Basophil Activating Factors in the Serum May Underlie the ‘Nonreleaser’ Basophil Status in the Basophil Activation Test

Abstract

Basophil activation test (BAT) has been increasingly used in allergy diagnosis. For certain food allergies, such as nut allergy, BAT has demonstrated high specificity and sensitivity in distinguishing allergic and tolerant subjects, which helps reduce the need for oral food challenges [1]. However, it has been reported that 10%–20% of subjects display ‘nonreleaser’ basophils due to spleen tyrosine kinase (Syk) deficiency [2, 3]. Although one study showed that ‘nonreleasers’ had a reduction in the incidence of allergic rhinitis [3], the status of ‘nonreleaser’ can change over time [4], and therefore, the clinical implications remain unknown.

The underlying cause of a ‘nonreleaser’ state remains unclear. The nonreleaser state is characterised as no response to IgE-mediated stimulation, via anti-FcεRI or anti-IgE engagement. In contrast, degranulation mediated by IgE-independent mechanisms remains intact, such as stimulation via G-protein–coupled fMLP receptors using fMLP [5]. The nonreleaser state is not a result of a genetic defect, as Syk is expressed normally in other granulocytes, such as eosinophils and neutrophils, and B cells, suggesting a separate regulation in basophils via mechanisms that remain largely unknown [4]. Although some researchers have speculated that it could be a protective mechanism for some individuals [3], a better explanation is needed as to why only basophils seem to be affected.

We hypothesise that a basophil-specific self-activating mechanism from the patient's own serum might be responsible for the observed ‘nonreleaser’ state. Loss of Syk in human basophils through IgE or non–IgE-dependent stimulation was reported previously [6, 7]. However, a clear link between nonreleaser basophils and basophil-specific self-activation mechanism was not established.

To investigate this hypothesis, we identified subjects most likely to carry basophils with impaired IgE-mediated degranulation function. To do this, we performed BAT using whole blood of subjects sensitised to at least one allergen (n = 30) and were not taking oral corticosteroids (Path 1 of Figure 1A). In parallel, subjects' sera were frozen, and batch tested using the progenitor cell derived basophils (PCB) [8]. This includes progenitor cell derived basophil activation test (PCBAT, Path 2 of Figure 1A) and serum-induced PCB activation test (Paths 3–4 of Figure 1A). Informed consent was obtained for all subjects (Rec reference: 20/NW/0302). Both BAT and PCBAT used 1 μg/mL anti-IgE as stimulant, and the percentage of degranulation was measured via CD63 expression compared with unstimulated control using flow cytometry.

While BAT results combine subject-dependent humoral and cellular factors, PCBAT only reflects the subjects' humoral responses via passive sensitisation of donor basophils using subjects' own sera (Figure 1A). Therefore, we propose that the ratio between the two assays' results could give a quantitative indication of the subjects' blood basophil cellular function. For example, a ratio of ‘1’ indicates equal degranulation capacity by blood basophils and progenitor-derived basophils (PCB), whereas a ratio of ‘0.2’ signifies that IgE-mediated degranulation by the subjects own blood basophils is five times less effective compared with PCB. This approach also helps exclude low responders resulting from low serum IgE levels, as they would display reduced responses in both BAT and PCBAT.

Three subjects from our cohort were characterised as BAT ‘nonreleaser’, as no degranulation could be observed after stimulation with anti-IgE in BAT(1.72%, 1.7% and 1.63% of CD63⁺ cells), but much stronger degranulation was seen using PCBAT(36.95%, 49.8% and 44.1%, respectively) [8]. These three subjects have the lowest BAT/PCBAT ratio of the entire cohort (ratio ≤0.05). The prevalence of nonreleaser (10%) is also in line with previously reported frequencies of nonreleaser basophils observed in BAT [3].

Since all the subjects were atopic, we speculated that IgE sensitisation might be important for interacting with the self-activating factors in the serum for basophil degranulation. Hence, we used PCB to assess the ‘self-activating’ property of each serum by stimulating PCB with 20% subjects' sera in two separate conditions: (i) PCB sensitised with recombinant IgE (rIgE 0.5 μg/mL) overnight and (ii) PCB without overnight rIgE sensitisation.

Some subjects could only degranulate under one of the experimental conditions (Figure 1B,C), suggesting different causative mechanisms for basophil activation. When selecting the maximum degranulation value from both IgE-sensitised and nonsensitised conditions and plotting against the calculated BAT/PCBAT ratio, we observed a stronger inverse association between the two parameters (Figure 1D, r = −0.65, p < 0.001). Sera from the identified three BAT nonreleasers could all induce PCB degranulation, but under three separate experimental conditions: serum from one subject induced PCB degranulation was found more potent in an rIgE-sensitised condition (rIgE 16.51% vs. no rIgE 2.42%), whereas another subject showed more potent degranulation in an rIgE nonsensitised condition (rIgE 4.57% vs. no rIgE 13.01%) and one subject degranulated in both conditions (rIgE 6.96% vs. no rIgE 8.65%; Figure 1B–D).

Our model bears a resemblance to BAT for chronic spontaneous urticaria (CSU-BAT), where basophils from healthy donors are stimulated with sera of patients with CSU (1:1 serum dilution), and positive basophil degranulation hints underlying type-IIb CSU. Furthermore, using atopic basophil donors in CSU-BAT generates stronger degranulation compared with nonatopic individuals [9], suggesting that IgE priming has a crucial role in self-reactive immune responses. Unfortunately, a history of CSU or autoimmunity from the tested subjects in our cohort is currently lacking.

In conclusion, we found a strong link between serum-associated self-reactive responses and the basophil ‘low/nonreleaser’ state in BAT. Although the mechanisms to induce basophil self-activation are heterogeneous and not yet well-defined, atopic subjects with low/no response in BAT should be further investigated for the presence of suspected circulating self-activating factors for basophil activation. The clinical implications of this finding are, however, yet to be determined.

J.W., A.S. and C.S.M. designed the study and wrote the manuscript. J.W., S.B.P. and C.T. designed experimental protocols. J.W. performed the experiments. R.W. performed subject selection and blood collection. J.W. and R.W. conducted subsequent data analysis and discussion. All authors contributed to revising the manuscript.

The authors declare no conflicts of interest.