T09 + vector, set to 100%) of 3 independent experiments. Significance versus T09-vector: *P ≤ .05.