FIGURE LEGENDS
Figure 1. Study design and experimental strategy. A) A total of n=71 subjects were enrolled from 11 clinical sites, and blood samples were collected and PBMC isolated and cryopreserved. B) PBMC were tested against a set of peptide pools consisting of overlapping peptides spanning the entire sequence of 11 different CR allergens, and T cell cytokine responses assessed by a combination of the Activation Induced Marker (AIM) and Intracellular Cytokine Staining (ICS) assays after 6 hours of stimulation. C) Dot plots depict the cytokine detection of IL-4, IFNγ, and IL-10 in CD154+ CD4+ T cells after stimulation with DMSO, PMA/Ion or sets of peptide pools for a given allergen. 3 representative participants are shown to illustrate cytokine secretion of IL-4, IFNγ, and IL-10 to a particular high-reactive CR allergen and the absence of response to a particular CR allergen as control. Specific allergen tested in each condition is indicated in dot-plots as well as the frequency of CD154+ cytokine+ events of total CD4+ T cells.
Figure 2. Baseline cytokine responses of CD154+ cells are IL-4 polarized and correlate with magnitude of responses. A) Graph bars show the overall magnitude of CD4+ T cell CR-specific responses as the sum of the 11 individual allergens tested. Each bar represents a participant (n=71) and the fraction of the response accounted for each cytokine (IL-4 (blue), IFNγ (red), or IL-10 (green)). B) Graph compares the IL-4, IFNγ and IL-10 allergen responses for all the individual participants combined. Geometric mean and p value are shown as statistically significant by Wilcoxon’s paired t test. C) Graphs depict the percentage of each individual cytokine contribution from total response, grouped as function of the IL-4, IFNγ, IL-10 dominant (>50% of total response) or non-dominant cytokine profile for a particular participant. Each dot and interconnected lines represents a participant and the number of participants in each group is shown. D) Graphs show the correlation of total cytokine response with the relative IL-4, IFNγ and IL-10 cytokine response. R values and p values are shown as statistically significant by nonparametric Spearman correlation test and best fit represented by a linear regression line (red).
Figure 3. Bla g 9 and Bla g 5 are the most immunodominant allergens and dominance is participant specific. A) Graph shows the overall response directed against the 11 allergens tested, calculated by summing each individual allergen response for all participants. Each bar represents an allergen and the fraction of the response accounted for each cytokine (IL-4 (blue), IFNγ (red), or IL-10 (green)). Table depicts the percentage of total or IL-4 responses for each allergen. B) Graphs show for each individual participant (dots and interconnected lines) the fraction of the response accounted for each allergen and grouped as a function of allergen immunodominance (-high) or lack of dominance. Bla g 1 and Chitinase not shown in graphs as no dominant responses were observed. The number of participants associated with each category is shown.
Figure 4. Treg numbers varies amongst participants and are inversely correlated with Teff responses. A) Dot plots show a representative participant for the Treg gating strategy. B) Graph show the correlation of total CD137+ cells and CD25+CD127low CD137+ cells in unstimulated cells by Pearson correlation test. C) Graph bars show the number of Tregs (CD137+ cells) across all participants (n=71). Figure insert depicts 2 representative participants with High or Low Treg populations, respectively. D) Graph shows the correlation of Tregs (Total CD137+ unstimulated cells) and Teff (cytokine+ CD154+ allergen-stimulated) cells by nonparametric Spearman correlation test. For both correlation graphics, each dot represents a participant (n=71). R values and p values are shown and best fit represented by a linear regression line (red).