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.