IL-9 is a key component of memory Th cell peanut-specific responses from peanut allergic children

52 53 Background: Differentiation between peanut allergic (PA) and peanut sensitized (PS) 54 individuals who tolerate peanut but have peanut-specific IgE and/or positive skin prick tests 55 represents a significant diagnostic difficulty. Previously, gene expression microarrays were 56 successfully used to identify biomarkers and explore immune responses during peanut 57 allergy immunotherapy. 58 59 Objective: Characterization of peanut-specific responses from PA, PS and atopic non60 peanut-allergic (NA) children. 61 62 Methods: A preliminary, exploratory microarray investigation of gene expression in peanut63 activated memory Th subsets from 3PA and 3NA children identified potential peanut allergy 64 diagnostic biomarkers. Microarray findings were confirmed using RT-qPCR in 30 individuals 65 (12PA, 12PS and 6NA). Flow cytometry was used to identify the Th subsets involved. 66 67 Results: Amongst 387 differentially expressed genes, IL9 showed the greatest difference 68 between PA and NA (45.59-fold change, p<0.001) followed by IL5 then IL13. . Notably, IL9 69 allowed the most accurate classification of PA and NA using a machine learning approach 70 using recursive feature elimination and the Random Forest algorithm. Skinand respectively 71 gut-homing Th cells from PA donors expressed similar Th2 and Th9-associated genes. RT72 qPCR confirmed that IL9 was the highest differentially expressed gene between PA and NA 73 (23.3-fold change, P<0.01) and PS children (18.5-fold change, P<0.05). Intracellular cytokine 74 staining showed that IL9 and the Th2-specific cytokine IL5 are produced by distinct Th 75 populations. 76