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Natural killer cell receptors (NKRs) are expressed on the surface of NK cells and T cells, participating in the regulation of downstream signal activation and inhibition, and actively involved in modulating immune responses. They possess C-type lectin-like domains that have lost the ability to bind Ca²⁺, instead participating in protein-protein interactions rather than carbohydrate binding.

  
The KLRB1 protein (NKR-P1A or CD161), a type II transmembrane protein belonging to the C-type lectin-like receptor family, is expressed on NK cells and subsets of CD4⁺ and CD8⁺ T cells. As an inhibitory receptor on mature NK cells, it inhibits NK cell cytotoxicity and cytokine secretion. Blocking or inactivation of CD161 can enhance T cell-mediated killing of gliomas, favoring the control of tumor growth in vivo.

  
Lectin-like transcript 1 (LLT1 or CLEC2D) is the ligand for the CD161 receptor expressed on natural killer cells and T cells. CD161 inhibits NK cell cytotoxicity and IFN-γ secretion by binding to LLT1 expressed on activated leukocytes. The LLT1/CD161 interaction shuts down NK cell activation while co-stimulating T cells.

The CLEC2D aAPC Cell reporter gene drug target model effectively simulates the signal transduction process of CD161/CLEC2D in vivo, as illustrated in the figure below.

Figure 1. Schematic of the CLEC2D aAPC Cell Model

Figure 2.  Dose Response of CD161 Blocking Antibody in CD161 Effector Reporter Cell (C8) with CLEC2D aAPC Cell.

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 CLEC2D aAPC 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.