Figure 2. Recombinant expression and surface detection of T cell receptors in YT-Indy cell line.

(a) Unmodified YT-Indy or YT-Indy cells transduced with lentivirus encoding HSDL1 L25V mutant-reactive TCRαβ followed by bicistronic CD3ζ/CD8α lentiviral helper cassette were analyzed by flow cytometry. Analysis confirms the robust expression of an RFP marker included with the TCRαβ transgene but no detection of TCR at the cell surface by antibody surface staining with FITC-conjugated anti-human TCR IP26 mAb clone (Abcam). Expression of CD8α at the cell surface was confirmed by staining with APC-conjugated anti-human CD8 HIT8a mAb (Biolegend). (b) YT-Indy cells previously transduced with mutant HSDL1-reactive TCRαβ encoding construct were double-infected with two helper cassettes designed to deliver complete CD3 and CD8 complexes. Detection of FITC fluorescence, indicative of surface TCRαβ binding, was observed to co-occur with robust RFP expression. In contrast, no TCR protein was detectable in YT-cells lacking helper cassettes despite expressing the RFP marker of transgene integration. (c-d) The TCR⁺ CD8⁺ double-positive population was isolated by FACS from triple transduced YT-rCTL shown in (a) and further characterized by surface expression. Strong expression, on the basis of mean fluorescence intensity (MFI) relative to primary human T cells comparator, was detected in CD8α, CD3ε, and TRBV channels. Surface staining of CD3ε was conducted using eFluor450-conjugated mAb clone UCHT1 (eBioscience). Surface staining of TCRβ was conducted using FITC-conjugated mAb clone JU74.3, specific for the known Vβ segment used by the HSDL1 TCR (Beckman Coulter).