Figure 2
Figure 2. Functional characterization of HIV-specific CD8+ T-cell clones with distinct antigen sensitivities. (A) Representative data showing the simultaneous and independent measurement of 5 separate functions in KK10-specific CD8+ T-cell clones using 8-color flow cytometry. Cells were stimulated for 6 hours in the presence of EBV-transformed HLA B*2705+ B cells and 10−8 M peptide before intracellular staining. Function plots are gated on CD8+ViViD– cells; percentages of cells in the different quadrants, gated with respect to the corresponding negative controls (displayed for reference in the left panels for the clone with low antigen sensitivity), are shown. (B) The pie charts depict the background-adjusted polyfunctional profile of 3 representative KK10-specific CD8+ T-cell clones with different antigen sensitivities (highest, C2A; intermediate, H10G; lowest, D8B). 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 represent the proportions of cells within each total clonal population that exhibited the number of functions indicated. (C) Polyfunctionality, defined as the percentage of cells displaying 3 or more functions simultaneously, is plotted as a function of antigen sensitivity. Each dot represents a distinct clone; dots framed by a square or hexagon indicate clones with identical TCRB sequences. The correlation was determined using Spearman rank test. (D) Representative staining for p24 and CD4 from primary CD4+ T cells 3 days postinfection with the replicative HIV-1 strain NL4.3 pseudotyped with vesicular stomatitis virus. (E) Polyfunctional profile of 2 CD8+ T-cell clones with different antigen sensitivity for KK10 (higher, C2A; lower, H8B) after 6-hour incubation with HLA-B27 primary CD4+ T cells infected with HIV-1 (1:10 CD8 to CD4 ratio). (F) Polyfunctional profiling of 3 representative KK10-specific CD8+ T-cell clones (highest, C2A; intermediate, H10G; lowest, D8B) along a peptide concentration gradient.

Functional characterization of HIV-specific CD8+ T-cell clones with distinct antigen sensitivities. (A) Representative data showing the simultaneous and independent measurement of 5 separate functions in KK10-specific CD8+ T-cell clones using 8-color flow cytometry. Cells were stimulated for 6 hours in the presence of EBV-transformed HLA B*2705+ B cells and 10−8 M peptide before intracellular staining. Function plots are gated on CD8+ViViD– cells; percentages of cells in the different quadrants, gated with respect to the corresponding negative controls (displayed for reference in the left panels for the clone with low antigen sensitivity), are shown. (B) The pie charts depict the background-adjusted polyfunctional profile of 3 representative KK10-specific CD8+ T-cell clones with different antigen sensitivities (highest, C2A; intermediate, H10G; lowest, D8B). 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 represent the proportions of cells within each total clonal population that exhibited the number of functions indicated. (C) Polyfunctionality, defined as the percentage of cells displaying 3 or more functions simultaneously, is plotted as a function of antigen sensitivity. Each dot represents a distinct clone; dots framed by a square or hexagon indicate clones with identical TCRB sequences. The correlation was determined using Spearman rank test. (D) Representative staining for p24 and CD4 from primary CD4+ T cells 3 days postinfection with the replicative HIV-1 strain NL4.3 pseudotyped with vesicular stomatitis virus. (E) Polyfunctional profile of 2 CD8+ T-cell clones with different antigen sensitivity for KK10 (higher, C2A; lower, H8B) after 6-hour incubation with HLA-B27 primary CD4+ T cells infected with HIV-1 (1:10 CD8 to CD4 ratio). (F) Polyfunctional profiling of 3 representative KK10-specific CD8+ T-cell clones (highest, C2A; intermediate, H10G; lowest, D8B) along a peptide concentration gradient.

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