Isolation and Expansion of Tumor-Infiltrating Lymphocytes

TIL were expanded as previously described (14 (link)). Primary bladder tumors or lymph node metastases were minced into ~1–3 mm³ fragments and plated in TIL media consisting of RPMI 1640, 2.05 mM L–glutamine (HyClone, Thermo Fisher Scientific, Waltham, MA), 10% heat-inactivated human AB serum (Omega Scientific, Tarzana, CA), 55 μM 2-mercaptoethanol (Invitrogen), 50 μg/ml gentamicin (Invitrogen), 100 I.U./ml penicillin, 100 μg/ml streptomycin, and 10 mM HEPES Buffer (Mediatech, Manassas, VA) in 24- or 48-well plates with 6000 I.U./ml rhIL-2 (Prometheus). Some cultures were supplemented with 1 ug/ml anti-CD137 agonistic antibody (Urelumab, BMS-663513). All cultures were expanded for 4 weeks and confluent wells were split into additional wells. TIL from each independent fragment was counted. Remaining tumor material was mechanically and enzymatically digested using media containing 2% Collagenase Type IV and a GentleMACS Dissociator (Miltenyi, 130–093-235). Cells were counted by trypan blue exclusion and subjected to subsequent analysis or cryopreserved as functional assay targets. Positive TIL growth was defined as confluency and expansion of the primary well into 2 wells.