Leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), also known as ILT4, LIR2, monocyte/macrophage immunoglobulin-like receptor 10, MIR-10, and CD85d, belongs to the leukocyte immunoglobulin-like receptor (LIR) family of transmembrane glycoproteins that negatively regulate immune cell activation.LILRB2 is expressed on both innate (monocytes, macrophages, basophils, and DCs) and adaptive (CD4+ T cells) immune cells and interacts with MHC-I on nucleated cells (unknown specific ligand). LILRB2 interacts with related ligands HLA-G, ANGPTLs, SEMA4A and CD1d in tumor microenvironment, resulting in myeloid cells promoting tumor growth and enhancing tumor immune escape. It has been shown pre-clinically that LILRB2 antagonism promotes macrophage maturation and activation. MK-4830 (Merck and Agenus) is an anti-LILRB2 fully human IgG4 monoclonal antibody, and phase II clinical studies have been initiated. In addition, IO-108, JTX-8064, and NGM707 are in phase I clinical studies.
Cell Line Name:
HEK293T LILRB2 isoform2 Cell Line
Host Cell Line:
Human HEK293T cell line
One vial of frozen cells (5X106 per vial)
Stable in culture over a minimum of 10 passages
Drug screening and biological assays
70% DMEM+20% FBS+10% DMSO
Split saturated culture 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL
37 °C with 5% CO2
Liquid nitrogen immediately upon receiving
Approximately 30 hours
Cell Line Generation
293T human LILRB2 isoform2 cell line was generated using a lentiviral vector expressing the human LILRB2 isoform2 sequence.
Characterization using PCR sequencing and WB
1. Prewarm culture medium (DMEM + 10% FBS + 1μg/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 1:3-1:6 every 3 days; seed out at about 1-3 × 105 cells/mL.
1. Prepare the freezing medium (70% DMEM + 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
1. Lentz RW, Colton MD, Mitra SS, Messersmith WA. Innate Immune Checkpoint Inhibitors: The Next Breakthrough in Medical Oncology? Mol Cancer Ther. 2021 Jun;20(6):961-974. doi: 10.1158/1535-7163.MCT-21-0041.
2. Chen HM, van der Touw W, Wang YS, Kang K, Mai S, Zhang J, Alsina-Beauchamp D, Duty JA, Mungamuri SK, Zhang B, Moran T, Flavell R, Aaronson S, Hu HM, Arase H, Ramanathan S, Flores R, Pan PY, Chen SH. Blocking immunoinhibitory receptor LILRB2 reprograms tumor-associated myeloid cells and promotes antitumor immunity. J Clin Invest. 2018 Dec 3;128(12):5647-5662. doi: 10.1172/JCI97570.
3. Zhao P, Xu Y, Jiang LL, Fan X, Ku Z, Li L, Liu X, Deng M, Arase H, Zhu JJ, Huang TY, Zhao Y, Zhang C, Xu H, Tong Q, Zhang N, An Z. LILRB2-mediated TREM2 signaling inhibition suppresses microglia functions. Mol Neurodegener. 2022 Jun 18;17(1):44. doi: 10.1186/s13024-022-00550-y.
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