GFP Reporter Cell Line - Jurkat

The αβ T cell receptor (TCR) is a receptor expressed on the surface of αβ T cells that recognizes antigens presented by major histocompatibility complex (MHC) molecules and triggers T cell immune responses. The researchers established a Jurkat cell line with a double knockout of TCRαβ and expressing CD8αβ molecules. After large-scale TCR sequencing of activation marker-positive CD8 T cells by NGS, reverse TCR cloning was performed using unique CDR3-specific PCR primers to directly obtain functional TCRs. The researchers successfully amplified 15 TCRα and 14 TCRβ chains from cDNA of 4-1BB-positive CD8 T cells restricted to HLA-A*02:01, HLA-A*02:06, HLA-B*07:02, and HLA-B*40:06. A panel of these TCRα and TCRβ gene combinations was studied using Jurkat reporter cell lines and artificial antigen-presenting cells (APCs). Their specificity and affinity were confirmed in two TCR pairs restricted by HLA-A*02:01, one TCR pair restricted by HLA-A*02:06, four TCR pairs restricted by HLA-B*07:02, and one TCR pair restricted by HLA-B*40:06. The TCR pairs of HLA-A*02:01/1-1 showed alloreactivity to HLA-A*02:06. One TCR pair showed a higher response to the naturally processed antigen than the peptide library. This reverse TCR cloning system not only provides functional information for NGS, but will also help in the development of TCR-T therapies.

Figure 1. Jurkat cells were cultured in RPMI-1640 medium and grown and detected in a 37°C, 5% CO2 environment. CD8αβ molecules and NFκB reporter systems were stably expressed by transducing lentivirus containing CD8β-T2A-CD8α and NFκB transcriptional reporter systems. After flow cytometry analysis and single-cell clone screening, Jurkat cell clones (J8G clones) expressing high levels of CD8αβ and GFP were finally obtained. (Hong CH, et al., 2022)