Discussion

We have developed a high-throughput progenitor cell derived basophil activation test (PCBAT), which was a better predictor of clinical reactivity to cat and peanut allergen (as measured on challenge testing) than conventional markers of allergy such as SPT or allergen specific IgE. PCBAT can be used with stored serum, removing the need for immediate access to expensive flow cytometry facilities not generally available in the clinic. By passively sensitising basophils with sera from our well characterised patient populations then culturing with the relevant allergen, we demonstrated dose dependent and allergen specific basophil activation with wide variability in trajectories. Importantly, PCBAT was negative amongst those reporting oral tolerance to peanut but with detectable specific IgE to peanut components.

Technical aspects of the PCBAT

The combined use of flow cytometry and immunostaining suggest the optimal window for maturation for this culture protocol was between day 16 and 21, similar to previous reports. (23), and reflecting the 5 days basophil lifespan in vivo (24). Our culture was highly enriched for mature basophils (25-50%), enabling high-throughput barcoding, improving efficiency.

PCB responsiveness and sIgE levels

The responsiveness of PCBAT was allergen specific showing a dose-dependent response with good association with levels of corresponding sIgE. In addition, patients receiving omalizumab treatment showed completely muted responsiveness in the PCBAT, in accordance with a previous study (25).
However, one subject, with low but positive sIgE to cat (0.5KU/L) did not show responsiveness in PCBAT; this patient was not on omalizumab treatment. As this subject had not undergone allergen challenge, it remains unclear whether this subject was sensitized but tolerant to cat. One non-sensitized control subject showed minor degranulation at the highest concentration of cat allergen used. We have confirmed that this subject had a positive dog sIgE (5KU/L); we speculate that this weak response to cat allergen might reflect cross-reactivity between cat allergen and dog sIgE, which has been previously reported (26-28). We also found quantifiable traces of Can f 1 in the cat allergen extracts used which may provide an alternative explanation (Figure E10).

The clinical relevance of PCBAT

We investigated the clinical relevance of PCBAT using two cohorts of patients who had undergone challenge testing—to inhaled cat allergen or to peanuts.
Although a significant association was observed between PCBAT AUC and inhalant challenge results, two subjects showed negative results in the PCBAT but positive in the inhalant challenge. Interestingly, these two subjects exhibited the smallest reaction in the skin test to cat at the time of challenge (2x2mm) and had significantly greater reactivity to other allergen including dust mite and grass (Table E4). In addition, for some of these subjects, the blood samples were collected up to 2 years after the inhaled allergen challenge, so a change in clinical reactivity during this time remains a possible explanation
We explored PCBAT in peanut allergy, as an exemplar of an allergic disease where clinical reactivity is not reliably predicted by serum sIgE, but where oral food challenge tests are used to confirm reactivity and identify thresholds of responsiveness. All 30 patients with physician diagnosed peanut allergy showed a positive PCBAT, and this correlated significantly with serum sIgE to whole peanut extract and to Ara h 1, 2 and 3. As half of the subjects had previously undergone oral food challenge to peanut, we were able to compare the dose of peanut at which the subject showed an objective reaction with the reactivity on the PCBAT (as AUC). We identified that AUC on PCBAT was a better predictor of clinical reactivity on oral food challenge than serum sIgE to whole peanut extract or to Ara h 2 and 3, and was similar to Ara h 1. Importantly we identified a small population with positive sIgE to 1 or more peanut allergen component that reported regularly eating peanuts, and found that none of them showed a positive PCBAT. This suggests that in principle, PCBAT may be a useful test in predicting clinical reactivity to peanut, but further testing on more subjects would be required.