CITRUS analyses for the stimulated cells, clustering on all 28 surface and intracellular markers and comparing median expression of the markers TNF-α, IFN-γ, IL-17A, and IL-2 between the three groups. a viSNE maps of PBMC of one representative subject of the control, IgEpos, and IgEneg allergy group, cells are colored by TNF-α, IFN-γ, IL-17A, and IL-2 expression respectively. b Median marker intensities for the various functional markers, shown as box plots and values for each individual within the groups of healthy controls, IgEpos, and IgEneg food allergic subjects, for each parent clusters identified in A. Differences between groups are shown as lines, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001 (Kruskal-Wallis and Dunn’s multiple comparisons test). c CITRUS histograms for the major parent clusters. For each surface marker, the histogram shows the marker expression on cells in the specific cluster (red) against the marker expression on all other cells (blue) – overlapping of histograms will result in purple areas. d Scatter plots of frequencies of polyfunctional CD4+ Th cells, CD8+ Tc cells, and NK cells simultaneously expressing TNF-α+ IFN-γ+ (left panel) and TNF-α+ IFN-γ+ IL-2+ (right panel) in the three groups of participants: controls, IgEpos food allergy patients, and IgEneg food allergy patients. Each dot represents a participant while the lines represent the group median. * denotes P ≤ 0.05 and ** denotes P ≤ 0.01 (Kruskal-Wallis and Dunn’s multiple comparisons test).