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Tumor cells can evade recognition and elimination by the host immune system by utilizing immune checkpoint receptors; consequently, blocking these receptors holds promise as a broadly effective strategy for tumor immunotherapy. Currently, while anti-PD-1/PD-L1 antibodies—much like anti-CTLA-4 antibodies—represent a relatively mature therapeutic modality, their overall efficacy in patients remains limited due to the prevalence of drug resistance. Therefore, the identification of novel targets for tumor immunotherapy has become a matter of urgent necessity.

Programmed Cell Death Protein 1 (PD-1) is a receptor expressed on activated T cells that negatively regulates immune responses by binding to its ligands, PD-L1 and PD-L2. PD-1 ligands are present in the majority of cancers; the interaction between PD-1 and its ligands (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 the activation of T lymphocytes and enhancing immune tolerance toward tumor cells, 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 and, in some instances, inducing T-cell apoptosis. PD-1/PD-L1 inhibitors function by releasing the immunosuppressive constraints on anti-tumor T cells, thereby promoting T-cell proliferation, infiltration into the tumor microenvironment, and the subsequent induction of anti-tumor immune responses. Furthermore, the PD-1/PD-L1/2 pathway plays a role in regulating autoimmune responses, rendering these proteins promising therapeutic targets for a wide range of conditions, including various cancers as well as autoimmune diseases such as multiple sclerosis, arthritis, lupus, and Type 1 diabetes. The tyrosine phosphatase SHP2 serves as a pivotal regulator of T-cell function, mediating both the downstream activation signals initiated by the T-cell receptor (TCR) and the downstream inhibitory signals triggered by PD-1.

The CHO-K1 Human PDL1 Target Cell (Adherent) Model—effectively simulates the signal transduction process of PD1&SHP2 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human PDL1 Target Cell (Adherent) Model

Figure 2. Blocking of PD1 induced PD1 SHP2 Reporter Cell (C17) Activity by PD1 Neutralizing Antibody with PDL1 Target cell (Adherent)(C11). Blocking of PDL1 induced PD1 SHP2 Reporter Cell (C17) Activity by Atezolizumab with PDL1 Target Cell (Adherent)(C11).

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 PDL1 Target Cell (Adherent) 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.