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KIR3DL3 (Killer-cell Immunoglobulin-like Receptor 3DL3) is a member of the Killer-cell Immunoglobulin-like Receptor (KIR) family and belongs to the inhibitory receptor subclass. Members of the KIR family facilitate both innate and adaptive immune responses through their expression on NK cells and T cells. KIR3DL3 comprises three extracellular immunoglobulin-like domains, a transmembrane region, and an intracellular segment containing an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM). KIR3DL3 is primarily expressed on Natural Killer (NK) cells and certain T-cell subsets (such as CD8+ T cells); it principally regulates NK cell activity and plays a balancing role in maintaining immune tolerance as well as in anti-tumor and anti-infection immunity.

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 featuring 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) and within inflammatory microenvironments. HHLA2 possesses dual immunomodulatory functions: it binds to the inhibitory receptor KIR3DL3 (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.

KIR3DL3 exhibits weak binding affinity for classical MHC Class I molecules, such as HLA-A and HLA-B. Upon binding to HLA, the intracellular ITIM motifs of KIR3DL3 are phosphorylated by Src-family kinases (such as Lck). These phosphorylated ITIMs recruit SH2-domain-containing tyrosine phosphatases (SHP-1/SHP-2), which subsequently dephosphorylate activating signaling molecules (such as Syk and Vav1), thereby blocking NK cell degranulation and cytokine (IFN-γ) secretion.

The Jurkat E6.1 Human KIR3DL3 Effector Reporter Cell Model—effectively simulates the signal transduction process of KIR3DL3 *in vivo*. The underlying principle is illustrated in the figure below.

Figure 1. Schematic Diagram of the Jurkat E6.1 Human KIR3DL3 Effector Reporter Cell Model

Figure 2. Recombinant KIR3DL3 Effector Reporter Cell stably expressing KIR3DL3.

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

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.