Fig. 2.

PD-1 can concentrate at the synapse of conjugates between infected 5C.C7 T cells and activated splenic DCs. (A) Splenic DCs from B10.A mice were purified by CD11c-positive selection and cultured in GM-CSF and either LPS or IL-4 for 16 h. The untreated DCs are in red, LPS-activated DCs are in blue, and the IL-4-cultured DCs are in green. (B) 5C.C7 T cells were infected with CD28-YFP (top row), CTLA-4-YFP (second row), or PD-1-YFP (bottom two rows). Conjugates with concentrated PKC θ (blue) at the synapse were scored for the localization of the costimulatory molecules (yellow) to the synapse. A representative cell with concentrated PD-1 is in the third row, and a cell with uniform distribution of PD-1 is in the bottom row. The DCs are visualized with anti-CD11c antibodies (red). (C) The statistics from three experiments with at least 30 conjugates are shown as mean and SD (error bars). The numbers after the bars represent the number of conjugates that had concentrated the molecule of interest of the total number of conjugates counted. (D) B7-H1 and B7-DC (red) accumulate at the immunological synapse of conjugates between 5C.C7 T cells infected with PD-1-YFP (yellow) and moth cytochrome c-pulsed DCs. PKC θ is in blue.