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).