Anti cd137 4b4 1

T cells were isolated from a leukoreduction system chamber from an HLA-A^∗02 positive healthy donor from the Stanford institutional blood bank using the RosetteSep human T cell enrichment cocktail (Stem Cell Technologies) and viably stored in liquid nitrogen. For T cell activation, T cells were thawed and stimulated with anti-CD3/CD28 beads (Life Technologies) in the presence of IL-2 (100 IU/mL). On days 1 and 2, activated T cells were retrovirally transduced using Retronectin (Takara) coated plates in media containing 100 IU/mL IL-2. anti-CD3/CD28 beads were removed on day 3 and media containing IL-2 were replenished once every 2 days. Following 8 days of in vitro expansion, T cells were co-cultured with 293A2 cells expressing full-length TMEM161A, EntS, LMP2, FluM1, or GFP alone at a 1:1 ratio. Following 18 h incubation, cells were stained with anti-CD3 (OKT3, Biolegend), anti-CD69 (FN50, Biolegend), anti-TCR⍺/β (IP26, Biolegend), anti-CD137 (4B4-1, BD Biosciences), and live/dead near-IR dye (Invitrogen). Data were acquired using FACS Fortessa (BD Biosciences) automated high throughput sampler and analyzed using FlowJo software (Treestar).

T cells were isolated from tumor single cell suspensions by antibody staining followed by cell sorting on a 5-laser FACSAria Fusion sorter (Stanford FACS Facility) purchased using funds from the Parker Institute for Cancer Immunotherapy. Tumor cell suspensions were stained in PBS with Zombie Aqua dye (Biolegend) for viability assessment. This was followed by staining in PBS with 2% FBS in Fc Blocking solution (Biolegend) plus the following antibodies: anti-CD4 (OKT4, Biolegend), anti-CD8 (SK1, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD45 (H130, Biolegend), anti-CD25 (BC96, Biolegend), anti-PD-1(EH12.2H7, Biolegend), anti-CD137 (4B4-1, BD Biosciences), anti-HLA-DR (L243, Biolegend). CD3⁺CD45⁺AquaZombie^- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at −80°C. Ectopic HLA-B^∗35 was detected with anti-HLA-BC monoclonal antibody (clone B1.23.2, Thermo Fisher Scientific). Transduced Jurkat 76 cells expressing exogenous TCRα/β chains were sorted on a FACSAria Fusion sorter at Stanford or a BD Biosciences Influx High Speed Cell Sorter at the Flow Cytometry Core Facility of the Cancer Institute of New Jersey.