DC positioning governs the location of MHC I and MHC II presentation and CD8⁺ and CD4⁺ T cell activation. (A) Animals were injected with ∼3 × 10⁶ OT-I and OT-II T cells labeled with CMFDA (green) and CMTMR (red); 1 d later, they were immunized in the footpad with 10 µg OVA, along with Lyve-1 antibody conjugated to quantum dot 705 for labeling the LS (blue). After 6 h, draining popliteal LNs were isolated, fixed, sectioned into 150-µm slices, and examined with a two-photon 800-nm-wavelength laser for T cell cluster formation (zoom-in insets demonstrate discrete OT-I and OT-II clusters in different areas, as highlighted by cyan and white arrowheads, respectively). Bar, 200 µm. (B) Quantification of the frequency of T cell clusters with respect to the distance to the closest LS. Distance was calculated from the cluster center to the nearest detected Lyve1-stained vessel for all detected clusters. Error bars represent standard error for data from three independent animals. (C) CD45.2⁺ OT-I and CD45.2⁺CMTMR⁺ OT-II T cells were injected into CD45.1⁺ recipients, which were then immunized with OVA 1 d later. 6 h after immunization, draining LNs were isolated for histo-cytometry analysis of the phenotype of DCs in direct contact with T cell clusters. Numbers in plots represent frequencies. Image analysis pipeline described in Materials and methods. Data represent two independent experiments, three draining LNs per experiment.