Antigen-specific proliferation of CCR7⁻ HIV-specific CD8⁺ T cells.

PBMCs were obtained from subject 15 and labeled with anti–CCR7 PE antibody (A). CCR7⁻ cells were sorted by flow cytometry (B), followed by CFSE labeling and 48 hours of HIV⁻peptide stimulation (C). Red and gray dots represent CCR7⁻and CCR7⁺ cells, respectively (A-B). Red, blue, and gray dots are as in Figure 1 (C-G). In a separate experiment, PBMCs were obtained from subject 15 (proliferating) and subject 17 (nonproliferating) followed by 6 hours of stimulation with HIV-peptides as described in “Patients, materials, and methods.” Cells were then labeled with anti–CD3 PerCp, anti–CD8 APC, anti-CCR7 (D-E), or anti–CD57 PE (F-G) and anti–IFN-γ FITC. The data are gated for CD3⁺CD8⁺ T cells. In panels D-G, blue dots represent CD8⁺ T cells that express the marker of interest but did not produce IFN-γ. Red dots correspond to CD8⁺ T cells that produced IFN-γ but did not express the marker of interest. Finally, green dots represent CD8⁺ T cells that expressed the marker of interest and produced IFN-γ. This experiment was repeated with PBMCs from the same subject to assure reproducibility.