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Evidence of pathway-specific basophil anergy induced by
peanut oral immunotherapy in peanut-allergic children
Ananth Thyagarajana, Stacie M. Jonesb, Agustin Calatronic, Laurent Ponsa, Mike Kulisa,
Caitlin S. Wood, Mohanapriya Kamalakannand, Brian P. Vickerya, Amy M. Scurlockb, A.
Wesley Burksa, and Wayne G. Shrefflerd,e,1
aDepartment of Pediatrics, Division of Pediatric Allergy and Immunology, Duke University Medical
Center, Durham, North Carolina
bDepartment of Pediatrics, Section of Allergy and Immunology, University of Arkansas for Medical
Sciences, Little Rock, Arkansas
cRho Federal Systems Division, Inc, Chapel Hill, North Carolina
dJaffe Food Allergy Research Institute, Mount Sinai School of Medicine, New York, New York
eMassachusetts General Hospital, Food Allergy Center and the Center for Immunology and
Inflammatory Disease, Boston, Massachusetts
Abstract
Background—In Westernized countries, over 1% of the population is allergic to peanuts or tree
nuts, which carries a risk of severe allergic reactions. Several studies support the efficacy of
peanut oral immunotherapy (OIT) for reducing the clinical sensitivity of affected individuals;
however, the mechanisms of this effect are still being characterized. One mechanism that may
contribute is the suppression of effector cells, such as basophils. Basophil anergy has been
characterized in vitro as a pathway-specific hyporesponsiveness; however, this has not been
demonstrated to occur in vivo.
Objective—To evaluate the hypothesis that basophil anergy occurs in vivo due to chronic
allergen exposure in the setting of a clinical oral immunotherapy trial.
Methods—Samples of peripheral blood were obtained from subjects during a placebo-controlled
clinical trial of peanut OIT. Basophil reactivity to in vitro stimulation with peanut allergen and
controls was assessed by the upregulation of activation markers, CD63 and CD203c, measured by
flow cytometry.
Results—The upregulation of CD63 following stimulation of the IgE receptor, either specifically
with peanut allergen or non-specifically with anti-IgE antibody, was strongly suppressed by active
OIT. However, OIT did not significantly suppress this response in basophils stimulated by the
distinct fMLP receptor pathway. In the subset of subjects with egg sensitization, active peanut OIT
also suppressed CD63 upregulation in response to stimulation with egg allergen. Allergen OIT
also suppressed the upregulation of CD203c including in response to stimulation with IL-3 alone.
Conclusion—Peanut OIT induces a hyporesponsive state in basophils that is consistent with
pathway-specific anergy previously described in vitro. This suggests the hypothesis that effector
cell anergy could contribute to clinical desensitization.
1,*Address correspondence to: Wayne Shreffler, MGH/CIID, 149 13th Street, Charlestown, MA 02129, USA. Phone: 617.726.6147;
Fax: 617.726.5651; wshreffler@partners.org.
NIH Public Access
Author Manuscript
Clin Exp Allergy. Author manuscript; available in PMC 2013 September 21.
Published in final edited form as:
Clin Exp Allergy. 2012 August ; 42(8): 1197–1205. doi:10.1111/j.1365-2222.2012.04028.x.
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Keywords
human basophils; desensitization; basophil anergy; CD63; CD203c; oral immunotherapy; peanut
allergy
Introduction
Peanut allergy affects over 1% of people in westernized countries [1–3]. The only accepted
treatment for food allergy is avoidance and ready access to emergency medications [4].
Unintentional ingestions occur frequently [5], and because peanut allergy is both persistent
and more often associated with severe reactions [6, 7], the need for new treatments is
substantial [8].
Basophils express the high affinity receptor for IgE (FcεRI) and represent a significant
population of antigen-specific cells in IgE-sensitized individuals that are capable of
releasing histamine, leukotrienes, cytokines and other mediators [9,10]. Basophils may also
be important modulators of adaptive immune responses [11–14]. A significant role for
basophils during in vivo allergen exposure is supported by studies documenting basophil
activation [15–17]. As such, basophils are both relevant ‘biomarkers’ of IgE-mediated
hypersensitivity and potential targets of immunomodulatory interventions.
Phenotyping of basophils during activation has revealed useful markers for flow cytometrybased studies [18]. Anaphylactic degranulation results in the translocation of lysosomalassociated membrane proteins (LAMPs), including CD63, from a predominantly
intracellular location to the cell surface [19, 20] resulting in a predominantly bi-modal
distribution of CD63 expression corresponding to cells that have undergone degranulation or
not. The ectonucleotide pyrophosphatase/phosphodiesterase (ENPP)-3, CD203c, is
upregulated during activation as well, but it is also constitutively expressed and its
upregulation is kinetically and pharmacologically distinct from the LAMPs [18, 21, 22].
Activation of circulating basophils has been shown to correlate with clinical disease in
several contexts including urticaria, anaphylaxis, asthma, food allergy, autoimmune disease
and helminth infection [23–27].
Clinical trials indicate that oral immunotherapy (OIT) reduces clinical sensitivity to peanut
[28–30]. We hypothesized that with chronic allergen exposure, basophils would become
refractory to signaling through the FceRI pathway, as has been shown to occur in vitro [31].
For this mechanistic study, we collected peripheral blood from a subset of peanut allergic
subjects enrolled in a double-blind, placebo-controlled trial of peanut OIT [30]. Our
objective was to study the effects of OIT on basophil responsiveness in order to better
understand mechanisms of OIT and evaluate basophil suppression as a biomarker for OIT.
Methods
Subject Characteristics and Treatment
Twenty-eight subjects from a clinical trial of OIT for peanut allergy that took place at Duke
University Medical Center and University of Arkansas for Medical Sciences were included
in this mechanistic study. Local Institutional Review Boards approved the protocol and
informed consent was obtained from the parents of all subjects. Subjects all had a history of
convincing clinical symptoms occurring within 60 minutes of ingesting peanut, a positive
skin prick test to peanuts (≥3 mm of negative control), and a peanut CAP-FEIA > 7 kUA/L.
The median age at enrollment was 5 years [range 2 — 10]. Clinical and several
immunological outcomes have been published separately [30].
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After enrollment, subjects were randomized in a 2:1 ratio to receive either partially defatted
peanut flour (Golden Peanut Company, Alpharetta, GA) or placebo (oat flour). Eighteen of
the subjects were randomized to receive active peanut treatment at the start of the trial. The
remaining ten subjects were randomized to placebo. The peanut OIT protocol consisted of
an Initial Escalation Day that was a modified rush desensitization starting at 0.1 mg of
peanut protein or placebo. This dose was doubled every 30 minutes until a 6 mg dose was
achieved. During the Buildup Phase, the peanut or placebo dose was increased
approximately every two weeks until a 4000 mg dose of peanut protein or placebo was
reached. The goal maintenance dose was 4000 mg once daily. One month after reaching the
maintenance dose all subjects underwent a 5000 mg double-blinded, placebo-controlled food
challenge to peanut. Upon completion of the challenge, the treatment status was revealed.
Those subjects on treatment continued on the maintenance peanut dose. The subjects on
placebo were given the option to undergo desensitization with open label peanut treatment.
At the time of this analysis, five of the placebo subjects elected to start open label active
treatment. Of the 23 subjects who received active treatment, three subjects dropped out
during build-up prior to a post-baseline assessment of basophil reactivity. Two other
subjects had not completed their post-treatment challenge at the time of this analysis; in
these cases treatment status was revealed only for this mechanistic analysis — subjects and
clinical investigators remained blinded. The median length of time on active therapy was
168 days [1 — 826]; the median length of time on placebo therapy was 358.5 days [68 —
423]. After the baseline assessment, basophil activation was assessed a median of 2 times [0
— 4] prior to unblinding, with a median 182 day interval [84 — 392]. For the entire study
(including the open phase), basophil activation was assessed a median of 3 [1 — 7] times.
The median interval between assessments for the total study time was 119 days [68 — 326].
Eleven subjects (39%) also had a clinical diagnosis of egg allergy based on a history of
convincing clinical reactions and persistent sensitization. All were strictly avoiding egg.
Nine of eleven received active peanut OIT treatment.
Reagents
RPMI 1640 with glutamine; N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Fisher
Scientific, Pittsburgh, PA); Recombinant human IL-3 (R&D Systems, Minneapolis, MN);
polyclonal rabbit anti-human IgE antibody (Bethyl Laboratories, Montgomery, TX); EDTA
(Promega Corp, Madison, WI); FACS lysing solution (BD Biosciences, San Jose, CA).
Antibodies
The following mAbs were used: FITC anti-CD63 (clone H5C6, BD Biosciences), PE antiCD203c (clone 97A6, Serotec, Oxford, UK), PE-Cy5 anti-CD123 (clone 9F5, BD
Biosciences), APC anti-CD41a (clone HIP8, BD Biosciences), and PE-Cy7 anti-HLA-DR
(clone L243, BD Biosciences).
Assays for IgE and IgG4
Peanut- and egg-specific IgE, IgG, and IgG4 levels were measured in serum by using the
ImmunoCAP 100 instrument (Phadia AB) according to the manufacturer’s instructions.
Basophil activation
After overnight shipment to Mount Sinai, whole blood (250 μL) was incubated with equal
volumes of basophil stimulation buffer (RPMI plus IL-3 at 2 ng/mL; IL-3 alone control)
alone or with the addition of dilutions of peanut antigen (from 1 × 101 to 1 × 10−5 μg/ml
total protein prepared from an aqueous extract of defatted roasted peanuts diluted in PBS),
dilutions of egg antigen (1 × 100 to 1 × 10−3 μg/ml total protein prepared from powdered
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egg white diluted in PBS), anti-IgE antibody (0.5 μg/mL, positive control), or fMLP (1 μM,
IgE-independent positive control) at 37°C for 30 minutes; RPMI alone was used as an
additional negative control. The reaction was stopped with 50 μL of cold PBS plus 20 mM
EDTA. Cells were then stained for expression of CD63, CD123, CD203c, CD41a, and
HLA-DR at 4°C in the dark for 30 minutes, then washed with cold PBS plus 0.5% BSA plus
2 mM EDTA. Red cells were then lysed and WBC fixed with 4 mL of FACS Lysing
Solution to the cell pellet for 20 minutes at room temperature.
Flow cytometry
Samples were analyzed on a BD LSRII flow cytometer. Single-color compensation samples
were prepared using antibody capture beads (BD Biosciences) according to the
manufacturer’s protocol. Fluorescence data were acquired and autocompensated on a
modified LSR-II configured for seven color parameters by using FACS Diva software
(version 4.0, BD Biosciences). Basophils were identified as CD123+CD203c+HLA-DRCD41a-. A minimum of 300 CD123+CD203c+HLA-DR-CD41a- events (i.e., basophils)
were recorded for each condition or the sample was excluded (83 of 1198 samples, 7%).
Statistical analysis
Values are presented as means and standard deviations. Continuous variables were
compared using Student’s t-test and the chi-square test for categorical variables. The
outcome of interest was the change over time for CD63 and CD203c between treatment and
placebo by stimulant. In particular, we hypothesized that the control group will remain
unchanged over time while the treatment group will decrease. This was accomplished using
a linear mixed effects model of the nlme package in R analysis (lme function) [32] enabling
us to take into account the fact that the measurements were made on the same participants
over time and the open-label design. The relationship between the CD63 and peanut
concentration was modeled by the modified four parameter log-logistic regression model.
The model was fitted to data by nonlinear regression analysis (drm function of the drc
package) [33] where EC50 is the concentration producing a response a halfway between the
minimum and maximum levels observed. Analyses were performed using the R statistical
computing environment [34], P values smaller than 0.05 were considered significant.
Results
Peanut oral immunotherapy suppresses basophil responsiveness to in vitro stimulation
with peanut allergen
At baseline, basophils incubated with RPMI or IL-3, which was included with all other
stimulants to prime degranulation, expressed low levels of CD63 (<1.5% CD63high). CD63
expression at baseline was strongly and equivalently upregulated in both treatment groups
by cross-linking the occupied high affinity IgE receptors (FcεRI) using the control stimulant,
anti-human IgE, the distinct G-coupled receptor for fMLP or with a range of soluble peanut
allergen concentrations from 10 μg/mL to 100 pg/mL (not shown).
Basophil responses were longitudinally assessed in these subjects during active or placebo
immunotherapy treatment (see Methods). As shown in Figure 1, in vitro basophil reactivity
to peanut allergen was strongly and significantly suppressed from baseline across the range
of allergen stimulation concentrations (see also Table 1). Maximal responses of ~40%
CD63high at 100 ng/mL were suppressed to ~10% over time (p <0.001). No significant
changes in peanut-allergen induced CD63 upregulation were observed in the placebo group
over time (p=0.34). In addition to suppressing the maximal responses, basophil sensitivity to
in vitro stimulation was suppressed, as measured by a shift in the half-maximal eliciting
concentration (EC50) by about 10-fold, from ~0.4 ng/mL to ~5 ng/mL (Figure 2). The
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reduced basophil reactivity to peanut allergen coincided with an induction of peanut-specific
IgG4, but there was no change in specific IgE (Figure 3).
Oral immunotherapy specifically suppresses FcεRI-induced upregulation of CD63
Active therapy not only suppressed basophil reactivity to specific allergen (peanut), but it
also significantly suppressed upregulation of CD63 in response to cross-linking of IgE with
anti-human IgE (p<0.001, Figure 4). There was no significant change in the placebo group.
Active therapy did not suppress basophil responsiveness to fMLP (p=0.19).
By study design, those subjects remaining on active therapy during the open-label portion of
the study were on treatment longer than any subject that received placebo. Additional data
collected beyond the placebo-controlled portion of the study are found in Table I. The
findings with respect to basophil suppression are the same: CD63 upregulation by activation
via the IgE receptor with both peanut allergen and anti-IgE was suppressed while activation
by fMLP was not (not shown).
Oral immunotherapy to peanut suppresses basophil reactivity to a bystander allergen
We also measured egg allergen-induced basophil activation in those subjects with a history
of clinical reactivity to egg and persistent egg sensitization (11/28, 39% of total subjects). Of
those subjects, ten were randomized to receive active peanut OIT treatment, but two dropped
out after the first visit. We therefore analyzed the longitudinal change in basophil reactivity
within the treated group. Although the subset of egg-sensitized subjects was small and the
response to egg more variable, peanut OIT significantly suppressed the in vitro basophil
response to egg allergen at the highest concentration (1 μg/mL; median CD63 high
expression from ~40% to <10%, p<0.05) and this trend was evident at lower concentrations
of egg allergen stimulation as well (Figure 5A). Within the egg sensitized subgroup, the
suppression of CD63 upregulation induced by peanut (Figure 5B) and anti-IgE (not shown)
was comparable to the overall group. Suppression of basophil activation by egg allergen in
this subset of individuals was not associated with any change in egg-specific IgE or IgG4
(Figure 6).
Oral immunotherapy also suppresses in vitro upregulation of CD203c
CD203c is constitutively expressed by human basophils and unlike CD63 is upregulated by
IL-3 alone [18, 20, 21] and further upregulated by FcεRI and fMLP receptor stimulation. We
found that the expression of CD203c following in vitro stimulation with IL-3 alone or
together with anti-IgE, allergen or fMLP was also suppressed by peanut OIT (Figure 7).
There was no change in the ex vivo CD203c expression as seen in the RPMI control
condition.
Discussion
Successful immunomodulatory may theoretically prevent mediator release from effector
cells such as basophils by suppressing signaling events upstream or at various stages
downstream of IgE binding. Induction of allergen-specific IgG, which occurs during both
subucutaneous and oral immunotherapy may competitively inhibit allergen interaction with
cell-bound IgE (upstream) or lead to co-ligation of FcγRIIb inhibitory receptors with FcεRI
(early downstream) and both of these mechanisms induce allergen-specific suppression of
effector cells due to the specificity of the inhibitory IgG [35, 36].
Several very general mechanisms of cellular adaptation downstream of repeated receptor
stimulation have also been demonstrated to occur in basophils and/or mast cells in vitro and
these vary in degree of specificity from highly allergen-specific, to pathway- but not
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allergen-specific [31, 37]. Our data suggest that OIT may induce basophil anergy — a
pathway-specific, but antigen non-specific, refractory state that is inducible in vitro by
FcεRI stimulation [31]. CD63 (LAMP-3) upregulation is a marker of basophil granule
fusion to the plasma membrane during degranulation [20]. Consistent with anergy, CD63
upregulation that was induced by specific allergen stimulation as well as non-specific
stimulation with anti-IgE and bystander allergen was suppressed by active OIT, while
fMLP-induced CD63 upregulation remained unaffected. A testable consequence of FcεRIstimulation induced anergy would be an expected loss of Syk expression [38], and we plan
to evaluate this in future mechanistic studies.
Consistent with previous reports, our data (Supplemental Figures 1 and 2) demonstrate that
CD203c is both constitutively expressed and further upregulated following activation [39].
The regulation of CD203c expression has been distinguished from CD63 in numerous ways
including by kinetics, pharmacological inhibitors and the capacity of IL-3 to independently
induce its expression [18, 22, 40]. Treatment with peanut OIT suppresses the IL-3 induced
CD203c upregulation to the level of resting basophils. Given a recent report that the IL-3
receptor complex includes the ITAM-containing FcRγ chain [41], reduced expression of Syk
following FcεRI stimulation could theoretically be involved in both the suppression of
CD63 upregulation downstream of FcεRI stimulation as well as the suppression of IL-3
induced upregulation of CD203c. To our knowledge, Syk dependence of CD203c
upregulation downstream of IL-3 stimulation has not been shown, though it has been
reported that IL-3 induced IL-13 expression is not sensitive to the Btk inhibitor, PCI-32765
[40] and that IL-3 does not significantly affect Syk phosphorylation [42]. Because IL-3
directly induces CD203c upregulation and because we chose, as is often done, to include it
as a priming agent with all stimulants when this study was designed, we cannot now
determine whether peanut OIT independently suppresses CD203c upregulation induced by
allergen, anti-IgE or fMLP. It must also be noted that it is possible that OIT suppression of
CD63 upregulation may be due to an effect on IL-3 signaling that is specific to its priming
of the IgE pathway. However, IL-3 primes basophil degranulation induced by multiple
secretagogues including anti-IgE, fMLP, C5a and calcium ionophore and, given the
differences of the signaling pathways involved, is thought to effect common distal signaling
mediators [43]. Based on this, we believe that our data are most consistent with the
interpretation that peanut OIT suppresses FcεRI signaling.
A significant limitation of the current study is our inability to correlate basophil reactivity
and suppression with clinical responsiveness to OIT, due to a lack of pre-treatment
challenge data, and an unanticipated absence of post-treatment response variability due to
the fact that all active treatment subjects tolerated the final 5000 mg challenge dose. In a
previously published study of milk allergic subjects, an association was found between
clinical sensitivity and basophil reactivity, and in that study IgE pathway-specific
suppression of basophils was also observed among individuals tolerating chronic oral
exposure of milk in the diet [27].
In conclusion, we show here evidence supporting the hypothesis that peanut OIT induced
pathway-specific basophil anergy, suggesting a novel mechanism that together with other
established immunomodulatory effects including the induction of specific IgG and changes
in antigen-specific T cell phenotypes may contribute to clinical desensitization. The sum of
these changes on the clinical response may be purely allergen-specific. However, if chronic
allergen exposure induces pathway anergy, there may be a measure of clinical nonspecificity with high dose continuous allergen exposure that has not been adequately
examined. If basophil responses are blunted at least in part due to anergy, and not only
secondary to the induction of allergen-specific IgG, this indicates that basophils are directly
engaging with allergen via specific IgE in vivo. Data in murine models support a role for
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basophils in capturing low concentrations of antigen and modulating B and T cell immunity
[11–14, 44]. Whether or not they can function as primary APCs [45, 46] – if they are
directly interacting with allergen, their participation in the immune response during OIT
deserves further exploration.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
Funding support from the Food Allergy & Anaphylaxis Network, Food Allergy Project, Gerber Foundation, NIH
Grant 1 R01-AI06874-01A1, NIH Grant 1 UL1 RR024128-01, NIH Grant R03-AI079544, Dorothy & Frank
Robins Family, NIH T32 Grant 5T32-AI007062-32, and the National Peanut Board.
Abbreviations
OIT oral immunotherapy
LAMP lysosomal associated membrane protein
fMLP N-formyl-methionyl-leucyl-phenylalanine
IL-3 Interleukin-3
ENNP3 ectonucleotide pyrophosphatase/phosphodiesterase (ENPP)3, CD203c
PN peanut
anti-IgE polyclonal anti-human IgE antibody
ITAM immune related tyrosine containing activation motifs
Syk spleen tyrosine kinase
Btk Bruton’s tyrosine kinase
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Figure 1. Allergen-induced CD63 upregulation is suppressed by OIT
The distribution of percent CD63high basophils after in vitro stimulation from subjects over
time by treatment status during the blinded portion of the OIT study. Peanut, active
treatment; Placebo, control treatment. Time intervals are indicated in days and were chosen
to include roughly equal numbers of assessments over time. Closed circles indicate mean
percent CD63high. Box plot represents the interquartile range (25th and 75th percentile) with
a line indicating the median. Whiskers extend to 1.5 times the interquartile range. Dashes
represent the outliers. Closed circles and lines indicate the mean change over time. P0 = day
0, P1 = day 21–156, P2 = day 157–423. *** for <0.001, ** for <0.01, * for <0.05, and ns for
=0.05.
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Figure 2. Both the maximum response and sensitivity of allergen-induced CD63 upregulation is
suppressed by OIT
The population fitted dose response for CD63 upregulation by treatment group is shown
over time in days (0, baseline; 21–156; 157–423) during the placebo-controlled study
period. Horizontal and vertical lines indicate half maximal and 50% eliciting dose,
respectively, for each treatment group. Active treatment is shown in blue; placebo is in red.
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Figure 3. Active OIT induces changes in peanut-specific IgG4
The peanut-specific IgE and IgG4 levels were determined using the Phadia UniCap system
from samples matched to time points analyzed for basophil reactivity. Box plot represents
the interquartile range (25th and 75th percentile) with a line indicating the median. Whiskers
extend to 1.5 times the interquartile range. Dashes represent the outliers.
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Figure 4. Allergen OIT suppresses polyclonal anti-IgE- but not fMLP-induced CD63
upregulation
The distribution of percent CD63high basophils after in vitro stimulation from subjects over
time by treatment status during the blinded portion of the OIT study. Anti-IgE w/IL-3, 0.5
μg/mL polyclonal rabbit anti-human IgE with 2 ng/mL human recombinant IL-3; fMLP, 1
μM N-formyl-methionyl-leucyl-phenylalanine with IL-3; IL-3 alone at the same 2 ng/mL
concentration; Medium alone is RPMI. Time intervals are indicated in days and were chosen
to include roughly equal numbers of assessments over time. Box plot represents the
interquartile range (25th and 75th percentile) with a line indicating the median. Whiskers
extend to 1.5 times the interquartile range. Dashes represent the outliers. Closed circles and
lines indicate the mean change over time. P0 = day 0, P1 = day 21–156, P2 = day 157–423.
*** for <0.001, ** for <0.01, * for <0.05, and ns for ≥0.05.
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Figure 5. Allergen OIT suppresses bystander allergen-induced CD63 upregulation
The distribution of percent CD63high basophils after in vitro stimulation from egg-allergic
subjects (n=8) receiving active treatment over time. A. CD63 upregulation induced by egg
white allergen (EGG1-4) at indicated concentrations. B. CD63 upregulation induced by
peanut allergen as in Figure 2 limited to the nine egg sensitized subjects. Box plot represents
the interquartile range (25th and 75th percentile) with a line indicating the median. Whiskers
extend to 1.5 times the interquartile range. Dashes represent the outliers. Closed circles and
lines indicate the mean change over time. P0 = day 0, P1 = day 21–156, P2 = day 157–423,
P3 = day 424–827. *** for <0.001, ** for <0.01, * for <0.05, and ns for ≥0.05.
Thyagarajan et al. Page 15
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Figure 6. Active OIT induced no changes in egg-specific IgG4 or IgE
The egg-specific IgE and IgG4 levels were determined using the Phadia UniCap system
from samples matched to time points analyzed for basophil reactivity. Box plot represents
the interquartile range (25th and 75th percentile) with a line indicating the median. Whiskers
extend to 1.5 times the interquartile range. Dashes represent the outliers.
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Figure 7. Expression of CD203c on basophils over time
The distribution of CD203c median fluorescence (MFI) on basophils after in vitro
stimulation of study subject basophils over time by treatment status. A. CD203c expression
of basophils stimulated with control stimulants as indicated above each panel. B CD203c
expression of basophils stimulated with peanut allergen. Box plot represents the interquartile
range (25th and 75th percentile) with a line indicating the median. Whiskers extend to 1.5
times the interquartile range. Dashes represent the outliers. Closed circles and lines indicate
the mean change over time. P0 = day 0, P1 = day 21–156, P2 = day 157–423, P3 = day 424–
827. *** for <0.001, ** for <0.01, * for <0.05, and ns for ≥0.05. IL-3, 2 ng/mL recombinant
human IL-3; anti-IgE, 0.5 μg/mL anti-human IgE with IL-3; 1 μM fMLP, N-formylmethionyl-leucyl-phenylalanine with IL-3; PN1-6, 1 × 101 to 1 × 10−4 μg/mL total peanut
protein with IL-3.
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