Polarization of IL-12 at the IS. (A) Kinetic of IL-12 production and secretion in DCs. DCs were stimulated with a combination of TLR agonist for the indicated times (time post TLR). The relative content of IL-12 p40 and p70 in cell culture supernatants (gray bars) and cell lysates (white bars) was determined by ELISA. Bars show means ± SEM of five independent experiments. (B) Intracellular localization of IL-12 in DCs. DCs stimulated with TLR agonist for the indicated times were immunolabeled with anti–α-tubulin (green) and anti–IL-12 p40/70 (red) antibodies. Images are z projections of confocal sections. (C) Association between IL-12–containing vesicles and MTOC. TLR-stimulated DCs (4-6 h) were immunolabeled as in B. The distance (μm) of IL-12 containing vesicles from the MTOC was measured in each cell in normal (control) or colchicine (colch)-treated DCs. Each dot represents the mean distance of vesicles/MTOC in a single cell. Mean values ± SEM of 25 cells/condition. (D) Confocal images showing recruitment of IL-12 at the DC–T cell interface. Experimental outline: DCs were stimulated with TLR agonist (5 h), loaded with peptide, and mixed with OT-I cells to induce synapse formation. (top) Confocal planes showing IL-12 (red) and α-tubulin (blue) distribution in DC–T cell doublets. T cells are in green (CFSE labeling). (E) DCs were treated with colchicine before synapse formation. The percentage of conjugates with IL-12 enriched at the contact in control and treated cells was measured in at least 50 conjugates/condition in three independent experiments (P < 0.001, Student’s t test). (F) Labeling with anti–VAMP-7 (Ti-VAMP) and anti–IL-12 antibodies on fixed DC–T cell synapses shows alignment of secretory organelles close to the DC–T cell interface. (G) DCs prelabeled with WGA (blue) to demark the membrane were mixed with CFSE-labeled T cells. The image shows one representative high-magnification projection of z stacks along the contact zone which depicts vesicles of IL-12 (red) crossing the DC plasma membrane.