Visualization of neo-transduced CD8⁺ T cells by confocal laser microscopy. Cells were prepared as described in Methods and shown in Figure 4. (a) Neo-marked CD8⁺ T-cell clones examined before patient infusions. More than 98% of the cloned cells stained for CD8 and neo was detected in more than 95% of these CD8⁺ T cells (shown is clone LN-2A3-5 from patient 1). Cells containing neo amplimers emit yellow-red fluorescence with a peak fluorescence intensity of 3327. Nontransduced (neo-negative) cells appear purple-blue with a peak fluorescence intensity of 256 (inset). (b) Pictured is a neo-positive cell recovered from a lymph node biopsy taken 4 days after the final T-cell infusion and visualized using the technique of Z-banding. Shown is a 1-μm section taken approximately through the center of a single cell. Note that signal is exclusively intranuclear and has a similar fluorescent intensity to the preinfusion CTL clones (patient 1). (c) For comparison, a similar in situ hybridization procedure, as described previously (6, 7), was used to identify cells in lymph node with transcriptionally active HIV. Gag-positive cells emit intense yellow-red fluorescence with intensities ranging from 2,048 to 3,327 (patient 1). However, unlike neo, the signal is found in both the nucleus and cytoplasm, as would be expected with productive infection. (d–f) Dual detection of neo⁺ and CD8⁺ T cells from (d) preinfusion and (e) postinfusion PBMC (patient 5). The CD8 surface antigen was detected using a PE-conjugated antibody and was the only fluorochrome observed (appears red) in preinfusion PBMC samples (d; day 0). In contrast, up to 3% of PBMCs taken immediately after infusion show both markers and appear yellow (e; day 8). Neo was not detected in cells that did not stain with antibodies to CD8 (f).