Measuring HIV Gag-specific immune responses: intracellular cytokine staining. (A) Flow cytometric gating strategy for RMCs. Doublet and dead cell discrimination is used to reduce background noise (second and third graphs). For CD8⁺ T cells, positive responses for each individual function (CD107a, IFN-γ, IL-2, MIP-1β, and TNF-α) are selected. Boolean gates are created from these 5 individual gates to divide responding cells into 31 distinct populations corresponding to all possible combinations of these functions. These are graphed using SPICE software to better visualize polyfunctional responses. (B) CD8⁺ T-cell responses in both peripheral blood and rectal mucosa from an elite controller using standard bivariate plots.

HIV Gag-specific polyfunctional CD8⁺ T-cell responses. (A) The overall polyfunctionality of the mucosal CD8⁺ T-cell response can be visualized with pie charts in which each slice represents a different functional category: black indicates 5 functions; purple, 4 functions; dark blue, 3 functions; light blue, 2 functions; green, 1 function. The number in the center of each pie represents the median total percentage of cells responding in any way to Gag stimulation. Elite and viremic controllers are combined into a single “controller” category. NC indicates noncontroller; HAART, HAART-suppressed. Statistical differences between groups are indicated above pie charts. (B) The bar chart shows the total Gag-specific response broken down into 31 individual response categories. Each bar represents the median and interquartile ranges for the frequency of responding cells for a different patient group as indicated by the colored rectangle beneath each pie (ie, blue indicates controllers; red, noncontrollers; green, HAART-suppressed). The colored bars below the x-axis relate to the pie slice colors in Figure 4A. Statistical differences between patient groups are marked by an asterisk (*) and color coded (blue indicates controllers; red, noncontrollers; green, HAART). All differences were significant at P < .01. (C) Expression of MIP-1β in mucosal CD8⁺ T cells as measured by MFI comparing polyfunctional cells to dual- and single-function cells in controllers and noncontrollers. Each data point represents the MFI from a separate population of cells (5+, 4+, 3+, 2+, or 1+). Horizontal bars represent the median for each group. ***P < .001.

Relationship between HLA class I genotype and CD4⁺ T-cell levels. (A) Percentage of CD4⁺ T cells as determined by flow cytometry in peripheral blood (PB, closed symbols) and rectal mucosa (RMC, open symbols) among controllers and noncontrollers with and without protective class I HLA alleles. (B) Percentages of CD4⁺ T cells in peripheral blood (PB, closed symbols) and rectal mucosa (RMC, open symbols) among persons with or without protective class I HLA alleles, defined as in Figure 5, irrespective of clinical status. Horizontal bars represent the median for each group. *P < .05, **P < .01, ***P < .001.

Percentage of CD4⁺ T cells in blood and mucosa. Percentage of CD4⁺ T cells, shown as a subset of CD3⁺ T cells, as determined by flow cytometry in peripheral blood (PB, closed symbols) and rectal mucosa (RMC, open symbols) across patient groups. Horizontal bars represent the median of each group. *P < .05, **P < .01, ***P < .001. All HIV-infected patient groups had significantly lower percentages of CD4⁺ T cells than seronegatives (P < .01).

HIV Gag-specific CD8⁺ T-cell responses in blood and rectal mucosa. (A) Total percentage of CD8⁺ T cells from peripheral blood (left panels) or rectal mucosa (right panels) able to respond in any way (CD107a, IFN-γ, IL-2, MIP-1β, or TNF-α) to Gag stimulation. Percentage of CD8⁺ T cells capable of producing IFN-γ (B), MIP-1β (C), TNF-α (D), and CD107 (E) in response to Gag stimulation. Horizontal bars represent the median for each group. *P < .05, **P < .01, ***P < .001.

CD8⁺ T-cell responses in controllers and noncontrollers with and without protective class I HLA alleles. (A) Patient immunogenetics. The bar graphs show the percentage of patients in this study with HLA-B57 (far left); any protective class I HLA (HLA-B13, B27, B57, B58, B81; second from left); HLA-DRB1*13 and/or DQB1*6 (third from left); and both protective class I and class II alleles (far right). (B) Total magnitude of the CD8⁺ T-cell response in rectal mucosa. Horizontal bars represent the median for each group. *P < .05, **P < .01, ***P < .001. (C) Overall polyfunctionality of the CD8⁺ T-cell response in rectal mucosa. Each pie slice represents a different functional category: black indicates 5 functions; purple, 4 functions; dark blue, 3 functions; light blue, 2 functions; green, 1 function. The number in the center of each pie represents the median percentage of responding cells responding in any way to Gag stimulation. Statistical differences between groups are written above pie charts. (D) Mucosal CD8⁺ T-cell polyfunctional responses broken down into 31 individual response categories. Each bar represents the median and interquartile ranges for the frequency of responding cells for a different patient group and class I HLA status as indicated by the colored rectangle beneath each pie (ie, blue indicates controllers/protective HLA). The colored bars below the x-axis relate to the pie slice colors in panel B. Statistical differences between controllers with protective class I alleles and other patient groups are marked by an asterisk (*) and color coded for each patient group (red indicates controller/nonprotective HLA; green, noncontroller/protective HLA; purple, noncontroller/nonprotective HLA). All differences are significant at P < .01.

CD8⁺ T-cell responses in persons with protective class I and II HLA alleles. (A) Total magnitude of the CD8⁺ T-cell responses in rectal mucosa in persons with or without protective class I and/or class II HLA alleles. Horizontal bars represent the median for each group. *P < .05, **P < .01. (B) Mucosal CD8⁺ T-cell polyfunctional responses broken down into 31 individual response categories. Each bar represents the median and interquartile ranges for the frequency of responding CD8⁺ cells in persons with or without protective class I and/or class II HLA alleles. Statistical differences between persons with both protective class I and class II alleles and other groups are marked by an asterisk (*) and color coded for each patient group (green indicates class II only; purple, no protective HLA). All differences were significant at P < .01.