CD4 T Cell Antigen-Specificity Assay

Antigen-specificity of the CD4 T cell lines generated above was determined using the “Combined OX40/CD25 and CD69/CD40L” approach described above and by ICS. For the ICS, briefly, 24 hr after IL-2 removal from the media (~Day 11 of culture) CD4 T cell line cells were collected, washed, and resuspended in X-VIVO + 10% HAB. The cell line was then restimulated with the appropriate antigen (0.5 μg/peptide/ml) for 6 hr. BFA/monensin was added after 1 hr. Cells were surface stained according to the panel in S6 Table, then fixed and permeabilized according to manufacturers instructions using eBioscience Intracellular Fixation and Permeabilization Buffer set (eBioscience, 88-8824-00). Cells were then stained intracellularly according to the panel in S6 Table, and resuspended in 1% FBS/PBS for acquisition.