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Human leukocyte antigen G (HLA-G) is a non-classical HLA-I molecule primarily expressed in extracellular trophoblasts of the placenta, mediating maternal-fetal immune tolerance during pregnancy. While HLA-G expression is limited in healthy tissues, pathological conditions can induce it. HLA-G expression has been observed in various cancers, including colorectal cancer, breast cancer, melanoma, and ovarian cancer. HLA-G plays an active role in regulating innate and adaptive immune responses and promoting tolerance, but an unfavorable role in inducing immune escape mechanisms. A common mechanism for evading immune surveillance is the loss or downregulation of classical HLA class Ia antigens, and the neo-expression of non-classical HLA class Ib antigens (such as HLA-E, -F, and -G).

The HLA-G primary transcript encodes seven different isoforms via alternative splicing: HLA-G1, -G2, -G3, and -G4 are membrane-bound, while HLA-G5, -G6, and -G7 are soluble isoforms. HLA-G1 and -G5 are the only isoforms that can bind to β2M. β2M serves as an additional binding site for the receptor, and it has been demonstrated that HLA-G1 and -G5 binding to the receptor does not necessarily require β2M binding.

The B2M-associated HLA-G aAPC cell, serving as the target cell for the LILRB2 Effector Reporter Cell, effectively mimics the in vivo signal transduction processes of LILRB1/2; the underlying principle is illustrated in Figure 1.

Figure 1: Schematic Diagram of the B2M-associated HLA-G aAPC Cell Model

Figure 1&2. Recombinant B2M-associated HLA-G/aAPC Cell stably expressing B2M.Recombinant B2M-associated HLA-G/aAPC Cell stably expressing HLA-G.

Figure 3. Dose Response of LILRB1 Blocking Antibody in LILRB1 Effector Reporter Cells (C13) with B2M-associated HLA-G/aAPC Cells (C15).

Figure 4.Dose Response of LILRB2 Blocking Antibody in LILRB2 Effector Reporter Cells (C24) with B2M-associated HLA-G/aAPC Cells(C15).

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 B2M-associated HLA-G/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.