Results

PCBAT Development

Characterization of PCBs maturity

Human primary PCBs were differentiated from CD34+hematopoietic progenitors following 28 days of culture. The maturity of PCBs was demonstrated using four methods - immunophenotyping (Figure1 A-C), immunofluorescence staining (Figure 1D), functional tests (Figure 1E) and morphological study (Figure E3) for PCBs obtained from 2 different donors (donor A in Figure 1 and donor B in Figure E4).
Mature PCBs were defined as 2D7+/FcεRI+/CD117-/HLADR-cells. The proportion of cells expressing the basophil marker 2D7 increased from day 7 (32.8%) peaked at day 16 (42.8%) and fell to 22.1% by day 28 (Figure 1A). When the 2D7+ cells were gated for FcεRI+, the majority were positive, around 80% of the 2D7+ cells were also FcεRI+ through the culture (Figure 1B). Furthermore, as the cells matured, the highest negativity for CD117- and HLADR- was seen at day 16 (Figure 1C). However, by day 28, the cells started to gain CD117 receptor expression, an additional indication that the culture was losing basophilic characteristics.
We performed immunofluorescence staining with another basophil marker, BB1. The BB1 positive cells were faintly visible at day 7, but clearly visible by day 16 and remained visible to day 28 (Figure 1D).
We tested the PCBs’ ability to degranulate upon engagement of the FcεRI by sensitising them with recombinant IgE followed by anti-IgE stimulation. The percentage of basophil activity, as measured by CD63 expression, increased as the cells matured, such that by day 16, 36.5% of cells showed degranulation (Figure 1E). This was repeated with PCBs from 4 other donors and showed a consistent percentage of activation between donors at day 16 (mean ± SEM 41.31% ± 3.34, Figure E5A and E5B). The maturation of PCBs and degranulation capability at each stage was very similar between the two donors (Figure 1, E4 and E5C).
Using this culture method, an average yield (n=9) of 2.27x107 cells (9x106-4.8x107, minimum-maximum) could be achieved at day 16 of culture of which 25-50% were basophils (Figure E6). May-Grünwald Giemsa staining performed on day 16 cells showed heavily granulated cells (Figure E3), a morphology resembling that of blood basophils previously reported (22).
The combined use of immunophenotyping, immunofluorescence, morphological characterization and functional tests suggested that the optimal window for PCBs was between day 16 and day 21. After day 21, cells began to lose basophilic features but still retained a high response to anti-IgE following IgE sensitization (Figures 1A and 1E).

High-throughput PCBAT with fluorescent barcoding

To increase the throughput, we incorporated fluorescent barcoding to the PCBAT. We simplified the gating strategies for selecting PCB population to minimize the interference between fluorescence dyes and the antibody panel. We selected CD203c+/FcεRI+cells for the degranulation assay, as CD203c+ cells were >99% 2D7+ (Figure E6). In addition, amongst CD203c+ cells, only FcεRI+population could degranulate in response to FcεRI crosslinking (Figure E7). In Figure 2 we showed a representative figure for the use of 16-plex fluorescence barcoding in PCBAT.

PCBAT predicts clinical reactivity to cat

Study groups and controls are described in Table I with individual subject PCBAT results and available skin test results are presented in Table E1-E4. All the samples responded to positive control stimulant (anti-IgE) and did not respond to negative control stimulant (medium only).
For adults with asthma sensitised to cat (Group 1, n=18), all but one subject showed a positive response in PCBAT (Figure 3A) with a broad range of trajectories and AUC. There was a significant correlation between AUC in PCBAT and sIgE to cat (Figure 3B p=0.001). Five of the six control subjects with asthma who were not sensitised to cat showed a negative response to cat (Figure 3C); one control subject (control 6) showed a weak positive response at the highest concentration.
Four asthma patients were undergoing treatment with the anti-IgE drug omalizumab, three were cat sensitized (Group 2, Figure 3D). No response was seen to cat extract amongst cat-sensitised asthma patients on omalizumab.
For 17 adults with airway reactivity to cat (Group 3) the range of cumulative dose of inhaled cat allergen required to cause a ≥20% drop in FEV1 was large (>2000-fold, Figure E8A), as were the DRS, and PC20 (Figure E8B-C). The DRS and PC20 were closely correlated (Figure E8D, p=0.0001 R2=0.94). Although specific IgE to cat was not available on these subjects, titrated skin test reactivity to cat showed a range of sensitivity to cat (7 doubling dilutions, Figure E8E). Fifteen subjects showed a positive response on PCBAT, with a broad range of trajectories (Figure 4A). There was no association between skin test reactivity to cat and DRS to cat allergen (Figure E8F, p=0.17). There was a significant correlation between PCBAT AUC and airway reactivity DRS (Figure 4B, p= 0.026) and the lnEARAUC0-2hrs after inhaled allergen challenge (Figure E8G, p=0.038) but not lnLARAUC3-7hrs (Figure E8H, p=0.29). All control subjects (n=6) showed no response in PCBAT (Figure 4C).

PCBAT predicts clinical reactivity to peanut

For adults with physician diagnosed peanut allergy (n=30, Group 4) all subjects showed a positive response on PCBAT which was dose-dependent (Figure 5A). The four negative control subjects did not respond to peanut on PCBAT (Figure 5B). There was a significant correlation between AUC in PCBAT and sIgE to whole peanut and Ara h 1, 2 (Figure 5C-E), 3 and 6 (Figure E9A and D). Serum sIgE to Ara h 8 and 9 were not significantly associated with AUC (Figure E9B and C, p>0.7).
Of the 30 subjects with physician diagnosed peanut allergy (Group 4), 15 had confirmed peanut allergy following double blind placebo control food challenge. There was a significant negative correlation between PCBAT AUC and results of oral food challenge test to peanut - subjects who showed a higher PCBAT AUC reacted to a lower dose on oral food challenge to peanut (Figure 5F p=0.001, R2=0.57). A trend towards a negative correlation was observed between sIgE to whole peanut and oral food challenge result (Figure 5G, p=0.094, R2=0.24). A significant negative correlation was also observed between sIgE to Ara h 1 and oral food challenge result (Figure 5H p=0.007, R2=0.55). There was no significant association between Ara h 2 sIgE and oral food challenge result (Figure 5I, p=0.125, R2=0.2).
Within a population-based birth cohort (Group 5), we identified 13 subjects who were sensitised to peanut (positive to 1 or more allergen component; 5 sensitised to Ara h 1, 2, 3 or 6, Table E6) who reported regular ingestion of peanut (i.e. sensitized but tolerant to peanut, Group 6). None of these 13 subjects showed responsiveness to even the highest concentration of peanut extracts in PCBAT (Figure 5J).