Polyfunctional profiling of HIV-specific CD4⁺ T-cell clones. (A) Representative data showing the simultaneous and independent measurement of 5 separate functions in a CD4⁺ T-cell clone specific for HIV-1 Gag. Cells were stimulated for 6 hours in the presence of autologous EBV-transformed B cells and cognate peptide before intracellular staining; αCD107 mAb was added to the assays immediately before stimulation. Function plots are gated on CD3⁺CD4⁺ViViD⁻ cells; percentages of cells in the different quadrants, gated with respect to the corresponding negative controls, are shown. (B) Representative peptide titration assays for the induction of individual functions in a CD4⁺ T-cell clone specific for HIV-1 Gag. (C) Polyfunctional profiles of HIV-1 Gag-specific CD4⁺ T-cell clones with lower or higher levels of antigen sensitivity along a peptide concentration gradient. Antigen sensitivity was determined in CD40L up-regulation assays with immortalized autologous B cells as targets. For simplicity, responses are grouped according to the number of functions (from CD107a, IFN-γ, TNF-α, IL-2, and MIP-1β) elicited in response to antigen encounter; individual segments of the pie charts represent the proportions of cells within each total population that exhibited the number of functions indicated.