2. Method for Cell Line Construction

The TCR Reporter Cell Line was developed by CRISPR/Cas9-mediated knockout of the TRAC and TRBC genes in Jurkat E6.1 cells, resulting in the Jurkat TCR knockout (K/O) construct. The knockout of TRAC/TRBC was confirmed via sequencing and the absence of surface TCR expression, verified by negative staining with a PE-labeled anti-human TRBC1 antibody (Figure 1).

Following a successful knockout, the Jurkat TCR K/O cells were transduced with a TCR reporter construct:
NFAT–mCMV–GFP–EF1–CD8 (Figure 2). Clonal selection was performed to isolate the cell line that showed the highest level of GFP expression—up to a 1,000-fold increase—after stimulation with PMA + ionomycin, indicating strong reporter activation in Jurkat-NFAT-GFP cells.

As illustrated in Figure 1, when the Jurkat NFAT-GFP reporter cells were transduced with a control EBV TCR construct, FACS analysis demonstrated positive staining with the PE-anti-TRBC1 antibody. This result confirmed the successful expression and surface integration of the EBV TCR construct in the reporter cells.

Fig 2. Construction of TCR Reporter Cell Line and Expression of Control EBV TCR Construct Jurkat NFAT-GFP cells, after transduction with the EBV TCR construct, regained surface TCR expression as shown by PE-anti-TRBC1 antibody staining. This confirms functional integration of the EBV TCR into the Jurkat cells.

Fig 3.Map for NFAT-mCMV-GFP Reporter Construct