Study strategy
PBMC were obtained from 71 study participants enrolled from 11 clinical sites prior to allergen IT and cryopreserved for subsequent analysis (Figure 1a ). To characterize the magnitude and polarization of allergen responses directed against Blatella germanica (Bla g) allergens, we tested sets of overlapping peptides, spanning the entire sequence of 11 different CR allergens, using Activation Induced Marker (AIM) assays 30,35,36, combined with Intracellular Cytokine Staining (ICS) (Figure 1b and see methods for more detail).
Figure 1c shows representative data for the detection of each cytokine in 3 different donors. Specifically, IL-4 response to Bla g 9, IFNγ response to Bla g 3, and IL-10 response to Bla g 2 allergen peptide pool stimulation. Strong cytokine production was observed in response to the positive control (PMA/Ion) but not in response to the negative control (DMSO) or peptide pools from allergens not recognized for a particular donor. For each participant, we determined the total number of effector T cells (Teff; antigen-specific CD154+ cells24,30) associated with production of individual IL-4, IFNγ, and IL-10 cytokines by summing the reactivity observed with each different pool.