CD4⁺ T cells polarize their secretory machinery toward IFN-γ/IL-4–primed PCMCs. IFN-γ/IL-4–primed PCMCs were pulsed or not with 10 μg/mL OVAp. After washing, APCs were cocultured with effector OT-II T cells for 10 minutes. Cells were settled onto polylysine-coated slides; fixed; and stained for tubulin, PKCθ, and IFN-γ; and analyzed by confocal laser scanning microscopy. (A) Representative staining for IFN-γ (red), tubulin (green), and PKCθ (blue) of a PCMC_IFN-γ/IL-4/OT-II T-cell conjugate, each staining is represented alone or merged with DIC images; PKCθ staining is also shown as pseudocolor intensity. Arrow 1 points to a synapse where IFN-γ is polarized toward the PCMCs and PKCθ is enriched at the OT-II T cell/PCMC contact site; arrow 2 points to a synapse where PKCθ only is enriched. (B) OT-II T-cell/PCMC conjugates were randomly selected and scored for the recruitment of PKCθ to the cell–cell contact site either alone or in combination with IFN-γ polarization. indicates conjugates exhibiting recruitment of PKCθ alone; ■, conjugates exhibiting polarization of both PKCθ and IFN-γ; □, conjugates exhibiting neither PKCθ nor IFN-γ polarization. Approximately 100 conjugates were analyzed per experiment. Histograms represent means ± SEM of 3 independent experiments. Bar = 5μm.