DOI: 10.5061/dryad.0gb5mkmch
title: Atopyrelated immune profiles are subject to genetic influence as evaluated using school-aged twin pairs

description: Both genetic and environmental risk factors have been described for atopic
 disease. However, the interaction of these with individual immune profiles
 remains unclear. Using flow cytometry, immune profiles of 93 school-aged
 twin pairs as well as in vitro responses were determined to evaluate
 immunological responses associated with atopy. Findings suggest a genetic
 impact on immune cell abundance, particularly for B cells. Whereas
 cytokine responses from in vitro stimulations appeared mainly shaped by
 environmental exposures. Relating to atopic traits, we observed increased
 abundance of both B cells and basophils in atopic individuals as well as
 increased expression of the IgE receptor across several subsets, including
 basophils and dendritic cells. Genetic background appeared central to
 regulating IgE receptor expression on basophils whereas expression on
 dendritic cells instead appeared sensitive to environmental exposures.
 Identifying environmentally regulated immune traits may facilitate the
 development of targeted therapies to limit the impact of atopic disease in
 the future.
description: # Atopy-related immune profiles are subject to genetic influence as
 evaluated using school-aged twin pairs Dataset DOI:
 [10.5061/dryad.0gb5mkmch](10.5061/dryad.0gb5mkmch) ## Description of the
 data and file structure **Study cohorts** A subset of school-age
 individuals (age range 7-13 years) from the Western Australian Twin Study
 of Child Health (WATCH). A total of 93 twin pairs (186 individuals) were
 included in the present study. **Clinical parameters** Atopy was defined
 as either (i) a positive skin prick test (SPT, wheal size ≥3mm) for any of
 the most common aeroallergens including house dust mite (HDM), cat dander,
 dog dander, fungi (*Alternaria alternata*, *Aspergillus spp.*), cockroach,
 perennial rye grass, mixed grass, and mixed mould or (ii) a total IgE
 level above 300 IU/mL as measured by a clinical pathology laboratory in
 Australia. **Flow cytometry analysis** Approximately 1x10^6 thawed PBMCs
 were stained. Briefly, thawed PBMC were washed using PBS supplemented with
 0.5% BSA and 0.1% NaN3 (Flow buffer), incubated with the extracellular
 antibody mixture at 4 °C for 30 min, washed and incubated in
 fixation/permeabilised solution for 30 min at 4 °C (Thermo Fisher)
 followed by intracellular FoxP3-PE staining at 4 °C for 30 min, washed and
 resuspended in Flow buffer for flow cytometric data generation. Flow data
 were acquired using the LSR-Fortessa II with FACSDiva software (BD
 Bioscience). Processing of samples was block-randomised, ensuring that all
 twin pairs were processed in the same batch, this to reduce technical
 variation in the paired analysis. **Cell culture and cytokine
 quantification** Thawed PBMC were resuspended in RPMI + 10% FCS and
 cultured at 0.25x10^6 cells per well, in 250 mL, in round-bottom 96-well
 culture plates. Cells were stimulated with 1 μg/mL Phytohemagglutinin
 (PHA, Remel-Thermo), 25 pg/mL Lipopolysaccharide (LPS, Alexis-Enzo), or
 0.5 mg/mL HDM (CSL) and incubated for 24 hours at 37 °C and 5% CO2.
 Following culture, supernatants were collected for cytokine assays and
 stored at -80 °C. From the HDM-stimulated cultures, cells were also washed
 and prepared for flow cytometry analysis, as above. For evaluation of
 cytokine content in cell supernatant, a 13-plexed cytokine panel
 (in-house) was used and analysed on a Luminex 100 System. Briefly,
 post-culture supernatants and in-house quality controls (one high and one
 low made from recombinant human standards) were diluted 1:2 in culture
 media. Pooled standards were also diluted in culture media, and cytokines
 were quantified as pg/mL concentration with reference to a standard curve.
 Antibodies were coupled to MagPlex beads (Luminex Corp), and the Luminex
 assay was performed using the manufacturer’s instructions. Standard curves
 and quantitation were determined using BioPlex Pro Manager software.
 In-house quality controls (high and low) were included across each plate
 and accepted if their values fell within 2 standard deviations of the
 mean. ### Files and variables #### File: ex_vivo.zip **Description:** Flow
 cytometry data files on ex vivo samples. These can be linked using the “Ex
 vivo FCS file” variable in the metadata file. #### File: HDM-stim.zip
 **Description:** Flow cytometry data on samples stimulated in vitro with
 HDM. These can be linked using the “HDM-stim FCS file” variable in the
 metadata file. #### File: Meta_data.csv **Description:** Twin- and
 Atopy-specific parameters, as detailed in the variable section, and also
 including cytokine production following stimulation with PHA, LPS, and
 HDM, cytokine concentrations are depicted in pg/mL. As an example, “LPS
 [IFNg]” depicts the concentration of IFNg following stimulation with LPS.
 ##### Variables * Subject ID (unique for each individual) * Family ID
 (common for each twin pair) * Zygosity (monozygotic and dizygotic) * Sex
 (male or female) * Age [yrs] (binned to avoid identification) - Current
 Atopy (yes=1, no=0, based on definition as detailed above) - HDM SPT+
 (yes=1, no=0, skin-prick test positive for HDM) - Technical Batch (batch
 in which samples were stained/cultured) - total IgE [plasma] (in IU/mL) -
 Ex vivo FCS file (file name to link with ex vivo flow cytometry data) -
 HDM-stim FCS file (file name to link with HDM-stim flow cytometry data) -
 LPS [IFNg] - LPS [IL1b] - LPS [IL5] - LPS [IL6] - LPS [IL8] - LPS [IL9] -
 LPS [IL10] - LPS [IL12] - LPS [IL13] - LPS [IL18] - LPS [IP10] - LPS
 [MIP1a] - LPS [TNF] - PHA [IFNg] - PHA [IL1b] - PHA [IL5] - PHA [IL6] -
 PHA [IL8] - PHA [IL9] - PHA [IL10] - PHA [IL12] - PHA [IL13] - PHA [IL18]
 - PHA [IP10] - PHA [MIP1a] - PHA [TNF] - HDM [IFNg] - HDM [IL1b] - HDM
 [IL5] - HDM [IL6] - HDM [IL8] - HDM [IL9] - HDM [IL10] - HDM [IL12] - HDM
 [IL13] - HDM [IL18] - HDM [IP10] - HDM [MIP1a] - HDM [TNF] ## Access
 information NA ## Human subjects data All participants have consented to
 that their research data will be published and the data has been
 de-identified prior to submission into the public domain.