Figure 3. Functional changes in HIV-specific CD8 T cell populations under conditions of reduced antigen load.

(A)Representative example of simultaneous multi-functional assessment of HIV-specific and CMV-specific CD8 T cells by multi-parametric flow cytometry at a given time point. Cells were stimulated for 6 hours with the corresponding cognate peptide before intracellular staining; αCD107a was present thoughout the assay to capture degranulating cells as described in the Materials and Methods. Percentages of function⁺tetramer⁺ cells are shown. Plots are gated on CD3⁺CD8⁺ cells. Dead cells were excluded from the analysis using a viability dye.(B) Multi-functional assessment of HIV-specific CD8 T cell responses by multi-parametric flow cytometry performed longitudinally for subject 1. After the gates for 4 functions were created (CD107a, IFN-γ, TNF and IL-2), the Boolean gate platform was used to create an array of 15 different positive combinations. The pies represent the functional profiles of HIV-specific CD8 T cell populations. The slices within each pie represent different functional combinations; 4⁺ (red), 3⁺ (orange), 2⁺ (yellow) and 1⁺ (green). The bars on the x-axis represent the response frequency for each combination. Numbers under the pies indicate the time points studied (m=month). Black, light grey and dark grey indicate pre-HAART or pre-escape, HAART or escape, and post-HAART time points, respectively.(C) Longitudinal flow cytometric analysis of HIV-specific CD8 T cell function for all 8 subjects. (D) Percentage of tetramer⁺ cellsexpressing CD107a, IFN-γ and TNF under conditions of high and low antigen load; the first time point before antigen decay and one time point after antigen decay, respectively, are shown. Ag=antigen.