Measuring the efficacy of T-cell activation by DCs in vivo. (A) Experimental setup. MHC class II^−/− female mice were injected in the footpad with various numbers of DCs and intravenously with transgenic Marilyn CD4⁺ T cells. On day 1, recipients were injected intravenously with the Dby peptide. Thirty minutes later, c-jun phosphorylation was assessed in Marilyn CD4⁺ T cells by flow cytometry. (B) Histograms showing the percentage of Marilyn CD4⁺ T cells displaying phosphorylated c-jun staining for the indicated number of injected DCs. (C) The graph compiles the percentages of Marilyn CD4⁺ T cells with phosphorylated c-jun staining as a function of the number of injected DCs. Each dot represents 1 recipient. (D) Confocal images of a popliteal LN section 24 hours after the injection of 1 × 10⁶ (left) or 2 × 10⁶ (right) GFP⁺ DCs. Scale bar represents 150 μm. (E) Two-photon imaging of T cell-DC encounters and interactions. MHC class II^−/− female recipients were injected in the footpad with GFP expressing DCs and intravenously with SNARF-labeled Marilyn CD4⁺ T cells. At 24 hours, recipient popliteal LNs were imaged using intravital 2-photon microscopy, and the Dby peptide was injected intravenously during image acquisition. Left: Time-lapse images showing the progressive recruitment of CD4⁺ T cells (red) by DCs (green). Right: The graph shows the percentage of T cells interacting with a GFP⁺ DCs over time. Each line corresponds to 1 individual experiment.