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Tumor cells can utilize immune checkpoint receptors to evade recognition and elimination by the body's immune system; consequently, blocking these receptors holds promise as a broadly effective strategy for tumor immunotherapy. Although anti-PD-1/PD-L1 antibodies—much like anti-CTLA-4 antibodies—represent a relatively mature class of therapeutics, 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 binds to its ligands, PD-L1 and PD-L2, to negatively regulate immune responses. PD-1 ligands are present in the majority of 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 tumor cells, thereby facilitating tumor immune evasion. The binding of PD-1 to PD-L1 attenuates T-cell-mediated immune surveillance, leading to an impaired immune response or even T-cell apoptosis. PD-1/PD-L1 inhibitors function by lifting the immunosuppression imposed on anti-tumor T cells, thereby promoting T-cell proliferation, infiltration into the tumor microenvironment, and the 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 key 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.

TheJurkat E6.1 Canine PD1 Effector Reporter Cell Model—effectively simulates the signal transduction process of PD1&PDL1 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the Jurkat E6.1 Canine PD1 Effector Reporter Cell Model

Figure 2. Recombinant Canine PD1 Effector Reporter Cell stably expressing Canine PD1.

Figure 3. Dose Response of Blocking Canine PD-L1 Blocking Ab in Canine PD1 Effector Reporter Cell ( C20) with Canine PDLl  aAPC Cell.

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.