Figure 3.

CD8⁺ cytokine responses to Ag85B. PBMCs were thawed, rested overnight, and stimulated for 5-6 hours with DMSO (negative control), SEB (positive control), or peptide pools corresponding to the vaccine antigens Ag85A, Ag85B, or TB10.4. Specimens were then stained for viability, phenotypic markers, and intracellular cytokine expression and evaluated by flow cytometry. Data was analyzed using Flow Jo software to generate cytokine Boolean gates. Each gate was subjected to DMSO subtraction to remove background. Negative results following background subtraction are set to zero. The sum of these gates was then used to determine the total cytokine response for vaccinated (A) and placebo control (B) groups or plotted by functionality to assess the polyfunctional response (C). Lines (A, B) are plotted for each individual subject. CD8⁺ Single-Gate (Boolean; C) ICS responses for Ag85B at Study Day are shown for vaccinated (gray bars) and placebo (black bars) groups. Bars represent the 25^th to 75^th percentile, with the cross bar representing the median response. The Mean is indicated by an “x” and the error bars represent the minimum and maximum responses.