Anti tcr β ip26

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).

Jurkat 76 cells expressing the exogenous TCR of interest were sorted on CD3/GFP double-positive populations (Figure S3A) and co-cultured with T2 cells in complete RPMI as detailed above. To find homologous sequences of the identified epitopes, netMHCpan was used to predict the binding affinity of the homologous peptide to a given HLA allele (Jurtz et al., 2017 (link); Reynisson et al., 2020 (link)). We used the BLASTP algorithm to perform the search for matching peptides in the UniParc protein database (Johnson et al., 2008 ). Peptides were dissolved in DMSO at 20 mM stock concentration and diluted to a final concentration of 2 μM. After 18 h of stimulation, cells were washed and stained with anti-CD3 (OKT3, Biolegend), anti-CD69 (FN50, Biolegend), and anti-TCR⍺/β (IP26, Biolegend) antibodies. Cells were acquired using FACS Fortessa (BD Biosciences) automated high throughput sampler or the Accuri C6 Plus flow cytometer (BD), and data analyzed using FlowJo software (Treestar).