Multiple T cells cluster on DCs. (A) Typical examples of DC–T-cell clusters. Bars represent 10 μm. (B) Confocal images of DC–T-cell clusters used to determine the binding frequency of CD4⁺ (red, Dil) and CD8⁺ (blue, DiD) T cells to DCs. DCs were pulsed with an influenza vaccine and coincubated with T cells previously in vitro expanded in the presence of the same vaccine. DCs, CD4⁺ cells, and CD8⁺ cells were incubated at the ratio of 1:3:3 for 4 hours when all cells were counterstained with CMFDA (green). Optical sections were 1.5 μm thick. Bars represent 10 μm. (C) Frequencies of single CD4⁺ cells bound to a DC, of single CD8⁺ cells bound to a DC, or of a CD4⁺ cell and a CD8⁺ cell bound to a single DC. Each dot represents a single experiment. In 3 independent experiments, we analyzed the total of 1070 DCs. The frequency of a CD4⁺ binding on a DC did not differ from CD8⁺ binding frequency (P = .2). (D) Numbers of DCs that bound 2 T cells (light columns) compared with the numbers predicted on the assumption of independent T-cell binding (dark columns). Each column pair represents an independent experiment. (E) Relative distribution of DCs in complex with 2 T cells: CD4⁺/CD4⁺, CD4⁺/CD8⁺, and CD8⁺/CD8⁺ observed in 3 independent experiments.