FIG. 5.

IFN-γ production of CD34 mAb-sorted T cells, but not peptide–MHC-sorted T cells, depends on both CD8⁺ and CD4⁺ T cells. Primary human T cells were transduced and MACSorted as described in the legend to Fig. 4. T cells were analyzed by flow cytometry after staining with APC-conjugated anti-CD8α mAb in combination with one of the following reagents: (A) PE-conjugated gp100/A2 tetramer (n=12); (B) PE-conjugated anti-TCR-V_β27 mAb (n=6); (C) FITC-conjugated anti-CD34 mAb (n=14); and (D) FITC-conjugated anti-IFN-γ mAb (n=2). In the case of IFN-γ stainings, T cells were stimulated for 6 hr with BLM (gp100^–/A2⁺) cells loaded with 10 μM gp100 peptide, permeabilized, and stained with anti-IFN-γ mAb, as described in more detail in Materials and Methods. Shown are representative dot plots, indicating percentages (all quadrants) and MFIs (upper and lower right quadrants when percent ≥5, in italic) of stained T cells.