Activation Induced Marker (AIM) Assay

Ex vivo T cell responses were measured based on T cell activation assays previously described (27, 28). This assay detects cells that are activated as a result of antigen specific stimulation by upregulation of activation-induced surface markers. Here, we assessed dual expression of OX40 (CD134) and PDL-1. Briefly, PBMC were thawed and rested overnight, plated at 1×10⁶ cells per well in a round-bottom 96-well plate. The next morning, cells were stimulated with peptide pools (2 μg/ml/peptide) for each individual allergen, PHA and PT (positive controls), or DMSO (negative control). Cells were incubated for 24 h. After the incubation, cells were labeled with a cocktail of antibodies ( Supplemental Table 2 ). After staining and washing, flow cytometry was performed. Cells were acquired using a BD LSR II flow cytometer and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA). Geometric mean values with geometric SDs are shown for each of the individual allergens.