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. 2007 Oct 1;204(10):2473–2485. doi: 10.1084/jem.20070784

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Copyright © 2007, The Rockefeller University Press

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Figure 2. — Phenotypic and functional assessment of B27-KK10–specific CD8⁺ T cells. (A) Representative staining for the expression of cell surface markers on B27-KK10–specific CD8⁺ T cells. HIV-specific cells identified using MHC class I–peptide complexes (black) are superimposed on the whole CD8⁺ T cell population in the same donor (red). Plots are gated on CD3⁺CD8⁺ cells. (B) Comparative marker expression between B27-KK10–specific CD8⁺ T cells (open diamonds) and other HIV-specific CD8⁺ T cells (closed circles). Horizontal bars show medians. (C) Representative example of simultaneous multifunctional assessment of B27-KK10–specific CD8⁺ T cells using nine-color flow cytometry. Cells were stimulated for 6 h in the presence of cognate peptide before intracellular staining. Percentages of cells in the different quadrants are shown. Plots are gated on CD3⁺CD8⁺ cells. (D) The pie charts depict the background-adjusted multifunctional behavior (one to five functions: CD107a, IFN-γ, TNF-α, IL-2, and MIP-1β) of B27-KK10–specific CD8⁺ T cell populations (n = 8) versus other HIV-specific CD8⁺ T cell populations (n = 7, including 2xA3-RY10, 2xB7-GL9, and 3xA2-SL9). For simplicity, responses are grouped by the number of functions (indicated by the numbers inside the pie charts and matched to the colored bars in E). The p-value was calculated by the permutation test. (E) Detailed functional responsiveness was analyzed for the B27-KK10–specific CD8⁺ T cell populations compared with the other HIV-specific CD8⁺ T cell populations. Each bar shows the mean percentages of cells displaying a particular combination of functions within the total of functional cells. p-values were calculated by the Mann-Whitney U test. (F) IFN-γ fluorescence of B27-KK10–specific CD8⁺ T cells and other HIV-specific CD8⁺ T cells upon stimulation with cognate peptides. Representative profiles are shown (left), and the IFN-γ MFI ratios (for antigen-specific CD8⁺ T cells upon stimulation/no activation) are presented for all populations studied (right). Horizontal bars represent means.