pSMAC formation and MTOC reorientation in pDC–T-cell conjugates. (A) pSMACs formed between OVA peptide–loaded mature pDCs (mpDC) and cDCs (mcDC) and blue-labeled OT-II CD4⁺ T cells. Plates show the maximal projection of confocal images from pDC and cDC ISs stained with phalloidin (green) and Arp2/3 (red), the differential interference contrast image, and a high magnification 3-dimensional reconstruction of the pSMAC. For Figures 2A, 3C, 5A, 6D, and S3A-C, a Leica TCS-SP5 confocal laser scanning unit attached to a Leica DMI6000 inverted epifluorescence microscope, with a HCX PL APO 63×/1.40-0.6 NA oil was used. Images were acquired with Leica confocal software, and figures were composed with Adobe Photoshop CS. (B) Matured Flt3-L–derived cells (mpDCs and mcDCs) were loaded with OVA peptide and conjugated with blue-labeled OT-II CD4⁺ T cells and double-stained for CD11b (red) and α-tubulin (green). cDCs and pDCs are indicated by red and white asterisks, respectively. For Figures 2B, 2C, and 3A, fixed cells were mounted on ProLong Gold (Invitrogen) and observed at 22°C on a Leica DMR photomicroscope (Leica, Wetzlar, Germany) with a HCX PL APO 63×/1.32-0.6 NA oil objective, coupled to a COHU 4912-5010 Camera (COHU, San Diego, CA). The acquisition software was Leica QFISH, version 2.1, and images were processed with Adobe Photoshop CS. (C) Immature and mature pDCs were conjugated with blue-labeled OT-II CD4⁺ T cells and stained for α-tubulin (green). (D) Estimation of MTOC translocation on conjugates formed between immature or mature pDCs or cDCs and OT-II CD4⁺ T cells either in the absence or presence of OVA peptide. Results are presented as the percentage of conjugates in which the MTOC was translocated to the IS. Data are arithmetic mean plus or minus SEM of 5 experiments. *P < .05 compared with the absence of antigen (Kruskal-Wallis test).