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\begin{document}

\title{Progenitor cell-derived basophils: a novel barcoded passive
degranulation assay in allergy diagnosis}



\author[1]{Jiakai Wu}%
\author[1]{Rajia Bahri}%
\author[2]{Marina Tsoumani}%
\author[1]{Aida Semic-Jusufagic}%
\author[1]{Clare Murray}%
\author[3]{A Custovic}%
\author[1]{George Guibas}%
\author[1]{Miriam Bennett}%
\author[4]{Gail Gauvreau}%
\author[4]{Ruth Cusack}%
\author[1]{Clare Mills}%
\author[1]{Silvia Bulfone-Paus}%
\author[1]{Angela Simpson}%
\affil[1]{The University of Manchester}%
\affil[2]{University of Manchester}%
\affil[3]{Imperial College London}%
\affil[4]{McMaster University}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Effector cells assays provide an overall measure of
responsiveness to allergen, but the lack of reliable, high-throughput
assays limits the clinical utility of this approach. The aim of this
study was to develop a high-throughput Basophil Activation Test (BAT),
based on human progenitor cell-derived basophils (PCB), and to
investigate the role of PCB activation test (PCBAT) in allergy
diagnosis. Methods: PCBs were differentiated from CD34+ progenitor
cells, and sensitized with sera from subjects sensitized to cat (n=35,
17 subjects clinical reactivity validated), peanut-allergic (n=30, 15
subjects clinical reactivity validated), peanut-sensitized but tolerant
subjects (n=13). Sensitized PCBs were then stimulated with a range of
concentrations of the corresponding allergens and degranulation was
measured using CD63 expression on flow cytometry. Results: Following
passive sensitisation of the mature PCB (2D7+/Fc\selectlanguage{greek}ε\selectlanguage{english}RI+/CD117-/HLADR-) with
serum and stimulation with allergen, we saw a dose-dependent increase in
CD63 expression which was allergen specific. In subjects sensititsed to
cat there was a positive correlation between PCBAT area under curve
(AUC) versus specific IgE (sIgE) to cat (p=0.001) and versus airway
responsiveness to inhaled cat allergen (p=0.026). There was a
significant negative correlation between PCBAT AUC for peanut allergen
and response to oral food challenge test to peanut - subjects with
higher PCBAT AUC reacted to a lower dose on the oral food challenge to
peanut (p=0.001), and had higher sIgE to Ara h 1 (p=0.007). All peanut
tolerant subjects showed no reaction to peanut on PCBAT. Conclusion:
PCBAT may confer a powerful alternative tool in allergy testing.%
\end{abstract}%



\sloppy


\section*{Keywords: basophil, allergy diagnosis, challenge tests, food
allergy, asthma, flow
cytometry}

{\label{keywords-basophil-allergy-diagnosis-challenge-tests-food-allergy-asthma-flow-cytometry}}

\section*{Introduction}

{\label{introduction}}

Sensitisation to inhalant allergens such as house dust mites, cats and
dogs, is commonly associated with asthma, but is neither necessary nor
sufficient for disease expression. Similarly, a positive skin prick test
(SPT) or IgE test to a food does not equate to clinical food allergy,
and false positive results are common. Challenge testing (either oral
food challenge or inhaled allergen challenge) can be offered to
patients, but is time consuming, carries the risk of severe reaction and
is not suitable for all patients. Therefore, tests with superior
diagnostic accuracy than IgE that are safe to conduct in all patients
would be of value in clinical practice, especially amongst patients
sensitised to many allergens.

Basophils and mast cells (MC) are the two primary effector cells in
allergic responses (1). Cellular degranulation triggers the release of
preformed and newly synthesized mediators inducing a potent biological
response in a sensitized person following allergen exposure (2). While
basophils are found in the circulation, MC are localized in peripheral
tissues. The two cell types may have different roles in an allergic
response but this is currently poorly understood (2, 3).

The more accessible circulating basophils have been used as cell models
for studying allergy (4). Basophils account for \textless{}1\% of blood
leukocytes however, making purification a challenge. To obviate the need
for purification, the basophil activation test (BAT) was developed using
immediately analysed fresh whole blood (4, 5). Following stimulation of
whole blood with allergen (or control), the responsiveness of the
basophils can be quantified using fluorochrome-coupled antibody markers
of basophil activation (e.g. CD63 and CD203c) by flow cytometry. The
advantage of the BAT is that it takes account of many factors which
influence basophil responsiveness to an allergen such as IgG4/IgE ratio
(6, 7), heterogeneity of sIgE to allergen components (8), medication (9)
and innate responsiveness of the cells (10). The disadvantages are that
blood needs to be analysed immediately after being drawn (5), requiring
the allergy clinic to have instant access to a staffed flow cytometry
facility. In addition, 10-20\% of people carry ``non-releaser''
basophils, which are non-responsive in the BAT, despite having clinical
allergy (11). Consequently, this test is not generally available for
clinical diagnostics, but used only in specialist laboratories for
hymenoptera venom and drug allergy testing.

The passive BAT, which uses basophils from a donor that are passively
sensitized with the serum from the patient, was developed as an
alternative method that circumvents some of these problems (12). Stored
serum samples from subjects can be analysed in batches, providing
greater flexibility and allowing humoral factors to be investigated
separately from cellular factors (7). However, the donors' basophils
must be stripped of endogenous IgE with a mild acid treatment before the
cells can be passively sensitized with patient serum samples, which can
damage the donor basophils and lead to auto-basophil activation (13) and
reduced sensitivity (14). Due to these limitations, passive BAT has only
been used in a few studies. Although the passively sensitized approach
has also been used on basophilic cell lines such as RBL-2H3, there are a
number of disadvantages, including the gradual loss of cell
responsiveness within weeks of cultures (15).

Recently, Bahri et al developed a robust and reproducible effector cell
assay based on human progenitor cell derived MC, the mast cell
activation test (MAT). This assay appeared to confer superior diagnostic
accuracy in distinguishing peanut allergic from peanut tolerant subjects
compared with existing diagnostics such as sIgE (to whole peanut or Ara
h 2), SPT and BAT (16). In this study we use a similar approach to
generate functional progenitor cell-derived basophils (PCB) and provide
detailed characterization of basophil differentiation, and demonstrate
the functionality and reproducibility of this technique. We then explore
the potential clinical application of progenitor cell basophil
activation test (PCBAT) by passively sensitizing the cells with
sera/plasma from five groups of patients with allergic asthma and food
allergy and testing degranulation to two allergens (cat and peanut).

\section*{Methods}

{\label{methods}}

\subsection*{Study design.}

{\label{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.

\subsection*{Materials}

{\label{materials}}

\subsection*{Development of progenitor cells-derived basophil activation
test
(PCBAT)}

{\label{development-of-progenitor-cells-derived-basophil-activation-test-pcbat}}

\subsubsection*{Generating PCBs}

{\label{generating-pcbs}}

Peripheral blood mononuclear cells (PBMCs) were isolated from leukocyte
cones (NHS Blood and Transplant Centre, Manchester) using Ficoll density
gradient centrifugation. CD34\textsuperscript{+} hematopoietic
progenitor cells were isolated by a magnetic bead method according to
manufacturer's instructions (MACS Miltenyl Biotec). Purified
CD34\textsuperscript{+} hematopoietic progenitor cells were diluted to
1x10\textsuperscript{5} cells/ml and cultured in
Stemspan\textsuperscript{TM} 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 10\textsuperscript{5}/ml up to day 28 at
37\textsuperscript{o}C with 5\% CO\textsubscript{2}.

\subsubsection*{PCBs characterization}

{\label{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.

\subsubsection*{Flow cytometry}

{\label{flow-cytometry}}

Cell staining was performed on a 96-well plate using approximately
5x10\textsuperscript{4} 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 Fc\selectlanguage{greek}ε\selectlanguage{english}RI (PE-Cy7) for
20 minutes at 4\textsuperscript{o}C. Detailed protocol and gating
strategy can be found in Figure E1-2.

\subsubsection*{Immunofluorescence
staining}

{\label{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).

\subsection*{Validation and performance of
PCBAT}

{\label{validation-and-performance-of-pcbat}}

\subsubsection*{Study subjects}

{\label{study-subjects}}

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

\subsubsection*{Measurement of sensitisation to cat and peanut
allergens}

{\label{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\textgreater{}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\textgreater{}0.3 ISU-E. Serum sIgE to cat was not
available for Group 3 and sensitisation was determined by titrated cat
allergen SPT (17).

\subsubsection*{Inhaled cat allergen
challenge}

{\label{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 (FEV\textsubscript{1})
dropped \textgreater{}20\% from baseline. To quantify airway
responsiveness to cat allergen, we calculated a dose response slope
(DRS) and also the PC\textsubscript{20} to cat allergen (described in
this article's Online Repository) (18). FEV\textsubscript{1} 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 (EARAUC\textsubscript{0-2hrs}) and the late
asthmatic response AUC between 3-7 hours post challenge
(LARAUC\textsubscript{3-7hrs}) (described in this article's Online
Repository).

\subsubsection*{Oral peanut challenge}

{\label{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.

\subsection*{PCBAT}

{\label{pcbat}}

PCBs were sensitized with either 20\% patients' sera or with human
myeloma IgE (1 \selectlanguage{greek}μ\selectlanguage{english}g/ml) overnight at 37\textsuperscript{o}C with 5\%
CO\textsubscript{2}. Sensitized PCBs were activated by incubating with
serial dilutions of extracts (roasted peanut extracts or cat allergen)
for 30 minutes at 37\textsuperscript{o}C. Anti-IgE (1\selectlanguage{greek}μ\selectlanguage{english}g/ml) and ``medium
only'' was included for every subject as positive and negative controls
respectively. PCBs were identified by staining the cells with
CD203c\textsuperscript{+}(FITC) and Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+}(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 0\selectlanguage{greek}μ\selectlanguage{english}g/ml) and Alexafluro 700
(4, 1.33, 0.43 and 0\selectlanguage{greek}μ\selectlanguage{english}g/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.

\subsection*{Statistics}

{\label{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.

\section*{Results}

{\label{results}}

\subsection*{PCBAT Development}

{\label{pcbat-development}}

\subsubsection*{Characterization of PCBs
maturity}

{\label{characterization-of-pcbs-maturity}}

Human primary PCBs were differentiated from
CD34\textsuperscript{+}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\textsuperscript{+}/Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+}/CD117\textsuperscript{-}/HLADR\textsuperscript{-}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\textsuperscript{+} cells were gated for
Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+}, the majority were positive, around 80\% of the
2D7\textsuperscript{+} cells were also Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+} through
the culture (Figure 1B). Furthermore, as the cells matured, the highest
negativity for CD117\textsuperscript{-} and HLADR\textsuperscript{-} 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\textbf{.}

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 Fc\selectlanguage{greek}ε\selectlanguage{english}RI
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 \selectlanguage{ngerman}± 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
2.27x10\textsuperscript{7} cells
(9x10\textsuperscript{6}-4.8x10\textsuperscript{7}, 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).

\subsubsection*{High-throughput PCBAT with fluorescent
barcoding}

{\label{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\textsuperscript{+/}Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+}cells for the
degranulation assay, as CD203c\textsuperscript{+} cells were
\textgreater{}99\% 2D7\textsuperscript{+} (Figure E6). In addition,
amongst CD203c\textsuperscript{+} cells, only
Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+}population could degranulate in response to
Fc\selectlanguage{greek}ε\selectlanguage{english}RI crosslinking (Figure E7). In Figure 2 we showed a representative
figure for the use of 16-plex fluorescence barcoding in PCBAT.

\subsection*{PCBAT predicts clinical reactivity to
cat}

{\label{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 FEV\textsubscript{1} was large (\textgreater{}2000-fold, Figure E8A),
as were the DRS, and PC\textsubscript{20} (Figure E8B-C). The DRS and
PC\textsubscript{20} were closely correlated (Figure E8D, p=0.0001
R\textsuperscript{2}=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 lnEARAUC\textsubscript{0-2hrs} after
inhaled allergen challenge (Figure E8G, p=0.038) but not
lnLARAUC\textsubscript{3-7hrs} (Figure E8H, p=0.29). All control
subjects (n=6) showed no response in PCBAT (Figure 4C).

\subsection*{PCBAT predicts clinical reactivity to
peanut}

{\label{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\textgreater{}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, R\textsuperscript{2}=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,
R\textsuperscript{2}=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, R\textsuperscript{2}=0.55). There was no significant
association between Ara h 2 sIgE and oral food challenge result (Figure
5I, p=0.125, R\textsuperscript{2}=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).

\section*{Discussion}

{\label{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.

\subsection*{Technical aspects of the
PCBAT}

{\label{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 \emph{in vivo} (24). Our culture was highly enriched
for mature basophils (25-50\%), enabling high-throughput barcoding,
improving efficiency.

\subsection*{PCB responsiveness and sIgE
levels}

{\label{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).

\subsection*{The clinical relevance of
PCBAT}

{\label{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.

\section*{Conclusion}

{\label{conclusion}}

By generating progenitor-cell derived basophils in high-yield we have
developed a flow cytometry-based basophil activation test for use with
stored serum which can be used to assess reactivity to both food and
inhalant allergens. By incorporating fluorescent barcoding, we have
increased the throughput of the assay, reducing costs significantly. We
identified wide variability in trajectories of response to allergen in
different subjects, and responses were muted in the presence of the
anti-IgE treatment omalizumab, indicating that this test better reflects
the overall immune milieu rather than just specific IgE. For subjects
who had undergone oral food challenge to peanut, we were able to use
results of PCBAT to predict clinical reactivity to peanut. The
correlation of PCBAT to clinical reactivity to inhaled cat allergen may
represent a safe and robust way of identifying those asthmatics who
might benefit from interventions for ongoing cat allergen exposure.
Although further evaluation is required, this proof of concept study
indicates that this test may have a role in food and inhalant allergy
testing as a means of identifying clinically important sensitisations.

\textbf{Table I} Demographic description of the study groups.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllllll@{}}
\toprule
& & & Sensitized subjects & Sensitized subjects & Sensitized subjects &
Sensitized subjects & Non-sensitized control subjects & Non-sensitized
control subjects & Non-sensitized control subjects & Non-sensitized
control subjects\tabularnewline
\midrule
\endhead
Group number & allergen used in PCBAT & Study group description & n &
Age (years, Mean, \selectlanguage{ngerman}±SD) & gender (\% male) & sIgE (KU/L, Mean, \selectlanguage{ngerman}±SD) & n &
Age (years, Mean, \selectlanguage{ngerman}±SD) & gender (\% male) & sIgE (KU/L)\tabularnewline
1 & Cat & Sensitised to cat & 18* & 41.2 (15.2) & 72.2\% & 12.10 (24.52)
& 6* & 61.0 (21.3) & 33.3\% & \textless{}0.4\tabularnewline
2 & Cat & Asthma, on Omalizumab, sensitised to inhalant allergens & 4* &
45.9 (14.7) & 100\% & 36.05 (47.52) & N/A & N/A & N/A &
N/A\tabularnewline
3 & Cat & Clinical reactivity quantified with inhaled allergen challenge
to cat & 17** & 38.1(15.6) & 52.9\% & N/A & 6* & 49.5 (9.9) & 33.3\% &
\textless{}0.4\tabularnewline
4 & Peanut & Doctor diagnosed peanut allergy (clinical reactivity
quantified using oral food challenge for 15 subjects, see OLS for
details) & 30* & 24.5 (7.3) & 46.7\% & 36.99 (38.60) & 4* & 44.8 (5.1) &
50\% & \textless{}0.4\tabularnewline
5 & Peanut & Sensitisation to 1 or more peanut allergen components, but
self-reported ingestion indicating oral tolerance & 13*** & 15.4 (0.9) &
69\% & N/A & N/A & N/A & N/A & N/A\tabularnewline
\bottomrule
\end{longtable}

\emph{* Patients recruited from ManARTS biobank (Rec reference:
15/NW/0409)}

\emph{** From McMaster University, Ontario, Canada}

\emph{*** From the population birth cohort---Manchester Asthma and
Allergy Study (MAAS, registration: ICRCTN72673620) detailed elsewhere
(29)}

\section*{References}

{\label{references}}

1. He SH, Zhang HY, Zeng XN, Chen D, Yang PC. Mast cells and basophils
are essential for allergies: mechanisms of allergic inflammation and a
proposed procedure for diagnosis. Acta pharmacologica Sinica.
2013;34(10):1270-83.

2. Kumar S, Verma AK, Das M, Dwivedi PD. Molecular mechanisms of IgE
mediated food allergy. Int Immunopharmacol. 2012;13(4):432-9.

3. Amin K. The role of mast cells in allergic inflammation. Resp Med.
2012;106(1):9-14.

4. MacGlashan DW, Jr. Basophil activation testing. The Journal of
allergy and clinical immunology. 2013;132(4):777-87.

5. Santos AF, Lack G. Basophil activation test: food challenge in a test
tube or specialist research tool? Clinical and translational allergy.
2016;6:10.

6. Okamoto S, Taniuchi S, Sudo K, Hatano Y, Nakano K, Shimo T, et al.
Predictive value of IgE/IgG4 antibody ratio in children with egg
allergy. Allergy, asthma, and clinical immunology : official journal of
the Canadian Society of Allergy and Clinical Immunology. 2012;8(1):9.

7. Santos AF, James LK, Bahnson HT, Shamji MH, Couto-Francisco NC, Islam
S, et al. IgG4 inhibits peanut-induced basophil and mast cell activation
in peanut-tolerant children sensitized to peanut major allergens. The
Journal of allergy and clinical immunology. 2015;135(5):1249-56.

8. Christensen LH, Holm J, Lund G, Riise E, Lund K. Several distinct
properties of the IgE repertoire determine effector cell degranulation
in response to allergen challenge. The Journal of allergy and clinical
immunology. 2008;122(2):298-304.

9. Hill DA, Siracusa MC, Ruymann KR, Tait Wojno ED, Artis D, Spergel JM.
Omalizumab therapy is associated with reduced circulating basophil
populations in asthmatic children. Allergy. 2014;69(5):674-7.

10. Youssef LA, Schuyler M, Gilmartin L, Pickett G, Bard JDJ, Tarleton
CA, et al. Histamine release from the basophils of control and asthmatic
subjects and a comparison of gene expression between ''releaser''. and
''nonreleaser'' basophils. Journal of immunology. 2007;178(7):4584-94.

11. Kepley CL, Youssef L, Andrews RP, Wilson BS, Oliver JM. Syk
deficiency in nonreleaser basophils. J Allergy Clin Immun.
1999;104(2):279-84.

12. Pruzansky JJ, Grammer LC, Patterson R, Roberts M. Dissociation of
IgE from receptors on human basophils. I. Enhanced passive sensitization
for histamine release. Journal of immunology. 1983;131(4):1949-53.

13. Ishizaka T, Ishizaka K. Mechanisms of passive sensitization. IV.
Dissociation of IgE molecules from basophil receptors at acid pH.
Journal of immunology. 1974;112(3):1078-84.

14. Moneret-Vautrin DA, Sainte-Laudy J, Kanny G, Fremont S. Human
basophil activation measured by CD63 expression and LTC4 release in
IgE-mediated food allergy. Annals of allergy, asthma \& immunology :
official publication of the American College of Allergy, Asthma, \&
Immunology. 1999;82(1):33-40.

15. Passante E. Mast cell and basophil cell lines: a compendium. Methods
in molecular biology. 2014;1192:101-13.

16. Bahri R, Custovic A, Korosec P, Tsoumani M, Barron M, Wu J, et al.
Mast cell activation test in the diagnosis of allergic disease and
anaphylaxis. The Journal of allergy and clinical immunology. 2018.

17. Heinzerling L, Mari A, Bergmann KC, Bresciani M, Burbach G, Darsow
U, et al. The skin prick test - European standards. Clinical and
translational allergy. 2013;3(1):3.

18. Cockcroft DW, Davis BE. Methacholine PC20: 1-point formula. Ann
Allergy Asthma Immunol. 2007;98(5):498-9.

19. Nitride C, Lee V, Baricevic-Jones I, Adel-Patient K, Baumgartner S,
Mills ENC. Integrating Allergen Analysis Within a Risk Assessment
Framework: Approaches to Development of Targeted Mass Spectrometry
Methods for Allergen Detection and Quantification in the iFAAM Project.
Journal of AOAC International. 2018;101(1):83-90.

20. Krutzik PO, Nolan GP. Fluorescent cell barcoding in flow cytometry
allows high-throughput drug screening and signaling profiling. Nature
methods. 2006;3(5):361-8.

21. Matthews JN, Altman DG, Campbell MJ, Royston P. Analysis of serial
measurements in medical research. Bmj. 1990;300(6719):230-5.

22. Lee JJ, McGarry MP. When is a mouse basophil not a basophil? Blood.
2007;109(3):859-61.

23. Kepley CL, Pfeiffer JR, Schwartz LB, Wilson BS, Oliver JM. The
identification and characterization of umbilical cord blood-derived
human basophils. J Leukoc Biol. 1998;64(4):474-83.

24. Ohnmacht C, Voehringer D. Basophil effector function and homeostasis
during helminth infection. Blood. 2009;113(12):2816-25.

25. Eckman JA, Sterba PM, Kelly D, Alexander V, Liu MC, Bochner BS, et
al. Effects of omalizumab on basophil and mast cell responses using an
intranasal cat allergen challenge. The Journal of allergy and clinical
immunology. 2010;125(4):889-95 e7.

26. Viander M, Valovirta E, Vanto T, Koivikko A. Cross-reactivity of cat
and dog allergen extracts. RAST inhibition studies with special
reference to the allergenic activity in saliva and urine. International
archives of allergy and applied immunology. 1983;71(3):252-60.

27. Madhurantakam C, Nilsson OB, Uchtenhagen H, Konradsen J, Saarne T,
Hogbom E, et al. Crystal structure of the dog lipocalin allergen Can f
2: implications for cross-reactivity to the cat allergen Fel d 4.
Journal of molecular biology. 2010;401(1):68-83.

28. Nilsson OB, Binnmyr J, Zoltowska A, Saarne T, van Hage M, Gronlund
H. Characterization of the dog lipocalin allergen Can f 6: the role in
cross-reactivity with cat and horse. Allergy. 2012;67(6):751-7.

29. Custovic A, Simpson BM, Murray CS, Lowe L, Woodcock A, Stu NMAA. The
National Asthma Campaign Manchester Asthma and Allergy Study. Pediatr
Allergy Immu. 2002;13:32-7.

\textbf{Figure Legends}

\textbf{Figure 1 Characterization of PCBs differentiation during day
7-28 of culture}

(A-C) Representative figures for non-stimulated controls showing
expression of basophil selection markers
2D7\textsuperscript{+}/Fc\selectlanguage{greek}ε\selectlanguage{english}RI\textsuperscript{+}/CD117\textsuperscript{-}/HLADR\textsuperscript{-}during
culture. (D) Parallel analysis of BB1 expression by immunofluorescence
staining. (E) Degranulation capability by stimulating IgE sensitized
cells with anti-IgE during culture. Gating strategies were the same as
illustrated in A-C.

\textbf{Figure 2 PCBAT with 16-plex fluorescent barcoding}

(A) Illustration of 16-plex fluorescent barcoding with Pacific blue and
Alexa Fluor 700. (B) A representative figure of a 16-plex fluorescent
barcoded sample in a PCBAT.

\textbf{Figure 3} \textbf{PCBAT with cat allergen extracts on cat
sensitized but clinical reactivity not validated subjects}

PCBAT using cat allergen extracts from ALK (dilution factor 1:250;
1:1,250; 1:6,250; 1:31,250; 1:156,250 and 1:781,250) were performed on
(A) 18 cat sensitized but clinical reactivity not validated subjects;
(B) the relationship between the PCBAT AUC from the sensitized groups
and the corresponding sIgE level were shown in scatter plot. (C) PCBAT
using cat allergen extracts were also performed on 6 atopic but non-cat
sensitized subjects and (D) 4 subjects who are sensitized to cat
allergen but under omalizumab treatment, spearman test and R square was
calculated and p\textless{}0.05 were considered significant.

\textbf{Figure 4 PCBAT with cat allergen extracts on cat allergic
subjects validated with inhalant allergen challenge}

PCBAT using cat allergen extracts from HollisterStier (dilution factor
1:250; 1:1,250; 1:6,250; 1:31,250; 1:156,250 and 1:781,250) were
performed on (A) 17 clinal reactivity validated cat allergic subjects;
(B) Scatter plot showed the relationship between the PCBAT AUC from the
allergic groups and the corresponding natural log transformed dosage
response slope. (C) PCBAT using cat allergen extracts were also
performed on 6 atopic but non-cat sensitized subjects, spearman test and
R square was calculated and p\textless{}0.05 were considered
significant.

\textbf{Figure 5 PCBAT with peanut allergen extracts on peanut allergic
and peanut tolerant subjects}

PCBAT using peanut allergen extracts (1, 10, 100 or 1000 ng/ml) were
performed on (A) 30 subjects with physician diagnosed peanut allergy,
and (B) on 4 atopic but non-peanut sensitized subjects. The relationship
between the PCBAT AUC from the sensitized groups and the corresponding
whole peanut sIgE level Ara h 1 sIgE level and Ara h 2 sIgE level were
shown in scatter plot (C-E respectively). Fifteen out of 30 sensitized
subjects underwent oral food challenge. The relationship between
cumulative peanut dose to show first objective sign and PCBAT AUC or
sIgE to whole peanut, Ara h 1 and Ara h 2 were shown in F-I
respectively. (J) 13 peanut sensitized but tolerant subjects. Anti-IgE
was used as positive control stimulant for all tested serum samples.
Spearman test and R square was calculated, p\textless{}0.05 were
considered significant.\selectlanguage{english}
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