Figure 1. Flow cytometric detection of CD4⁺ T-cell cytokine expression in PBMCs.

PBMCs from adults (n=11) and young children (n=17) were stimulated with various pneumococcal and NTHi protein antigens and analyzed by ICS. Individual cytokine producing CD4⁺ T-cells in each sample were sequentially gated on total CD45RA followed by CD69⁺ cells as shown in the example figure from a child. Several Boolean gate combinations were produced for cytokine positive cells and CD4+ T cell frequencies producing individual cytokines but not others were quantified as described earlier [25] (A). Individual cytokine combinations as produced by Boolean gating were further validated by back-gating on CD69-high cells (shown in red) (B).