Signaling at the contact zone between plasmacytoid DCs and T lymphocytes. (A) PKC-θ and pY174Vav are relocated to the IS between mature pDCs and T lymphocytes. OVA peptide–loaded immature and mature pDCs were conjugated with blue-labeled OT-II CD4⁺ T cells and double-stained for pY174-Vav or PKC-θ (green) and phalloidin (red). (B) The chart shows quantification of PKC-θ localization at the IS of immature or mature Flt3-L–derived cDCs and pDCs as well as pDCs isolated from lymph nodes (LNpDC), in either the absence or the presence of OVA peptide. Results are presented as the arithmetic mean plus or minus SEM of PKC-θ contact-zone localization from 5 experiments. *P < .05 compared with the absence of antigen. (C) pY174-Vav is localized throughout the pDC–T-cell contact zone, whereas PKC-θ is concentrated at the IS cSMAC and surrounded by actin. pDC–T-cell conjugates were generated in the presence of OVA and double-stained for phalloidin and pY174-Vav or PKC-θ. Plates show a confocal section, the maximal projection of confocal sections, and the differential interference contrast image. (D) Western analysis of ZAP-70, LAT, PKC-θ, and Vav phosphorylation in T cells interacting with immature and mature pDCs and cDCs. One representative experiment is shown of 3 performed. Densitometric analyses were performed, and the ratio between the phosphorylated protein and corresponding total protein expression was determined. Results are expressed as the arithmetic mean plus or minus SD of the fold induction, using as denominator the immature pDC condition, of 3 experiments, and statistical analysis was by the Kruskal-Wallis test (*P < .05). ipDC indicates immature pDCs; mpDC, mature pDCs; icDC, immature cDCs; mcDC, mature cDCs.