Allergen-induced activation of NK cell genes represents an early life immune response in development of allergic asthma

Peripheral blood samples from children enrolled in the URECA cohort were collected at age 2. In short, at each URECA research center, within 16 hours of blood collection, mononuclear cells were separated and incubated (48 hours, 37C, 5% CO2) in the presence of German cockroach extract (10mg/mL; CR), dust mite extract (Dermatophagoides pteronyssinus, 10mg/mL; DM), tetanus toxoid (5mg/mL; TT), or medium alone (NS). For the initial case-control cohort (cohort 1), cases (n=21) were URECA participants who met the following inclusion criteria: a) ≥2 aeroallergen sensitizations at age 3, 5, and 7 including sensitization to DM and/or CR at each age, b) a diagnosis of asthma at age 7, and c) had PBMC RNA samples collected at age 2. Controls (n=30) were URECA participants with: a) no aeroallergen sensitization through age 7, b) did not have asthma at age 7, and c) had PBMC RNA samples collected at age 2. Controls were selected to match to cases on at least a bivariate level according to the following variables: gender, race, URECA site, birth season, and levels of Bla g 1, Der f 1, and Def p 1 measured in the home 3 months after birth. All available NS, CR, DM, and TT stimulation PBMC samples from these 51 individuals were selected for RNA-sequencing. For cohort 2, seven additional URECA participants were selected who met the following inclusion criteria: a) ≥1 aeroallergen sensitization by age 7 including CR sensitization, b) a diagnosis of asthma at age 7, and c) PBMC RNA samples collected at age 2. Fourteen additional controls were selected to match these cases by the same criteria as the controls of cohort 1. Furthermore, all URECA participants with PBMC RNA samples collected at age 2 and who had sufficient year 7 outcome data for group classification were added to the sequencing run to allow a 4 group comparison based on CR sensitization (age 3, yes/no) and asthma (age 7, yes/no). For the latter analyses, only NS and CR stimulation PBMC samples were used for RNA-sequencing.