Generation of TCR-transduced CD4+ T Cells

Lentivirus vector construction and lentivirus production were established as previously described (Hu et al., 2021 (link)). To generate TCR-transduced-CD4⁺ T and TCR-transduced-CD4⁺ Jurkat cells, CD4⁺ T cells were stimulated with 1 μg/ml OKT3 antibody and 1 μg/ml anti-CD28 antibody (Miltenyi Biotec) and cultured in a complete medium containing 100 IU/ml IL-2 for 48 h. CD4⁺ Jurkat cells and stimulated CD4⁺ T cells were seeded into 24-well-plates and incubated with 500 μl lentivirus and 500 μl RPMI 1640 for 6 h. Next, 500 μl complete medium was added to each well. After 24 h, the original medium was replaced with a 2-ml complete medium and incubated for 48 h. Then, the cells were harvested and used for subsequent assays.