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Programmed Cell Death Protein 1 (PD-1), a receptor expressed on activated T cells, binds to its ligands—PD-L1 and PD-L2—to negatively regulate immune responses. PD-1 ligands are present in most cancers; the interaction between PD-1 and PD-L1/2 suppresses T-cell activity and enables cancer cells to evade immune surveillance. The PD-1/PD-L1 signaling pathway constitutes a critical component of tumor-induced immunosuppression, inhibiting T-lymphocyte activation and enhancing immune tolerance within the tumor, thereby facilitating tumor immune evasion. The binding of PD-1 to PD-L1 attenuates T-cell-mediated immune surveillance, leading to a compromised immune response or even T-cell apoptosis. PD-1/PD-L1 inhibitors can reverse the immunosuppression of anti-tumor T cells, thereby promoting T-cell proliferation, infiltration into the tumor microenvironment, and the induction of anti-tumor responses. The PD-1/PD-L1/2 pathway is also involved in regulating autoimmune responses, making these proteins promising therapeutic targets for various cancers, as well as for conditions such as multiple sclerosis, arthritis, lupus, and type 1 diabetes. The tyrosine phosphatase SHP2 is a key regulator of T-cell function, mediating both downstream activation signals from the T-cell receptor (TCR) and downstream inhibitory signals from PD-1.

The CHO-K1 Human PDL2/TCR Activator Cell Model—effectively simulates the signal transduction process of PD1&PDL2 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human PDL2/TCR Activator Cell Model

CHO cell line expressing full length human PDL2. Expression is confirmed by flow cytometry.

Figure 2.Recombinant PDL2/TCR Activator/CHO constitutively expressing PDL2.

Figure 3. Dose Responese of anti-PD1 neutralizing antibody in PD1 NFAT-Luc Jurkat (C16) with PDL2 TCR Activator CHO(C1).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  CHO-K1 Human PDL2/TCR Activator Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.