Detection of 5 concurrent T-cell functions and characterization of HIV-specific CD8⁺ T-cell functionality in HIV-infected progressors. (A) Gating scheme for identification of multifunctional CD8⁺ T-cell responses. Shown are representative data of the HIV Gag-specific response from subject A26, an HIV-infected progressor, after a 6-hour in vitro stimulation. See “Patients, materials, and methods” for a detailed explanation of the procedure. (B) MIP-1β is the dominant HIV-specific CD8⁺ T-cell response (P < .001 for all stimulation conditions). Box plots represent the 10th, 25th, 50th, 75th, and 90th percentiles of the contribution of the indicated functional response (x-axis) toward the total CD8⁺ T-cell response against the indicated HIV peptide mix (color coded as shown) within the 79 progressors. The responses from each subject were standardized to allow comparison of the proportions of the total response expressing each function irrespective of the others. (C) Total CD8⁺ T-cell response frequency does not correlate with viral load. The red dots represent the total response summed across all functional combinations for each antigen for each progressor (n = 79). The x-axis denotes CD8⁺ T-cell frequency, and the y-axis denotes log₁₀ viral RNA load. (D) The CD8⁺ T-cell response to HIV-Gag is composed of multiple functional subpopulations and is largely restricted to cell populations with limited functionality. The black bars represent the total CD8⁺ T-cell response frequency to Gag in subject A26 expressing the particular combination of functions shown. Each dot denotes CD107a, IFN-γ, MIP-1β, IL-2, and/or TNF-α positivity. The panel also contains horizontal bars of different colors showing those combinations of 5, 4, 3, 2, or 1 function for reference. Responses shown are background subtracted using the 28/49d negative control. (E) The functional profile of HIV-specific CD8⁺ T-cells is limited in HIV-infected progressors. Each pie chart represents the mean response across the 79 subjects to the 5 different HIV-antigen stimulations. For simplicity, responses are grouped by number of functions, matched to the colored bars in panel D. As shown, more than 75% of the average response to each antigen expresses fewer than 4 functions.