Activation-Induced Marker (AIM) Assay

The AIM assay was performed as described previously.^78,79 Briefly, PBMC were thawed, washed in Roswell Park Memorial Institute 1640 Medium (RPMI), re-suspended in complete RPMI media with 5% human serum, and rested overnight. The cells were plated at concentration of 1×10⁶ cells/well in a 96-well round bottom plate. For each stimulation, peptides were added to the well at a final concentration of 1 μg/mL, containing less than 0.1% DMSO. Peptide pools containing CEF or CMV pp65 were used as positive controls and media with 0.1% DMSO was used as a negative control. After 24 hours, the cell supernatant was removed, and the cells were collected for additional analysis. Stimulation experiments were performed twice at independent times.