Mouse tcrβ specific antibody

After bulk TCR sequencing, top rank alpha chains and beta chains were selected and randomly paired to obtain a library of 116 candidate TCR constructs. Detailed information on the library can be found in Table S3. The TCR library was cloned into lentiviral vectors for T‐cell transduction. Multiplicity of infection (MOI) was controlled at 0.2 to ensure single lentivirus integration into the genome of each transduced T cell.
Lentiviruses were generated using the pMD2.G and pSPAX2 envelope plasmids in HEK293T cells. Freshly isolated PBMCs were activated with anti‐CD3 and anti‐CD28 (Miltenyi Biotec) overnight, and proceeded to virus infection. Viral supernatant mixed with 400 U/ml IL‐2 (R&D System, USA) and 8 μg/ml protamine (Sigma Aldrich) was added onto peripheral blood lymphocytes (PBLs) and centrifuged at 800 g and 25°C for 1.5 h. Transduced T cells were maintained in GT551‐H3 medium (TaKaRa) supplied with 5% human serum (GemCell), 1% penicillin/streptomycin (HyClone), and 400 IU/ml IL‐2. The expression of TCR genes was routinely examined using a mouse TCRβ‐specific antibody (BD Biosciences) by FACS.

TCR sequences were codon-optimized and cloned into the lentiviral vector pLVX (Clontech). Mouse TCR constant regions were used in replacement of human TCR constant regions to reduce endogenous TCR mismatch and improve TCR surface expression.^{43 (link)} Lentiviruses were generated with HEK293T cells, and PBMCs from healthy donors were transduced as described previously.^{44 (link)} TCR-engineered T cells were maintained in GT551-H3 medium (Takara) supplied with 5% human serum (GemCell), 1% penicillin/streptomycin (Hyclone), and 400 IU/mL IL-2. The expression of TCR gene was routinely examined with a mouse TCRβ specific antibody (BD Biosciences) by FACS.