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.