Detecting Antigen-Specific T Cell Activation

The AIM assay was previously described (31 (link)). This assay detects cells that are activated as a result of antigen-specific stimulation by upregulation of activation-induced surface markers. Here, we employ 4-1BB (CD137) enrichment (CD137 MicroBead Kit, Miltenyi, Germany) followed by staining with a cocktail containing CD4-APC-ef780, CD3-AF700, CD8/CD14/CD19-V500, CD45RA-eF450, CCR7-PerCPCy5.5, 4-1BB-APC, and OX40-PECy7 (see Table S2 in Supplementary Material). Cryopreserved PBMCs were thawed, and 1 × 10⁶ cells/condition were stimulated with LoMo megapool (2 µg/ml) or high molecular weight (PUR) urine extracts (10 µg/ml) in 5% human serum (Gemini Bioproducts) for 24 h. DMSO (0.25%) with medium was used as negative control. For intracellular cytokine staining (ICS), PBMCs were incubated with LoMo megapool or PUR extract for 24 h. After 20 h, BFA [5 μg/ml (BD Bioscience, San Diego, CA, USA)] was added for an additional 4 h. Cells were then washed, stained for extracellular markers for 30 min, washed again, fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin (Sigma), and stained for intracellular IL-4-BV421, IL-5-PE, IL-17-FITC, and IFN-γ-PerCPCy5.5 (Table S2 in Supplementary Material). PMA and ionomycin (1 and 0.1 µg/ml) were used as positive control. Samples were acquired on a BD LSRII Flow Cytometer and analyzed using FlowJo X Software.