KC-6333

A204-HHLA2-KO Cell Line

64698

Home » A204-HHLA2-KO Cell Line

Background of A204-HHLA2-KO Cell Line

HHLA2 (HERV-H LTR-Associating 2) is a Protein Coding gene. This gene encodes a member of the B7/CD28 family of immune co-regulatory molecules. Members of this family share immunoglobulin-like domains and play pivotal roles in regulating T-cell activation, immune tolerance, and tumor immune evasion. The encoded type I transmembrane protein, also known as B7-H7, contains extracellular immunoglobulin V-like (IgV) and C-like (IgC) domains and is constitutively expressed on monocytes, macrophages, and dendritic cells, with aberrant overexpression observed in a wide range of human cancers. HHLA2 serves as a dual-function immune checkpoint by engaging two distinct receptors: it delivers a co-stimulatory signal to T cells and NK cells through TMIGD2 (also known as CD28H), and simultaneously transmits an inhibitory signal via the killer cell immunoglobulin-like receptor KIR3DL3. Through these interactions, HHLA2 can both enhance and suppress immune responses depending on the receptor context, making it a critical modulator of anti-tumor immunity and a promising target for cancer immunotherapy. Alternative splice variants have been described but their full-length sequences and functional characteristics have not been fully determined.

Specifications

Catalog Number	KC-6333
Cell Line Name	A204-HHLA2-KO Cell Line
Clone Number	1B2
Host Cell Line	A204
Description	Stable A204 clone with human HHLA2 gene knockout, No.1B2
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% DMEM+20% FBS+10% DMSO
Propagation Medium	90% DMEM+10% FBS
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

A204-HHLA2-KO cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of A204-HHLA2-KO cell line stable clone using PCR sequencing.

Figure 2: Characterization of A204-HHLA2-KO cell line stable clone using RT-PCR sequencing.

Cell Resuscitation

Prewarm culture medium (90% DMEM + 10% FBS) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:5-1:8 every 2-3 days.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

Zhao R, Chinai JM, Buhl S, Scandiuzzi L, Ray A, Jeon H, Ohaegbulam KC, Ghosh K, Zhao A, Scharff MD, Zang X. HHLA2 is a member of the B7 family and inhibits human CD4 and CD8 T-cell function. Proc Natl Acad Sci U S A. 2013;110(24):9879-9884. doi:10.1073/pnas.1303524110. PMID: 23716685; PMCID: PMC3683725.
Bhatt RS, Berjis A, Konge JC, Mahoney KM, Klee AN, Freeman SS, Chen CH, Jegede OA, Catalano PJ, Pignon JC, Bueno R, Zhu B, Huang J, Wu CJ, Freeman GJ, Hammerman PS, El-Gabry EA. KIR3DL3 is an inhibitory receptor for HHLA2 that mediates an alternative immune checkpoint pathway. Nat Immunol. 2021;22(2):126-138. doi:10.1038/s41590-020-00834-7. PMID: 33398179.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.