Methods

Study design.

We developed a new high-throughput Basophil Activation Test, using basophils generated from peripheral blood progenitor cells from healthy donors—the PCBAT. To assess the potential clinical utility, serum samples from study participants were used to passively sensitise the basophils, which were then incubated with allergen before assessing basophil activation using flow cytometry. The association between the degree of basophil activation and clinical characteristics of the study participants was then assessed.

Materials

Development of progenitor cells-derived basophil activation test (PCBAT)

Generating PCBs

Peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte cones (NHS Blood and Transplant Centre, Manchester) using Ficoll density gradient centrifugation. CD34+ hematopoietic progenitor cells were isolated by a magnetic bead method according to manufacturer’s instructions (MACS Miltenyl Biotec). Purified CD34+ hematopoietic progenitor cells were diluted to 1x105 cells/ml and cultured in StemspanTM supplemented with 10ng/ml IL-3, 100ng/ml SCF, 50ng/ml IL-6, 5mg/ml human LDL and penicillin/streptomycin (100U/ml). This was day 0 of culture, cell density was then maintained between 2-5 x 105/ml up to day 28 at 37oC with 5% CO2.

PCBs characterization

To monitor the differentiation process, the culture was sampled at day 7, 10, 16, 21 and 28. The cells were characterized using flow cytometry, immunofluorescence and metachromatic staining and by functional assay (PCBAT). This was repeated on two separate donors.

Flow cytometry

Cell staining was performed on a 96-well plate using approximately 5x104 cells/well. For PCB characterization, cells were stained with the following antibodies: CD63 (APC), CD123 (Percp-Cy5.5), CD117 (BV605), CD203c (FITC), HLADR (eFluro450) and FcεRI (PE-Cy7) for 20 minutes at 4oC. Detailed protocol and gating strategy can be found in Figure E1-2.

Immunofluorescence staining

Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% tween and 10% goat serum. Cells were then stained in mouse anti-BB1 antibody (1:10) followed by Alexa Fluor 555 goat anti-mouse secondary antibody (1:200). Slides were mounted with fluoroshield mountant containing DAPI for cell nuclear staining and examined under a Leica DM IL LED microscope using Leica Application Suite software (Leica, UK).

Validation and performance of PCBAT

Study subjects

Five groups with different clinical characteristics were identified and described in Table 1. All subjects provided written informed consent.

Measurement of sensitisation to cat and peanut allergens

Serum sIgE was measured for Groups 1 and 2 to cat and for Group 4 to peanut and peanut components allergens Ara h 1, 2, 3, 8, 9 (Immunocap, ThermoFisher Scientifc, Sweden); sIgE>0.35 kU/L indicated a positive test. For Group 5, serum sIgE to Ara h 1, 2, 3, 6, 8, 9 was measured using ISAC (ThermoFisher Scientifc, Sweden). A positive result was indicated by sIgE>0.3 ISU-E. Serum sIgE to cat was not available for Group 3 and sensitisation was determined by titrated cat allergen SPT (17).

Inhaled cat allergen challenge

Seventeen cat sensitized adults underwent inhaled cat allergen challenge at McMaster University, Ontario. Participants inhaled cat allergen at increasing concentrations until lung function (FEV1) dropped >20% from baseline. To quantify airway responsiveness to cat allergen, we calculated a dose response slope (DRS) and also the PC20 to cat allergen (described in this article’s Online Repository) (18). FEV1 was measured for 7 hours after the last dose of inhaled allergen and recovery was measured as AUC; the early asthmatic response AUC between 0-2 hours post challenge (EARAUC0-2hrs) and the late asthmatic response AUC between 3-7 hours post challenge (LARAUC3-7hrs) (described in this article’s Online Repository).

Oral peanut challenge

Of thirty physician confirmed peanut allergic patients, 15 underwent oral food challenge to peanut as part of iFAAM project (19), (challenge protocol in the article’s Online Repository). Participants ingested increasing quantities of peanut protein until objective signs of an allergic reaction were shown. The cumulative dose of peanut required to show first objective sign was used as a measure of clinical reactivity to peanut allergen.

PCBAT

PCBs were sensitized with either 20% patients’ sera or with human myeloma IgE (1 μg/ml) overnight at 37oC with 5% CO2. Sensitized PCBs were activated by incubating with serial dilutions of extracts (roasted peanut extracts or cat allergen) for 30 minutes at 37oC. Anti-IgE (1μg/ml) and “medium only” was included for every subject as positive and negative controls respectively. PCBs were identified by staining the cells with CD203c+(FITC) and FcεRI+(PE-Cy7). CD63 (PE) was used as a degranulation marker. After cells were stained with antibodies and viability dyes, Fluorescent barcoding (16-plex) was performed using methods previously described (20). Briefly, cells were fixed with 1.6% formaldehyde then permeabilized with methanol containing pacific blue (40, 13.3, 4.43 and 0μg/ml) and Alexafluro 700 (4, 1.33, 0.43 and 0μg/ml). Cells stained with different combinations of pacific blue and Alexafluro 700 were then pooled before flow cytometry analysis. A minimum 5% of CD63 positive cells were required to indicate a positive PCBAT response. To depict the responsiveness of the PCBAT, we present results as AUC for CD63 expression at increasing allergen concentrations; results for sensitivity (EC50 and CDsens) and reactivity (CDmax) of the PCBAT are presented in Table E1-E5.

Statistics

Demographic variables were presented as means and standard deviation. The AUC was calculated using the trapezoidal rule on logarithmically transformed allergen concentrations to quantify the responsiveness of a degranulation assay (21), as previously described. Methods for EC50, CDsens and CDmax calculation is described in this articles’ Online Repository. Correlation coefficients were calculated by using the Spearman R test in (SPSS v22, IBM, Armonk, USA). A 2-sided P value ≤ 0.05 was considered statistically significant.