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HHLA2 (also known as B7H5, B7H7, or B7y) is a member of the B7 family. It is a type I transmembrane protein consisting of an extracellular region containing tandem IgV1-IgC-IgV2 domains, a transmembrane region, and a cytoplasmic tail. HHLA2 is expressed at low levels in tissues such as the placenta and intestinal epithelial cells, but is highly expressed in various solid tumors (including lung cancer, breast cancer, colorectal cancer, and renal cancer) as well as in inflammatory microenvironments. HHLA2 exerts dual immunomodulatory functions: it binds to the inhibitory receptor KIR3DL3 (found on NK cells) and an unidentified T-cell receptor to transmit inhibitory signals, thereby attenuating anti-tumor immune responses; conversely, it activates the PI3K-AKT pathway via the receptor TMIGD2, thereby promoting T-cell survival and memory formation.

The CHO-K1 Human HHLA2(B7H7) aAPC Cell Model—effectively simulates the signal transduction process of KIR3DL3/ HHLA2(B7H7) or TMIGD2/HHLA2(B7H7) *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the CHO-K1 Human HHLA2(B7H7) aAPC Cell Model

CHO cell line expressing full length human . Expression is confirmed by flow cytometry.

Figure 2.Recombinant HHLA2(B7H7) aAPC Cell stably expressing HHLA2(B7H7).

Figure 3. Dose Response of HHLA2 Blocking Abs in KIR3DL3 Effector Reporter Cell (C1) with HHLA2(B7H7) aAPC Cell(C1).

Figure 4. Dose Response of HHLA2 Blocking Abs in TMIGD2 Effector Reporter Cell(C1) with HHLA2(B7H7) 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 HHLA2(B7H7) 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.