Adoptive T-cell immunotherapy (TCR-T) is a cancer treatment strategy that enhances the tumour recognition and destruction capabilities of genetically modified T-cells by introducing antigen receptors to specifically target tumour antigens. TCR-T involves engineering allogeneic or autologous T-cells to express transgenic TCRs. TCRs have evolved to effectively respond to relatively low levels of antigens and can recognize almost any protein through the major histocompatibility complex (MHC) system, making them highly sensitive to altered self (tumour-associated) or pathogen-derived antigens.The TCR complex is formed by the combination of TCR alpha (α) and beta (β) chains with auxiliary signalling molecules such as CD3. A single T-cell typically expresses one α chain and one β chain due to TCR gene rearrangement during development. Introducing exogenous TCR α and β chains can lead to the formation of mixed TCR complexes (e.g., an α chain from the endogenous TCR and a β chain from the exogenous TCR). This mixed αβ TCR alters antigen specificity. Additionally, mismatching between endogenous and exogenous TCR chains reduces the pairing of the “preferred” exogenous TCR, thereby decreasing recognition of the corresponding antigen.CD4 and CD8 are T-cell co-receptors that bind to non-polymorphic regions of MHC, enhancing the signal and sensitivity of TCRs with low affinity or those responding to low levels of antigens. CD4 is typically expressed on helper and regulatory T-cells and binds to MHCII expressed by professional antigen-presenting cells (APCs). Conversely, CD8 is usually expressed on cytotoxic T-cells and binds to MHCI expressed by all nucleated cells. MHCII generally presents exogenous (e.g., pathogen-derived) antigens, while MHCI presents endogenous (e.g., self (tumour) or viral) antigens.Conventional methods for detecting TCR-T are time-consuming and yield variable results due to reliance on donor T-cells, complex experimental protocols, and unverified kits. Moreover, endogenous TCR expression in primary T-cells can interfere with results.The TCR Knockout Reporter Cell Line (CD4+) overcomes these limitations by knocking out endogenous TCR α and β chains in Jurkat T-cells and additionally expressing a luciferase reporter gene driven by a TCR signalling pathway-dependent promoter. Researchers simply need to introduce the TCR complex under investigation into these cells to form transgenic TCR cells. These transgenic TCR cells can then be activated by homologous peptides and APCs expressing MHC, followed by luciferase quantification after a certain period. This cell line provides an efficient, stable, and convenient method to assess the efficacy of transgenic TCR complexes in activating T-cells without the influence of endogenous TCR expression.