Specific reactivity of TIL was assessed by interferon-γ (IFN-γ) release assay. TIL were washed prior to use to remove IL-2, then cultured overnight with autologous, HLA-matched, or HLA-mismatched tumor cells at a ratio of 1:1. Single cell suspensions of fresh tumor digests were prepared as targets from autologous or allogeneic melanoma specimens by overnight digestion of macerated tumor fragments in media containing collagenase, hyaluronidase, and DNAse. The single cell suspension was washed twice with HBSS and aliquots were cryopreserved. Targets were thawed on the day of coculture, and viable tumor cells were assessed by trypan blue exclusion. The supernatant from each coculture was then assayed for IFN-γ by ELISA (Peirce/Endogen) according to the manufacturer recommendations. A TIL culture was defined as possessing specific reactivity if IFN-γ release was twice background (coculture of TIL with HLA-mismatched tumors) and at least 200 pg/mL unless otherwise noted.