Fig. 4.

PBMC intracellular cytokine detection by flow cytometry. PBMC intracellular cytokine detection by flow cytometry was performed following overnight stimulation with Staphylococcal enterotoxin B (SEB; positive control), the M72 peptide pool or medium. a Cytokine production by M72-specific CD4⁺ T cells was determined by discriminating the CD4⁺ and CD8⁺ T cells from the “morphological” gating window. b CD4⁺ T cells were analyzed with respect to the production of CD40L, IL-2, TNF-α and IFN-γ. The unstimulated (medium) samples show background levels of cytokine production. When restimulated with the M72 peptide pool, the production of CD40L, IL-2 and TNF-α by CD4⁺ T cells and lower production of IFN-γ was detected. The numbers in the quadrant gates of the plots denominate each distinct population based on their cytokine production. Samples from the same subject are shown, with responses at 1 month post second vaccination. Results shown are representative of the range of responses seen with all subjects studied. The lowest, highest and median numbers of acquired viable CD4⁺ T cells examined were 16975, 75000 and 75000 cells respectively