Exploratory analysis of CD63 and CD203c expression in basophils from hazelnut sensitized and allergic individuals
Our results show that the BAT is a useful tool to determine biological activity against a food allergen such as HN. The correlation between CD203c/CD63 expression and the sensitization results measured by sIgE and SPT indicates basophil activity can be linked to sensitization status. Moreover, the CD203c-based BAT may distinguish between sensitized and symptomatic individuals but only if a careful dose–response analysis is performed.
The literature is controversial about the advantages and disadvantages of basophil surface markers [20]. CD63, compared to CD203c, is not a basophil-specific marker and has been shown to be less specific and less sensitive (in grass pollen and house dust mite allergy, latex allergy, wasp venom hypersensitivity, peanut allergy) [21–23]. On the other hand, the up-regulation of CD63 expression has been reported to provide similar results to CD203c expression in cat-allergic subjects [24] and to be more sensitive and specific in egg-allergic children [23]. All these data suggest a high variability of sensitivity and specificity depending on the allergen and cohort that was studied.
In this study, measuring CD203c expression was superior to CD63 when sensitized and symptomatic groups were considered (Fig. 2c, d; Table 2). However, with the BAT, we were not able to distinguish a patient with systemic reactions from a patient with OAS. The ability to differentiate between the symptom severities was limited either by the diagnostic assessment, which was based on clinical history, or by the Cor-a-1-based HN allergy in our cohort. It is known that Cor a 1 can induce severe allergic symptoms but may require the intake of larger amounts of the Bet v 1 homologues such as it is the case for Gly m 4 in soy-containing dietary food products, or it may need allergen-protective matrix effects [12]. The biological activity of Cor a 1 might be very high in birch pollen endemic regions, [25] thus, the difference between OAS and more severe allergic reactions are not detected in the BAT.
CD203c as well as CD63 expression values at a HN concentration of 0.01 µg/ml correlated with the SPT and sIgE results (Table 3). Considering CD-sens, a correlation was detected for CD203c but not CD63. Thus, the BAT can be used as a functional assay to detect sensitization status, which had been shown previously [26]. However, in our hands, CD203c seems to be superior compared to CD63. Moreover, the BAT has the potential to more closely resemble the clinical phenotype of patients [27]. In previous studies, we have shown the usefulness of CD203c expression in demonstrating differences of the allergenicity between native and roasted HN extracts [2] and between two different tomato cultivars [18]. Previous reports, where CD63 and CD203c expression were analyzed in combination, support our findings [28–30]. The data of Wanich et al. [28] indicate less basophil activity in tolerant versus symptomatic cow’s milk-allergic subjects. The BAT could also differentiate between tolerant and symptomatic peanut-sensitized subjects [30]. Santos et al. [29] demonstrated the utility of basophils as biomarkers for severity and threshold of allergic reaction in a pediatric cohort suffering from peanut allergy. Whether a combined analysis is suitable is a matter of discussion due to different expression kinetics [16, 31].
Differences regarding kinetics might be a reason for the findings when comparing CD63 and CD203c in our cohort. The maximal up-regulation of CD63 occurs within 25–30 min, whereas CD203c requires only 10–20 min [32]. The stimulation time used in this study averaged 15 min to capture both markers.
For practical reasons, basophil activation experiments should be restricted to a single allergen concentration [16, 32]. However, an individual, highly heterogeneous basophil response has been described previously [33] and was also present in our cohort. Thus, a single allergen concentration is not sufficient to analyze basophil responses.
The BAT offers many advantages in food allergy. In contrast to sIgE, the BAT provides a biological readout [34]. A wide range of food, raw material, purified or recombinant allergens can be analyzed [35]. Unspecific positive reactions are less frequent in BAT compared to SPT. In addition, SPT bears the risk of sensitization [36]. Moreover, basophils can be analyzed even when the subject receives anti-allergic treatment [37]. However, other studies demonstrated an inhibitory effect of immunosuppressants such as cyclosporin A [38] or other drugs (statins) [39]. Therefore, a careful history regarding a possible drug intake is mandatory if a BAT is considered.
The diagnosis of food allergy is mainly based on patient history, analysis of IgE and/or SPT, ideally combined with DBPCFC, which is still the gold standard in the diagnosis of food allergy [11, 12]. However, DBPCFC is expensive and time-consuming for both the physician and the patient and is often refused by the patient. Additionally, there is a risk for patients to experience severe allergic reactions. Thus, in this study, a detailed case history including allergy-focused diet history assessment was used to define the status of HN allergy, which had previously been shown to have a high diagnostic values [40, 41].
However, an ex vivo but highly diagnostic method to reliably predict clinical reactivity is desirable. Neither the presence of sIgE in the circulation nor the presence of biologically active IgE on mast cells are suitable to differentiate between sensitization and clinical allergy [35], with perhaps the exception of Cor a 9 and Cor a 14 in children cohorts [41, 42]. For both allergens, an age-dependent variability seems to exist, as allergic adults are less sensitized to both components [43]. In our cohort, no sensitization to Cor a 9, Cor a 11 or Cor a 14 was found. Thus, in our cohort Cor a 1 is the predominate allergen. The sIgE to Cor a 1 was lower in the sensitized group compared to the symptomatic groups (significant for OAS group), which was more pronounced when calculated as a sIgE/total-IgE-ratio. Glaumann et al. [19] investigated the utility of BAT in an Ara h 8-sensitized cohort. Ara h 8 is a Bet v 1 homologue in peanut like Cor a 1 in HN. They could show that the BAT adds safety information if sIgE and DBPCFC results were controversial. Thus, the BAT might be useful if clinical history, SPT and/or sIgE measurement are inconsistent or in addition an oral food challenge is not possible [42]. Furthermore, the use of recombinant allergens in the BAT might have added value in discriminating between clinically relevant and mere sensitization [44, 45] and should be considered in future studies.
Most BAT protocols are not standardized. For the future, protocol optimization is required and should consider preanalytical conditions and well-defined flow cytometry gating protocols [46]. The timeframe of analysis is relevant as well; ideally, the BAT should be performed immediately after a blood draw [37, 46] but at least within 24 h, as longer storage time can lead to a loss of basophil reactivity and false negative results [37].
A weakness of this study is the small number of analyzed subjects and the results should be regarded explorative. However, we were able to differentiate between non-allergic, sensitized and symptomatic subjects. The inclusion of HN-sensitized subjects is a strength of the study, as such a group is essential to validate a diagnostic test in allergy [33]. Still, due to the small sample size, necessary diagnostic parameters (sensitivity, specificity, positive and negative predictive value) were not calculated. Furthermore, the diagnosis of a symptomatic HN allergy was based on clinical history, as mentioned above. Moreover, we did not prove that the activation of the basophils with the HN extract was exclusively via cross-linking surface-bound sIgE against HN (Cor a 1) or via cross-reaction of surface-bound sIgE against birch homologues (Bet v 1).
The sensitivity and specificity of the BAT is influenced by the rate of non-responders. These patients do not show activation after anti-IgE stimulation [33]. The reason for this non-responsiveness of the basophils is a selective decrease in Syk expression, which is a downstream event of Fc?RI activation [33]. The proportion of non-responders depends on the BAT protocol and varies between 5 and 25% [32, 33]. In this study, 3.25% of patients when considering both expression markers and up to 6.5% of patients when considering only CD63 expression were non-responders.