HS cells are not activated by cell-free virions or by HIV-infected lymphocytes. (A) Reactivity of HS clone L11 to cell-free virions. L11 cells were exposed to HIV_NL-AD8 (8 nM p24) or the cognate gag2 peptide (44 nM) for 6 hours. Activity of L11 cells was tested by measuring intracellular cytokine production (IL-2, TNFα, and IFNγ) by flow cytometry. Cells were also stained with anti-CD3 mAbs. The percentage of cytokine⁺ cells is indicated. Isotypic mAbs were used as negative controls to set the quadrant position. Similar results were obtained after overnight incubation. Data are representative of at least 3 independent experiments. (B) Reactivity of HS clone L11 to HIV-infected lymphocytes. L11 cells previously activated by PHA and grown with IL-2 were infected with HIV_NL-AD8 (8 nM p24). A few days later, the presence of productively infected cells was assessed by intracellular Gag staining (upper panel). The percentage of Gag⁺ cells is indicated. NI indicates control noninfected cells. Lower panels: These cells were then used as stimulators and cocultivated overnight with uninfected L11 cells, previously stained with CFSE. Cytokine production by stimulators (CFSE^-) and effectors (CFSE⁺) was then assessed as described in panel A. As a positive control, stimulators were pulsed with the gag2 peptide (44 nM). The percentage of cytokine-positive cells among CFSE⁺ effectors is indicated. Data are representative of 3 independent experiments.