Fig. 6.

FRET-shift/amplicon sequencing using tumor-infiltrating T cells as input effectors. B16F10-Ova tumor masses were engrafted into wild-type C57BL/6 mice and boosted by vaccination with Ova prior to isolation of tumor-infiltrating CD8⁺ T cells by FACS, further expanded by stimulation with plate-bound anti-CD3/28, and co-cultured with random minigene library cells spiked with Ova minigene-expressing cells at 1:10,000 abundance. Three replicate mice were prepared, and TIL samples from each were independently screened against the spiked library cell population. Limited outgrowth ex vivo of the TIL populations necessitated that co-cultures be conducted at reduced E:T ratios, ranging from 0.5:1 to 0.7:1 across the replicates (4 h length). In parallel to TIL samples, a matched negative control sample was prepared by performing FRET-shift FACS/amplicon sequencing on spiked library target cells that were not subjected to co-culture with any T cells. The Δ relative abundance of all detected minigenes are displayed for the three replicate TIL screens as well as the no-T-cell control. The dashed line represents 10σ above the mean Δ relative abundance value