KC-2848

Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG Cell Line

26805

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Background of Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG Cell Line

In cancer-immunity cycle, the immune checkpoint PD1 and its ligand PDL1 act as accomplices to help tumors resist to immunity-induced apoptosis and promote tumor progression. Immunotherapy targeting PD1/PDL1 axis can effectively block its pro-tumor activity. Anti-PD1/PDL1 therapy has achieved great success in the past decade.TIGIT interacts with CD155 expressed on antigen-presenting cells or tumor cells to down-regulate T cell and natural killer (NK) cell functions. TIGIT has emerged as a key inhibitor of anti-tumor responses that can hinder multiple steps of the cancer immunity cycle. Pre-clinical studies indicated that TIGIT blockade may protect against various solid and haematological cancers. Several monoclonal antibodies (mAbs) that block the inhibitory activity of human TIGIT have been developed. Clinical trials are ongoing, investigating TIGIT blockade as a monotherapy or in combination with anti-PD1/PD-L1 mAbs for the treatment of patients with advanced solid malignancies. PVRIG, also known as CD112R, is a transmembrane protein that can act as a coinhibitor to suppress T-cell receptor mediated signals. Studies have already shown that a PVRIG gene can inhibit CD8+-T-cell function, thus making it a potential novel checkpoint for human T cells.

Specifications

Catalog Number	KC-2848
Cell Line Name	Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG Cell Line
Host Cell Line	Jurkat-NFAT-Luc2-PD1-TIGIT
Description	Stable Jurkat-NFAT-Luc2 cell line expressing exogenous PD1, TIGIT and PVRIG gene.
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS +0.5µg/mL Puromycin + 300µg/mL Hygromycin B + 500µg/mL G418
Selection Marker	Puromycin, Hygromycin B, G418
Morphology	lymphoblast
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

Jurkat NFAT Luc2 PD1 TIGIT PVRIG Cell Line was generated using a lentiviral vector expressing PD1 & TIGIT & PVRIG sequence.

Characterization

Figure 1. Characterization of human TIGIT & human PD1 overexpression in the Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG stable clone using FACS.

Figure 2. Characterization of human PVRIG overexpression in Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG stable clone using FACS.

Figure 3. Jurkat-NFAT-Luc2-PD1-TIGIT-PVRIG cells and KC-2777 293T-OS8-PVRL2-PD1 cells were seeded into 96-well plates, treated with different antibodies for 6 hours, and then read out using Bright-Glo Detection System.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10%FBS, 300µg/mL Hygromycin B, 500µg/mL G418 and 0.5µg/mL Puromycin)in a 37°C water bath. 2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes. 3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol. 4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium. 5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet. 6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask. 7. Incubate the flask at 37°C, 5% CO₂ incubator. 8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use. 2. Keep the freezing medium on ice and label cryovials. 3. Transfer cells to a sterile, conical centrifuge tube, and count the cells. 4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium. 5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium. 6. Aliquot 1 mL of the cell suspension into each cryovial. 7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer. 8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Lei Q, Wang D, Sun K, Wang L, Zhang Y. Resistance Mechanisms of Anti-PD1/PDL1 Therapy in Solid Tumors. Front Cell Dev Biol. 2020 Jul 21;8:672. doi: 10.3389/fcell.2020.00672. PMID: 32793604; PMCID: PMC7385189. 2.Russell FA, King R, Smillie SJ, Kodji X, Brain SD. Calcitonin gene-related peptide: physiology and pathophysiology. Physiol Rev. 2014 Oct;94(4):1099-142. doi: 10.1152/physrev.00034.2013. PMID: 25287861; PMCID: PMC4187032. 3.Whelan S, et al. PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+- T-cell Function. Cancer Immunol Res, 7, 257-268 (2019).