Generation and Characterization of Primary T Cell Lines

The previously published primary T cell lines A25Salmonella, which contains the clones PG10 and PG90 (33 (link)), LDN5 (26 (link)), BC24A, BC24B, and BC24C (34 (link)) were grown by stimulation with 30 ng/mL anti-CD3 antibody and 25 × 10⁶ irradiated PBMC and 5 × 10⁶ irradiated Epstein-Barr virus-transformed B cells, and 1 ng/mL IL-2, which was added on day 2 of the culture. To study tetramer binding to the GEM42 TCR, we used a TCR-transduced 5KC-78.3.20 hybridoma (14 (link), 15 (link)). To generate primary TRBV4–1⁺ and TRBV4–1⁻ T cell lines, PBMC from random blood bank donor D43 were stained with anti-CD3 (555342, BD) and anti-TRBV4–1 (IM2287, Beckman Coulter). Cells were sorted on a BD FACSAria (BD Biosciences), and lymphocytes were selected based on forward scatter and side scatter. 1×10⁶ CD3⁺TRBV4–1⁺ and 1×10⁶ CD3⁺TRBV4–1⁻ cells were sorted. After 2 weeks of stimulation as described above both cells were stained with anti-CD3 and anti-TRBV4–1 antibodies to check purity.