Intravital pDCs and T-cell dynamics. (A) Immature Ly5.2⁺ Flt3-L–derived pDCs were intravenously or subcutaneously injected into Ly5.1⁺ mice. After 20 hours, pDCs in spleen or draining popliteal lymph nodes (LN) were analyzed by flow cytometry. Density plots indicate the percentage of Ly5.2⁺ pDCs that migrated into each lymphoid organ. (B) The bar charts show MHC class II and CD86 expression on immature pDCs before (□) or after intravenous () or subcutaneous injection () and on in vitro–matured (CpG treated) pDCs (■). Data are mean fluorescence intensities (MFI) plus or minus SEM. (C) Mature or immature Ly5.2⁺ pDCs loaded with OVA peptide and Ly5.2⁺ OT-II CD4⁺ T cells were intravenously injected into mice. After 48 hours, the activation of transferred T cells was assessed by forward scatter pattern and CD25 expression (). Negative controls in which only OT-II CD4⁺ T cells were injected are overlaid (). The chart shows geometric MFI plus or minus SEM of T-cell CD25 expression. *P < .05 by Mann-Whitney test. (D) Flt3-L–derived pDCs from GFP mice were intravenously injected into C57BL/6 recipients. After 16 hours, the recipients were intravenously injected with OT-II CD4⁺ cells labeled with CMTMR. After 4 hours, mice were killed and spleen sections were stained for CD3 or MOMA-1 (yellow) and Hoechst for nuclear staining (blue). Plates show maximal projections of confocal sections. (E) pDCs from GFP mice were either left immature or matured with CpG (24 hours) and then loaded as indicated with OVA peptide and intravenously injected into C57BL/6 recipients. After 16 hours, the recipients were intravenously injected with OT-II CD4⁺ cells labeled red with CMTMR. Mice were anesthetized 4 hours later and spleens were microsurgically exposed for intravital 2-photon imaging. Intravital time-lapse 2-photon laser scanning microscopy images of GFP (pDCs) and CMTMR-labeled T cells (red) are presented as average intensity projections along the z-axis. Long-lasting interactions are indicated (circles). Images were average-projected using ImageJ (National Institutes of Health), and manual tracking of individual cells was performed with Metamorph software. (F) DC–T-cell interactions were classified into 3 categories according to their duration: short (< 5 minutes), medium (5-15 minutes), and long (> 15 minutes). The histogram shows the proportions in each category. *P < .05 compared with immature pDCs by χ² test. Results represent more than 100 interactions per condition from duplicates from 2 independent experiments (n = 144 for ipDCs [−OVA], n = 212 for ipDCs [+OVA], and n = 109 for mpDCs [+ OVA]).