Large frequency of memory CD4⁺ T cells after CAF09 immunization.

<p>PBMCs isolated from vaccinated animals 7½ weeks after first immunization were stimulated with vaccine antigens (fusion protein + MAP3694c) and surface stained for the CD4 receptor and intracellular localized IFN-γ, TNF-α and IL-2 cytokines. Boolean gating was used to determine the frequencies of the seven possible CD4⁺ T cell subpopulations expressing any combination of the IFN-γ, TNF-α and IL-2 cytokines. The frequencies are shown for each of the three vaccination groups after subtraction of background from non-stimulated adjuvant controls. Pie charts illustrate the fraction of CD4⁺ T cell expressing three cytokines (3+), any two cytokines (2+), or any one cytokine (1+) for each vaccination group (CAF01, CAF09, and ISA 61 VG). Pie chart color-coding and the subpopulation association for each color is shown below the bar graph (<b>A</b>). The mean fluorescence intensity (MFI) for the IFN-γ-, TNF-α-, or IL-2 producing CD4+ T cells in each vaccination group was calculated as a mean of all cytokine producing cell regardless if a cell produce one, two or three of the measured cytokines. Bars represent mean ± SD of three calves. Statistical analysis: unpaired t test ***<i>p</i> < 0.001 (<b>B</b>).