Recombinant Human Leukocyte immunoglobulin-like receptor subfamily B member 3 (LILRB3)

What is LILRB3 and what are its structural and functional characteristics?

LILRB3, also known as CD85a, ILT5, or LIR3, is an immunoglobulin superfamily member involved in immune regulation . The mature form of LILRB3 is a highly polymorphic 85-95 kDa glycoprotein consisting of a 420 amino acid extracellular domain (ECD) with four Ig-like domains, a 21 amino acid transmembrane segment, and a 167 amino acid cytoplasmic domain containing three immunoreceptor tyrosine-based inhibitory motifs (ITIMs) .

The ITIMs in LILRB3's cytoplasmic domain are crucial for its inhibitory function. When activated, these motifs recruit phosphatase SHP-1, which inhibits signaling events and provides negative regulation of immune responses . LILRB3 belongs to the subfamily B of leukocyte immunoglobulin-like receptors, which are characterized by their inhibitory functions, in contrast to the activating subfamily A members that signal through association with FcR gamma .

Alternative splicing generates an isoform with a 17 amino acid insertion in the juxtamembrane extracellular domain, adding to the complexity of LILRB3 regulation and function . This structural diversity may contribute to the receptor's varied functions across different cell types and contexts.

What is the expression pattern of LILRB3 across immune cell types?

LILRB3 expression is restricted to cells of the myeloid lineage. It is expressed on the surface of peripheral monocytes, neutrophils, eosinophils, basophils, and mast cell progenitors . Recent research has also demonstrated LILRB3 expression on in vitro differentiated mast cells and osteoclasts .

Interestingly, while LILRB3 is typically considered myeloid-specific, studies have detected LILRB3 expression on T cells in rheumatoid arthritis (RA) patients. The percentage of both CD4+ and CD8+ T cells expressing LILRB3 was significantly higher in patients with RA compared to controls or systemic lupus erythematosus (SLE) patients . Within the RA patient group, LILRB3 expression on T cells correlated with disease activity, being highest in active RA compared to inactive RA (DAS28<3.2) .

Expression analysis of LILRB3 can be recapitulated during differentiation of PLB-985 cells into neutrophil-like cells, providing a model to study the regulation and function of this receptor . This model system helps overcome the challenges associated with studying primary human neutrophils, which have a short lifespan and limited manipulability.

How does LILRB3 modulate neutrophil function and immune responses?

LILRB3 serves as an important checkpoint to control human neutrophil activation and their anti-microbial effector functions during resting and early-activation stages of the neutrophil life-cycle . Continuous ligation of LILRB3 has been shown to inhibit key IgA-mediated effector functions in neutrophils, including:

• Production of reactive oxygen species (ROS)

• Phagocytic uptake of microbes

• Microbial killing capacity

This inhibitory activity suggests that LILRB3 functions as a negative regulator of neutrophil activation, potentially preventing excessive immune responses that could lead to tissue damage.

On osteoclast precursors, LILRB3 ligation inhibits RANK L/TRANCE or M-CSF induced differentiation, indicating a role in bone homeostasis regulation . LILRB3 can also bind to ligands exposed on necrotic tumor cells, which may impact immune responses in the tumor microenvironment .

Interestingly, both LILRB3 and its murine relative PIR-B have been identified as receptors for Staphylococcus aureus, and their activation by bacteria influences innate immune responses triggered by Toll-like receptors (TLRs) . This suggests a complex role in pathogen recognition and response modulation.

What methodologies are available for production and purification of recombinant LILRB3?

Researchers studying LILRB3 frequently require purified recombinant protein for functional studies, binding assays, or antibody production. Based on published protocols, the following methodology has been successfully employed:

Expression vector construction:

• Amplify signal peptides and extracellular domains of LILRB3 from cDNA vectors using PCR

• Design forward primers containing an overhang for the expression vector (e.g., pcDNA3.4), a BamHI restriction site, Kozak sequence, and LILRB3-specific region

• Design reverse primers with an overhang for the expression vector, a NotI restriction site, stop codon, and 6xHis tag

PCR conditions:

• Use Phusion High Fidelity Taq polymerase

• Thermocycling parameters: 1 cycle (98°C for 2 mins), 35 cycles (98°C for 15 seconds, 62°C for 30 seconds, 72°C for 30 seconds), 1 cycle (72°C for 10 minutes)

Expression and purification:

• Express recombinant LILRB3 in EXPI293F cells cultured in EXPI media at 37°C with 5% CO₂

• Package 50 μg of vector into 250 μg PEI-MAX 40K in 5 ml of Opti-MEM media

• After 20 minutes of incubation at room temperature, add the mixture to 50 ml of EXPI293F cells at 2×10⁶ cells/ml

• Harvest supernatants after 72 hours of culture

• Dialyze against 50 mM Tris, 300 mM NaCl, pH 8

• Purify using affinity chromatography (e.g., ÄKTA Pure system) with Nickel columns for His-tagged proteins

This methodology produces C-terminal His-tagged recombinant LILRB3 that can be used for various experimental applications, including binding studies, functional assays, and structural analyses.

What techniques can be employed to study LILRB3 expression in human samples?

Several complementary techniques can be used to assess LILRB3 expression in human samples:

Flow cytometry:

• Incubate cells (approximately 5×10⁶ cells/ml) with anti-LILRB3 monoclonal antibodies (5 μg/ml) for 1 hour at 4°C

• For detection, incubate with fluorochrome-conjugated secondary antibodies (e.g., PE-conjugated goat anti-mouse-IgG) for 1 hour at 4°C

• Analyze by flow cytometry, gating on appropriate cell populations based on forward and side scatter properties

Immunoprecipitation and Western blotting:

• Lyse cells (3×10⁷) in ice-cold 0.3% saponin containing protease inhibitors (1:1000 AEBSF and Leupeptin) for 30 minutes at 4°C

• Clear lysates by centrifugation (15 minutes at 4°C)

• Incubate supernatants with Dynabeads Protein A pre-coated with anti-LILRB3 antibodies for 2 hours at 4°C

• Wash beads thoroughly in ice-cold lysis buffer

• Elute proteins by heating in sample buffer at 95°C for 5 minutes

• Separate by SDS-PAGE, transfer to membranes, and probe with rabbit anti-LILRB3 polyclonal antibodies

• Detect using appropriate secondary antibodies (e.g., goat anti-rabbit-HRP)

Mass spectrometry:
This approach can confirm LILRB3 expression in immunoprecipitated samples from cell lysates, providing unambiguous identification of the receptor and potentially revealing post-translational modifications or binding partners .

A combination of these methods provides robust validation of LILRB3 expression and can differentiate between LILRB3 and the closely related LILRA6, which has high similarity in the extracellular domain and may cross-react with some antibodies .

How does LILRB3 expression correlate with disease states such as rheumatoid arthritis?

The relationship between LILRB3 expression and disease states, particularly autoimmune conditions like rheumatoid arthritis (RA), has been investigated through comparative analyses of patient samples. Researchers studying this correlation have employed the following methodological approach:

Sample collection and preparation:

• Obtain heparinized blood samples from:

  • Healthy blood donors (controls)

  • RA patients with various disease activity scores (DAS28 <3.2 for inactive RA; DAS28 >3.2 for active RA)

  • SLE patients for comparison with another autoimmune condition

Staining and flow cytometry:

• Isolate peripheral blood mononuclear cells (PBMCs)

• Stain with antibodies against:

  • LILRB3 (e.g., Biolegend, PE)

  • T cell markers: CD3 (e.g., Biolegend, APC-Cy7), CD4 (e.g., BD, PerCP-Cy5.5), CD8 (e.g., Biolegend, V500)

  • Additional markers as needed: CD25 (e.g., Biolegend, PE/Cy5), CD28 (e.g., Biolegend, Pacific Blue)

• Include appropriate isotype controls

• Analyze by flow cytometry, gating on lymphocyte populations

Statistical analysis:
Perform ANOVA and non-parametric tests (e.g., Mann-Whitney Test) to compare LILRB3 expression between groups.

Key findings:
The percentage of both CD4+ and CD8+ T cells expressing LILRB3 was significantly higher in both inactive and active RA compared to controls or SLE patients (p=0.0397) . Within the RA patient group, LILRB3 expression correlated with disease activity, being significantly higher in active RA compared to inactive RA (p=0.0287) .

These findings suggest that LILRB3 expression may serve as a biomarker for RA disease activity and could potentially be involved in disease pathogenesis. The increased expression of this inhibitory receptor in active RA may represent a compensatory mechanism to reduce disease activity, as LILRB3 functions to inhibit immune cell activation .

What is the role of LILRB3 in cancer immunotherapy and how can it be targeted?

LILRB3 has emerged as a potential target for cancer immunotherapy, particularly due to its expression on immunosuppressive myeloid cells that inhibit antitumor immunity. Recent research has revealed several important aspects of LILRB3's role in cancer:

LILRB3 function in cancer immunity:

• LILRB3 is functionally expressed on immunosuppressive myeloid cells, including myeloid-derived suppressor cells (MDSCs)

• It supports the immunosuppressive activity of myeloid cells in the tumor microenvironment

• Galectin-4 and galectin-7 have been identified as activators of LILRB3

• LILRB3 signaling helps cancer cells evade immune surveillance

Experimental approaches to target LILRB3:

• Development of antagonistic antibodies that block LILRB3 signaling

• Assessment of antibody efficacy on patient-derived samples:

  • Isolation of myeloid cells from patients with solid cancers

  • Treatment with anti-LILRB3 antagonistic antibodies

  • Evaluation of immunosuppressive activity modulation

• In vivo validation using myeloid-specific LILRB3 transgenic mice:

  • Tumor challenge experiments

  • Treatment with anti-LILRB3 antibodies

  • Assessment of tumor development and immune cell infiltration/function

Experiments have demonstrated that blockade of LILRB3 signaling by antagonistic antibodies inhibits the activity of immunosuppressive myeloid cells in samples from some patients with solid cancers . Furthermore, anti-LILRB3 treatment has been shown to impede tumor development in myeloid-specific LILRB3 transgenic mice through a T cell-dependent mechanism .

These findings suggest that LILRB3 blockade may represent a novel approach for immunotherapy of solid cancers, particularly in combination with existing T cell-centered immune checkpoint blockade therapies to overcome resistance mechanisms involving immunosuppressive myeloid cells.

What experimental approaches can differentiate between LILRA6 and LILRB3 functions?

LILRA6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) are paired receptors with high similarity in their extracellular domains, making it challenging to distinguish between them in experimental settings. Antibodies generated against these receptors often cross-react, creating uncertainty about which receptor is truly expressed and functioning in specific cell types . The following experimental approaches can help differentiate between LILRA6 and LILRB3:

Immunoprecipitation and mass spectrometry:

• Lyse cells of interest (e.g., neutrophils) using 0.3% saponin with protease inhibitors

• Perform immunoprecipitation using antibodies against LILRA6, LILRB3, or isotype controls

• Analyze immunoprecipitated proteins by mass spectrometry to definitively identify the receptor present

This approach has successfully demonstrated that LILRB3, but not LILRA6, is detected in human neutrophil lysates .

Functional assays with specific signaling pathway inhibitors:

• Design experiments to distinguish between ITAM-mediated signaling (LILRA6) and ITIM-mediated signaling (LILRB3)

• Use SHP-1/SHP-2 inhibitors to block LILRB3 signaling

• Use Syk inhibitors to block LILRA6 signaling

• Assess functional outcomes such as cytokine production, phagocytosis, or ROS generation

Genetic approaches:

• Use siRNA or CRISPR-Cas9 to specifically knock down LILRA6 or LILRB3

• Validate knockdown specificity using RT-PCR with gene-specific primers

• Assess functional consequences of receptor-specific knockdown

Receptor-specific stimulation:

• Use recombinant ligands with differential binding preferences for LILRA6 versus LILRB3

• Generate receptor-specific agonistic or antagonistic antibodies using careful screening for specificity

• When using mouse monoclonal antibodies, include FcγR inhibitors (e.g., FLIPr-like) to ensure that observed effects are due to Fab-mediated receptor binding rather than Fc-FcγR interactions

By combining these approaches, researchers can more confidently attribute observed functions to either LILRA6 or LILRB3, advancing understanding of their distinct roles in immune regulation.