Fig. 4: RSV-F-specific ICS responses in PBMC and polyfunctionality post boost.

RSV-F-specific IFNγ responses were determined in peripheral CD4+ T cells using polychromatic flow cytometry after stimulation with RSV-F peptides. a–c Shown are arithmetic group means of IFNγ expression in CD4+ T cells with upper 95% confidence limit (upper whisker) of individually background subtracted values. Animals received a homologous prime-boost with the Ad26/Ad35 mix a, a homologous prime-boost with Ad26 b, or a heterologous prime-boost with Ad26 and Ad35 c. Arrows indicate immunization time points. The pie charts below summarize the polyfunctional composition of the cytokine response in individual animals two weeks post boost (study week 14). Functional subsets were determined by Boolean gating of IFNγ, TNFα, and IL-2 expressed after RSV-F peptide stimulation and each slice represents the fraction of the total RSV-F-specific cytokine response that consists of T cells expressing all three functional markers (red), any combination of two functional markers (yellow), or individual functional markers (gray). Positive responses to RSV-F peptide stimulation within each functional T-cell population were determined per animal in comparison to the corresponding negative control, using a one-sided Fisher’s exact test with Bonferroni adjustment for multiple comparisons. If peptide stimulation induced a significant response for at least one cytokine, the animal is considered a responder, marked by and asterisk under the pie in this figure.