Generation of TRAC-1928z T Cells

TRAC-1928z T cells were generated as previously described^{28 (link), 57 (link)}. In brief, αβTCR-T cells were purified from PBMCs with the Pan T cell Isolation kit (Miltenyi Biotec) on the AutoMACS Pro according to manufacturer instructions. Purified cells were activated with CD3/CD28 Dynabeads (1:1 beads:cell) in X-Vivo 15 media (Lonza) supplemented with 5% Human Serum (HS) (Gemini Bioproducts) with 5ng/mL rhIL-7 (R&D Systems) and 5ng/mL rhIL-15 (R&D Systems). 48 h after αβTCR-T cell activation, CD3/CD28 beads were magnetically removed, and T cells were transfected by electrotransfer of TRAC ribonucleoprotein using the Nucleofector II device (Lonza). Then 2×10⁶ T cells were resuspended in P3 buffer (Lonza) and mixed with 60pmol TRAC ribonucleoprotein in a total volume of 20μL. Following electroporation and considering 66.7% viability, cells were diluted into culture medium and 1×10⁶/mL and incubated at 37°C, 5% CO₂. Recombinant AAV6 donor vector pAAV-TRAC-1928z^{28 (link)} was added to the culture 30min after electroporation at a multiplicity of infection of 3×10⁵ genome copies. Twenty-four h after targeting, T cells were reprogrammed as described above (WT-TiPS) and TRAC-1928z-TiPS colonies were established and cloned on MEF feeder cells. PCRs were performed to determine biallelic, specific target transgene integration into the TRAC locus.