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 CTLA-4 is a T-cell co-receptor, also known as CD152. Compared to CD28, CTLA-4 exhibits a higher binding affinity for molecules of the B7 family (including CD80 and CD86) on antigen-presenting cells (APCs). Although CTLA-4 binds to B7 co-stimulatory receptors, it plays a negative role in T-cell activation. After the T cell receptor (TCR) recognizes an antigen presented by the major histocompatibility complex (MHC) on an APC, the T cell’s CD28 binds to the APC’s B7. This inhibits T cell activation by blocking T lymphocyte responses, reducing T lymphocyte proliferation, suppressing Treg lymphocyte activity, and decreasing cytokine secretion, thereby suppressing the immune response.

The CTLA4-IL2 Promoter Reporter Jurkat cell line effectively mimics the in vivo signaling pathways of CTLA4 and IL2; the mechanism is illustrated in the figure below.

Figure 1. Schematic diagram of the CTLA4/ IL-2 promoter Reporter cell model

Figure 2. Recombinant CTLA4/IL2 Promoter Reporter/Jurkat stably expressing CTLA4.

Figure 3. Dose Response of Ipilimumab in CTLA4 IL2-Promoter Reporter Jurkat(C39) with TCR Activator Raji (C1).

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.