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Immun checkpoint inhibitors (anti-CTLA4, anti-PD1, anti-PDL1, and combination therapy of anti-PD1/PDL1 with anti-CTLA4) represent a major breakthrough in oncology in recent years and are a novel form of cancer immunotherapy.
Programmed Cell Death Protein 1 (PD-1), a receptor expressed on activated T cells, binds to its ligands PD-L1 and PD-L2, negatively regulating immune responses. PD-1 ligands are present in most cancers, and the PD-1-PD-L1/2 interaction inhibits T cell activity, allowing cancer cells to evade immune surveillance. Binding of PD1 to PDL1 can weaken T cell-mediated immune surveillance, leading to impaired immune responses, and even T cell apoptosis. PD1-PDL1 inhibitors can relieve immune suppression of anti-tumor T cells, resulting in T cell proliferation, infiltration into the tumor microenvironment, and induction of anti-tumor responses. The PD1-PDL1 signaling pathway is an important component of tumor immune suppression, which can inhibit T lymphocyte activation, enhance tumor cell immune tolerance, thereby achieving tumor immune escape.

  
CTLA4 is a T cell co-receptor also known as CD152. Compared to CD28, CTLA4 has a stronger binding affinity for B7 family molecules (including CD80 and CD86) on antigen-presenting cells (APCs). Although CTLA4 binds to B7 co-stimulatory receptors, it plays a negative role in T cell activation. After the T cell receptor (TCR) recognizes antigens presented by the major histocompatibility complex (MHC) on APCs, T cell CD28 binds to B7 on APCs, leading to T cell activation. This activation, however, results in the inhibition of the immune response through mechanisms such as blocking T lymphocyte reactions, reducing T lymphocyte proliferation, suppressing Treg lymphocyte activity, and decreasing cytokine secretion.

  
Combined blockade of CTLA4 and PD1 signaling pathways has been shown to extend animal survival in B16 melanoma and K7M2 metastatic osteosarcoma models, whereas monotherapy with antibodies blocking CTLA-4 or PD-1 alone has limited efficacy. Clinically, combination therapy with antibodies targeting PD-1/L1 and CTLA4 has been extensively studied and applied. The combination of Nivolumab and Ipilimumab was the first to receive approval from the U.S. FDA for the treatment of melanoma, becoming the world's first approved combination regimen. Subsequently, multiple indications have been approved (including renal cell carcinoma, hepatocellular carcinoma, and colorectal cancer).

PDL1 CD80&CD86 aAPC Cell serves as target cells for PD1&CTLA4 Dual Effector Reporter Cells, effectively mimicking the signal transduction process of PD1&CTLA4 in vivo. The principle is shown in the figure below.

Figure 1. Schematic of the PDL1 CD80&CD86 aAPC Cell Model.

Figure 2. Dose Response of Blocking Antibodies in PD1&CTLA4 Dual Effector Reporter Cell (C16) With PD-L1 CD80&CD86 aAPC Cell (C14).

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.