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T cell-mediated immune responses are initiated when the T cell receptor (TCR) recognizes antigenic peptides bound to the Major Histocompatibility Complex (MHC). Subsequently, the TCR transmits the antigenic signal via its associated co-receptor (CD3) to the intracellular ITAM domains of CD3, thereby triggering a cascade of intracellular immune signaling pathways within the T cell. For instance, in the context of cancer or infection, the TCR recognizes these aberrant protein fragments; upon activation, the receptor stimulates the T cell to destroy or suppress the abnormal cells.

The TCR-CD3 complex is formed through the non-covalent association of the TCR with the γ, δ, ε, and ζ subunits of CD3. The binding of MHC to the TCR induces LCK-mediated phosphorylation of the ITAM domains within the TCR-CD3ζ subunits. This phosphorylation of CD3ζ triggers signal transduction—involving ZAP70 and LAT—leading to the recruitment of numerous downstream adaptor and signaling molecules and the formation of the LAT signalosome. The assembled LAT signalosome activates various signaling pathways linked to transcription factors—such as AP-1, NF-κB, and NFAT—thereby driving T cell proliferation, cytokine production, and effector functions.

The CD3ε KO NFAT-Luc Jurkat reporter gene model serves as an excellent mimic of the in vivo TCR signal transduction process; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the Jurkat E6.1 Human CD3E KO NFAT-Luc Cell Model

Figure 2. Sequencing results of CD3E KO [CD3E(NM_000733.4):c.397_398ins4bp/CD3E:p.I133Kfs]

Figure 3. CD3E KO NFAT-Luc Jurkat cells, CD3E knockout in NFAT-Luc Jurkat cells.

Figure 4. Dose response of CD3 Agonist Antibody in CD3E KO NFAT-Luc Jurkat (2C24).

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.