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T cells play a central role in cell-mediated immunity, mediating long-lasting, antigen-specific effector and memory responses. T cells serve as targets for numerous immunotherapies, including checkpoint inhibitors, bispecific T-cell engagers, and immune agonists. Furthermore, T cells themselves have been investigated as therapeutic agents—specifically through the use of chimeric antigen receptors (CARs) or autologous adoptive transfer—for the treatment of hematological malignancies. In recent years, various immunotherapeutic strategies aimed at inducing, enhancing, and/or engineering T-cell responses have emerged as promising approaches for treating diseases such as cancer and autoimmune disorders.

The T-cell receptor (TCR) is the molecular structure responsible for the specific recognition and binding of antigen-peptide/MHC complexes by T cells. The T-cell activation mediated by the TCR plays a pivotal role in thymic T-cell development, the differentiation of T-cell subsets, and the execution of effector T-cell functions. The TCR specifically recognizes antigen peptides presented by MHC molecules on the surface of antigen-presenting cells, translating this extracellular recognition into intracellular signals. This process involves inducing the activation of tyrosine kinases proximal to the TCR, facilitating the assembly of signal transduction complexes, and activating downstream signaling pathways—such as MAPK, PKC, and calcium signaling cascades—ultimately leading to the activation of specific transcription factors, the regulation of effector protein expression, and the completion of T-cell activation.

The TCRαβ KO CD4+ NFAT-Luc Jurkat reporter cell model features the knockout of the endogenous TCR receptor, coupled with the overexpression of CD4 and a downstream NFAT signaling pathway reporter gene; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the Jurkat E6.1 Human TCRαβ KO CD4+ NFAT-Luc Cell Model

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.

Figure 2. TCRαβ KO CD4+ NFAT-Luc Jurkat cells, overexpressing CD4 in TCRαβ KO NFAT-Luc Jurkat cells. The flow cytometry data above confirms the overexpression effect of CD4 and the knockout effect of TCRαβ.

Figure 3. Dose response of CD3 Agonist Antibody in TCRαβ KO CD4+ NFAT-Luc Jurkat (2C10C12C10).