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B and T Lymphocyte Attenuator (BTLA) is a member of the CD28 superfamily. The protein structure of BTLA is similar to that of Programmed Cell Death Protein 1 (PD-1) and Cytotoxic T-Lymphocyte-Associated Protein 4 (CTLA-4), comprising an extracellular domain, a transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain contains a Growth Factor Receptor-Bound Protein 2 (Grb-2) association motif, an Immunoreceptor Tyrosine-based Switch Motif (ITSM), and an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM). BTLA is widely expressed in lymph nodes, the thymus, and the spleen, but is expressed at low levels—or not at all—in organs such as the heart, kidneys, brain, and liver.

HVEM belongs to the Tumor Necrosis Factor Receptor superfamily and is the only detectable ligand for BTLA in human cells. HVEM can interact with BTLA in either a *cis* or a *trans* manner. On cells that co-express both BTLA and HVEM, BTLA can interact with HVEM in *cis*; conversely, when BTLA and HVEM are expressed on different cells, a *trans* interaction occurs. In addition to BTLA, HVEM can also interact with CD160, Lymphotoxin-α, LIGHT (TNFSF14), and Synaptic Adhesion-like Molecule 5 (SALM5). Notably, BTLA and CD160 compete for the same binding site within the CRD1/CRD2 region of HVEM, whereas LIGHT binds independently to the opposite side of HVEM within the CRD2/CRD3 region.

The binding of HVEM to BTLA triggers the tyrosine phosphorylation of the ITIM within BTLA, leading to the recruitment of the Src homology 2 (SH2)-domain-containing protein tyrosine phosphatases SHP-1 and SHP-2; these protein tyrosine phosphatases typically mediate immunosuppressive effects. However, the binding of the Grb-2 association motif to Grb-2 results in the recruitment of the p85 regulatory subunit of PI3K and subsequent T-cell activation. The BTLA/SHP2 Reporter Cell model effectively mimics the *in vivo* signal transduction processes of BTLA and SHP2; the underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the BTLA/SHP2 Reporter Cell Model

Figure 2. Recombinant BTLA/SHP2 Reporter Cell constitutively expressing BTLA.

Figure 3. Dose Response of BTLA agonist Ab Induced SHP2 Recruitment in BTLA SHP2 Reporter Cell(C11) with FCGR2B(FcγRIIb) CHO.

Figure 4. Inhibtion of HVEM Induced SHP2 Recruitment In BTLA SHP2 Reporter Cell(C11) By lcatolimab with HVEM U2OS (C7).

Cell Passage Procedures

1.This cell line grows in suspension.
2.Upon receipt, cells should be thawed immediately or stored in liquid nitrogen until use.
3.Before thawing, pre-warm the water bath and culture medium to 37 °C, and prepare a small amount of dry ice.
4.Remove the cryovial from storage and transport it to the cell culture laboratory on dry ice.
5.Rapidly thaw the cells in a 37 °C water bath. Once the cells are completely thawed, spray the cryovial with 70% ethanol for disinfection and transfer it to a biosafety cabinet.
6.Add 10 mL of pre-warmed culture medium into a 15 mL centrifuge tube. Transfer the contents of the cryovial into the tube and centrifuge at 1000 rpm for 5 minutes.
7.Carefully discard the supernatant. Resuspend the cell pellet in 5 mL of pre-warmed culture medium by gentle pipetting. Immediately perform cell counting and adjust the cell density to 3–6 × 10⁵ cells/mL based on the counting results, then transfer the cells into a culture flask.
8.Count the cells every 1–2 days. When the cell density exceeds 1 × 10⁶ cells/mL, passage the cells promptly or add fresh culture medium. Maintain the cell density between 2 × 10⁵ and 1 × 10⁶ cells/mL.

Suspension Cell Cryopreservation Procedure:

1.Collect 8 × 10⁶ cells, centrifuge, and discard the supernatant.
2.Add 1 mL of cell freezing medium (90% FBS + 10% DMSO) and gently pipette to mix thoroughly. Transfer the suspension into a cryovial.
3.Immediately place the cryovial into a controlled-rate freezing container (Nalgene 5100-0001), fill with isopropanol up to the indicated level, and store at −80 °C.
4.After 24 hours, transfer the cryovial to liquid nitrogen for long-term storage.