Multiparameter flow analysis of the polyfunctional profile of KLRG-1+ and KLRG-1− HIV-1–specific CD8+ T cells. Epitope-specific CD8+ T-cell responses of 20 subjects with chronic-progressive or controlled HIV-1 infection directed against 23 HIV-1 epitopes were tested (A3-RK9, B8-EI8, B8-FL8, B27-KK10, B57TW10, B57-KF11, B7-GL9). All 16 possible combinations of the 4 antigen-specific functions studied for each epitope are shown on the x-axis, and the contribution of each epitope-specific CD8+ T-cell population exhibiting the respective combination of functions is indicated as bars (see bar graphs on lower portion of figure). Responses are grouped and color-coded according to the number of functions (1 = yellow, 2 = green, 3 = blue, 4 = red). Blue bars show the results from the KLRG-1+ CD8+ T-cell responses, and red bars show the results from the KLRG1− responses. The data are summarized by pie charts in which each slice of the pie represents the fraction of the total epitope-specific response. Pie charts on the left show the responses of the KLRG1+ CD8+ T cells, and pie charts on the right show the KLRG1− fraction. (A) The optimal epitope HIV-1–specific CD8+ T-cell responses were predominantly monofunctional. Although the KLRG1− CD8+ T-cell responses appeared to contain a larger fraction of dual-functional and trifunctional responses, these differences were not statistically significant. To tease out differences in the responsive ability of KLRG1+ versus KLRG1− CD8+ T cells, we stimulated PBMCs of the same 20 subjects with phorbol 12-myristate 13-acetate and ionomycin (B). The ability to respond to this stimulus between KLRG1+ and KLRG1− cells was significantly different. Although KLRG1+ CD8+ T cells responded predominantly with monofunctional responses, we observed significantly more polyfunctional responses from KLRG1− CD8+ T cells. Significant differences are indicated; *P < .05. We also noted that most TNF-α secretion was from KLRG1− CD8+ T cells, whereas relatively small amounts of TNF-α were produced by KLRG1+ CD8+ T cells (P < .0001). The horizontal lines at each variable indicate the mean (C).