To the Editor:
Peanut allergy is the most common cause of anaphylaxis in
childhood.1 The acute IgE-mediated allergic reaction
is initiated by basophils or mast cells via cross-linking of surface
bound IgE through high affinity IgE receptors (FcεRI). Unlike mast
cells, basophils circulate in the bloodstream and can be used to
simulate the early events of allergen stimulation, with correlations to
clinical outcomes.2 Current approaches largely focus
on peripheral blood derived mononuclear cells, which lack a significant
percentage of basophils and limits interrogatory research on
granulocytes. Less is known about the subtle molecular and cellular
mechanisms that affect the innate immune system, specifically basophils,
in peanut allergy and during oral immunotherapy (OIT). High dimensional
immune profiling via mass cytometry (CyTOF) offers new possibilities to
better understand innate cell populations in the context of food
allergy. We aimed to a) develop a whole-blood stimulation mass cytometry
approach to characterize basophil activation patterns and b) use this
dataset to identify dynamics of basophil subsets, based on differences
in expression of cellular markers and phosphorylation patterns upon
allergen-specific stimulation. Such an approach could be applied to
monitor desensitization during OIT at a high resolution.
We established a CyTOF panel to study differences in allergen-specific
signaling pathways upon peanut stimulation in whole blood using
barcoding. Fresh blood from clinically proven peanut-allergic (n=6) and
non-allergic (n=3) children were obtained from The Hospital for Sick
Children, Toronto, Canada. Samples were immediately stimulated with
0.01-100 ng/ml peanut extract for 3 or 15min. Phorbol 12-myristate
13-acetate-Ionomycin (PMA/Iono) and FcεRI-crosslinker (anti-FcεRI) were
included as positive controls. Barcoded samples were pooled and stained
with a panel of surface markers and anti-phosphorylation antibodies for
p38, ERK, mTOR and Akt. Samples were acquired on a CyTOF Helios2.
Relative basophil abundance did not differ between allergic and
non-allergic individuals (Figure S1A). Moreover, there were no
differences in expression of IgG receptors (CD16, CD32, CD64), IgE
receptors (FcεRI, CD23) and other granulocyte markers (CCR3, CRTh2, IgE,
CD123, Siglec8) (Figure S1B). The %CD63+ expression
increased exclusively on basophils from peanut-allergic patients in
response to peanut allergen stimulation, in a dose-dependent manner
(Figure S2A-E).
Protein phosphorylation of Akt, MAP-kinases (ERK1/2, p38), and the mTOR
pathway play crucial roles for the synthesis and secretion of
cytokines,3 especially in the context of
allergy.4 Phosphorylation of Akt, ERK1/2, p38 and mTOR
were monitored at 3 and 15min after stimulation to measure basophil
activation downstream of the FcεRI receptor. Allergen-induced
phosphorylation of ERK and p38 occurred in a dose-dependent manner at
3min, whereas phosphorylation of Akt and mTOR significantly increased at
15min (Figure 1A-B). Phosphorylation was significantly lower to absent
in the non-allergic patients (Figure 1A-B). Phosphorylation status was
subsequently assessed in both a successfully poly-desensitized
pistachio-cashew-allergic (Figure S3A) and walnut-peanut-allergic
(Figure S3B) OIT patient. From baseline, there was an initial increase
in allergen-induced ERK phosphorylation in basophils at the end of OIT
up-dosing. This change was followed by a partial return to baseline
phosphorylation at the end of OIT, which may reflect the changes in
basophil reactivity reported during OIT (Figure S3A-B).
To expand on existing knowledge in the basophil compartment, we
developed a bioinformatic approach that allows changes to be monitored
within the basophil subsets by complex normalization, pooling and
clustering using the Leiden algorithm (Figure 2A-D).5During stimulation, shifts in the proportion of stimulated basophils in
subsets, as well as increased pERK and pmTOR marker expression (Figure
2E-F), were found and confirmed via gating.
In conclusion, we developed a whole-blood-based mass cytometry approach
to assess basophil activation pathways that matched clinical reactivity
and created an approach to investigate changes in basophil subsets
during interventions for food allergy.