Methods
Study population. Sera from 112 patients, who were
prospectively enrolled in the MONA study and had clear clinical data on
the peanut allergic status (80 peanut allergic children and adolescents
and 32 non-allergic controls) were used to assess reactivity to peanut
extract with the Hoxb8 MAT in a blinded setting. Allergic patients were
classified based on positive OFC, or a pre-test probability of
>90% allergic reaction (i.e. SPT ≥8 mm and/or sIgE
≥15kUA/L). OFC was performed in an open-label fashion as previously
described and according to internally standardized clinical protocols.
Non allergic controls were defined as patients who either passed an oral
food challenge due to a suspicion of peanut allergy or consumed peanut
regularly and were enrolled due to suspicion or proven tree nut allergy
in the MONAS cohort as reported . Amongst the 112 samples, BAT results
were available for a total of 96 individuals including 69 allergic
samples and 27 non-allergic control samples. Research ethics board (REB)
approval was obtained (REB 1000053791). Patient provided informed
consent and studies were performed in accordance with the Helsinki
Declaration. Serum samples were received at University of Bern
completely blinded under an MTA.
Skin prick test (SPT) and allergen-specific IgE
measurements (sIgE). Previously acquired SPT and sIgE results from
MONAS have been used in this study for direct comparison to Hoxb8 MAT
results. From the 96 samples, for which BAT data were available, SPT
data for peanut extract (ALK-Abello) was previously collected for 47
individuals. Furthermore, sIgE measurements were performed using
ImmunoCAP (Thermo Fisher) allergen testing as part of the clinical
workup in MONAS. ImmunoCAP data from a total of 67 individuals was
available (Fig. 1a ).
BAT. Previously acquired BAT results from the MONA study
have been used and bioinformatically re-analyzed in this study for
direct comparison to Hoxb8 MAT results. BAT has been performed as
previously described . Briefly, heparinized whole blood was drawn and
stored at room temperature until processed within <6 hours.
Whole blood was stimulated with up to 7 serial 10-fold dilutions
(0.001-1000 ng/mL) with peanut extract (ALK-Abelló). The BAT was
performed according to the manufacturer’s instructions (BÜHLMANN
Laboratories AG, Basel, Switzerland). Flow cytometric analysis was
performed with a CytoFLEX (Beckman Coulter, USA). Additionally, sera
from 7 BAT non-responders, who were previously identified in MONAS, were
transferred to the University of Bern for this study.
Hoxb8 MAT. Hoxb8 MC progenitors that were cultured in
RPMIc/IL3 medium containing 4-hydroxytamoxifen (4-OHT) were washed in
PBS, resuspended in RPMIc/IL-3 medium without 4-OHT and reseeded at 7.5
x 104 cells/ml in a culture flask for 5 days to
differentiate mature Hoxb8 MCs. For the Hoxb8 MAT, 5 x
104 Hoxb8 MCs per condition were seeded in a 96-well
round bottom plate. Cells were centrifuged at 600 x g for 5 minutes and
resuspended in pre-processed serum samples for overnight passive
sensitization. Pre-processing included a buffer exchange into activation
medium using 2 mL ultrafiltration spin columns with an MWCO of 100kDa
(VivaSpin, Sartorius, Germany). The columns were used according to the
manufacturer’s instructions. Subsequently, cells were stimulated with 6
serial 10-fold dilutions (0.001-1000 ng/mL) with peanut extract
(provided by ALK-Abelló) plus a staining antibody for anti-CD107a
(clone: 1D4B, BioLegend). Stimulation was performed for 25 minutes at
37°C in the presence of 5% CO2. After washing the cells
with FACS buffer, they were resuspended in 200µl FACS buffer and
acquired on a CytoFLEX S 4L 13C (B2-R3-V4-Y4) plus 96 DW plate loader
(Beckman Coulter Life Sciences, CA, USA) and results were evaluated with
FlowJo Version 10.1 (FlowJo, OR, USA).