FIG 2.

CD4⁺ T-cell licensing of DCs is necessary to prime naive CD8⁺ T cells in vitro. (A) Gating strategy and pentamer staining of CMV-pp65-specific CD8⁺ T cells. SSC, side scatter; FSC, forward scatter. (B) Gating strategy and pentamer staining of primed CB-derived CD8⁺ T cells. DCs were loaded with 10 μg/ml BSA (left graph) or 10 μg/ml pp65 (middle and right graph) and cocultured with donor-matched naive CD8⁺ T cells in the absence (middle graph) or presence (left and right graphs) of CD4⁺ T cells. High-level pentamer staining events (>log₄ intensity) were single-cell sorted and clonally expanded for 4 to 6 weeks (representative of 6 independent experiments). (C) Representative pentamer staining of two individually derived CD8⁺ T-cell clones. (D) Characteristics of 6 clones from 3 independent experiments. Shown are mean fluorescence intensity (MFI) values of pentamer stainings and TCR sequences. Clone 6 was produced using a CD40 agonist antibody. TRBV, T-cell receptor beta variable gene; TRBJ, T-cell receptor beta joining gene. (E and F) Phenotype analysis of pp65-specific CD8⁺ T-cell clones. (G) Cytokine production (IFN-γ, TNF, and IL-2; pg/ml) of CD8⁺ T cells after PMA/ionomycin stimulation.