Recombinant Human Leukocyte immunoglobulin-like receptor subfamily A member 6 (LILRA6), partial

What is LILRA6 and what is its basic structure?

LILRA6, also known as ILT8 and CD85b, is a transmembrane molecule belonging to the Leukocyte Immunoglobulin-like Receptor (LILR) family of immune regulatory proteins. The mature human LILRA6 protein consists of a 424 amino acid extracellular domain (ECD) containing two immunoglobulin-like domains, a 21 amino acid transmembrane segment, and a 13 amino acid cytoplasmic domain. The protein contains a positively charged arginine residue in the transmembrane segment that likely mediates association with the signaling protein Fc epsilon RI gamma. Alternative splicing can generate a shorter isoform that is truncated near the middle of the ECD .

How does LILRA6 differ from LILRB3?

LILRA6 and LILRB3 represent a pair of activating/inhibitory receptors with nearly identical extracellular domains (94% amino acid sequence identity). They are considered "paired receptors" as they have almost identical extracellular domains but differ significantly in their signaling activities. While LILRB3 is inhibitory and signals through immunoreceptor tyrosine-based inhibition motifs (ITIMs) in its cytoplasmic tail, LILRA6 is activating and signals through association with FcRγ, which contains an immunoreceptor tyrosine-based activation motif (ITAM). This structural arrangement allows these receptors to deliver opposing signals despite recognizing the same ligands .

What are the primary cellular expression patterns of LILRA6?

LILRA6 is primarily expressed on cells of myelomonocytic lineage, with monocytes showing the highest expression levels. This expression pattern is similar to that of LILRB3, which is detected on monocytes, macrophages, dendritic cells, granulocytes, and some T cells. Importantly, previous studies have had difficulty distinguishing between LILRB3 and LILRA6 expression due to their structural similarity, but recent gene-specific analyses have confirmed monocytes as the primary cell type expressing LILRA6. The expression level of LILRA6 on these cells correlates directly with the gene copy number .

What genetic variations of LILRA6 have been identified and how can researchers account for them in experimental design?

LILRA6 exhibits extensive genetic polymorphism and copy number variation (CNV). In a study of Caucasian populations, approximately 32% of individuals possessed more than 2 copies of LILRA6, while 4% had only one copy per diploid genome. Copy numbers can range from one to six per individual. When designing experiments with LILRA6, researchers should consider:

• Genotyping subjects for LILRA6 copy number using quantitative PCR methods

• Sequencing to identify specific allelic variants

• Including appropriate controls that account for CNV diversity

The CNV status can significantly affect expression levels and potentially influence receptor function and signaling capability. Researchers should therefore stratify experimental cohorts based on LILRA6 copy number when analyzing functional outcomes .

How should researchers approach LILRA6 expression analysis considering its polymorphism and CNV?

When analyzing LILRA6 expression, researchers should implement a comprehensive approach that accounts for both polymorphism and copy number variation:

• Use gene-specific primers that distinguish LILRA6 from the highly similar LILRB3

• Employ multiple reference genes (e.g., ZNF80, GPR15, or TERT) for accurate normalization

• Combine quantitative PCR with genomic DNA analysis to correlate expression with copy number

• Consider analyzing multiple gene regions (e.g., 3'UTR and intronic fragments)

• When possible, perform allele-specific expression analysis to identify differential expression of variants

A study examining LILRA6 mRNA expression in peripheral blood mononuclear cells found that expression levels correlate with copy number of the gene, suggesting a gene dosage effect that should be accounted for in experimental design and data interpretation .

What approaches can be used to differentiate between LILRA6 and LILRB3 in expression studies?

Differentiating between LILRA6 and LILRB3 presents a significant challenge due to their 94% sequence identity in the extracellular domain. Researchers should consider:

• Gene-specific PCR primers targeting divergent regions, particularly in the 3'UTR

• Allele-specific oligonucleotide probes for quantitative PCR

• Genomic DNA assays to determine copy number alongside expression analysis

• Custom antibodies targeted to the few divergent extracellular residues

• Functional assays that distinguish between activating (LILRA6) and inhibitory (LILRB3) activity

It is critical to note that most commercially available antibodies cannot distinguish between these receptors, and many previous studies reporting "LILRB3 expression" likely detected both LILRB3 and LILRA6. Researchers should explicitly acknowledge this limitation when interpreting published data .

What signaling pathways are activated downstream of LILRA6 and how do they differ from LILRB3?

LILRA6 engages several key signaling pathways that contribute to immune cell activation:

LILRA6 associates with the FcεRIγ adaptor protein through its transmembrane arginine residue, leading to ITAM phosphorylation and downstream signal propagation. In contrast, LILRB3 contains cytoplasmic ITIMs that recruit phosphatases like SHP-1 and SHP-2, resulting in negative regulation of the same pathways. This molecular arrangement allows these paired receptors to fine-tune myeloid cell responses based on the relative expression levels and engagement of each receptor .

What are the known ligands for LILRA6 and how can researchers investigate novel binding partners?

Several potential ligands for LILRA6 have been identified:

• Cytokeratins 8, 18, and 19 - exposed on necrotic epithelial cells

• Angiopoietin-like 7 - demonstrated in binding assays

• MHC class I molecules - suggested by functional studies

To investigate novel binding partners, researchers can employ:

• Immunoprecipitation followed by mass spectrometry

• Recombinant LILRA6-Fc fusion proteins for pull-down assays

• Reporter cell assays (e.g., 2B4 reporter system) to validate functional binding

• Surface plasmon resonance to quantify binding affinities

• Cell-based binding assays using allele-specific LILRA6 variants

Importantly, different LILRA6 alleles show varying binding affinities to ligands. For instance, research on the closely related LILRB3 found that specific alleles (e.g., LILRB312) displayed particularly strong binding to necrotic cells compared to others (e.g., LILRB301). Given the extensive sequence similarity in the extracellular domains, similar allelic binding variation likely exists for LILRA6 .

How does LILRA6 interact with MHC class I molecules and what are the functional consequences?

LILRA6 has been shown to associate with and activate MHC class I molecules and β2-microglobulin. This interaction appears to induce the expression of transporters associated with antigen processing. The functional consequences include:

• Enhanced antigen presentation capacity in myeloid cells

• Modulation of CD8+ T cell responses

• Regulation of NK cell education through altered MHC-I presentation

• Potential cross-presentation of exogenous antigens

Unlike LILRB1 and LILRB2, which recognize a broad range of MHC-I molecules through conserved α3 domain and β2-microglobulin interactions, LILRA6 binding appears more selective and may be influenced by specific peptide-MHC complexes. This interaction represents a mechanism by which innate immune receptors can modulate adaptive immune responses .

What role does LILRA6 play in autoimmune and inflammatory conditions?

LILRA6 has been implicated in several autoimmune and inflammatory conditions:

• Atopic dermatitis - Human LILRA6 polymorphisms correlate with disease susceptibility

• Inflammatory bowel disease - Altered expression observed in patient samples

• Rheumatoid arthritis - Potential role in pathogenesis

The activating nature of LILRA6 suggests that enhanced signaling through this receptor could contribute to inappropriate inflammatory responses. Additionally, the variable copy number of LILRA6 may influence disease susceptibility by altering the balance between activating (LILRA6) and inhibitory (LILRB3) signals. Individuals with higher LILRA6 copy numbers might experience enhanced inflammatory responses upon receptor engagement .

How does LILRA6 CNV correlate with cancer susceptibility and what are the potential mechanisms?

Genome-wide association studies have shown that duplications at the LILRA6 locus are associated with high-grade serous ovarian cancer susceptibility. The mechanisms potentially linking LILRA6 CNV to cancer may include:

• Altered immune surveillance of transformed cells

• Modified tumor microenvironment through cytokine regulation

• Enhanced recognition of damage-associated molecular patterns (DAMPs) released by tumor cells

• Interaction with cytokeratins exposed on necrotic cancer cells

Researchers investigating this connection should consider stratifying cancer cohorts by LILRA6 copy number and examining functional differences in myeloid cells from individuals with varying copy numbers. Higher LILRA6 expression may lead to altered immune responses against nascent tumors, potentially influencing cancer progression and therapeutic outcomes .

What is the role of LILRA6 in antimicrobial immunity and how can it be leveraged for immunomodulatory approaches?

LILRA6 appears to play significant roles in antimicrobial immunity:

• Activation of macrophage-mediated immune responses

• Induction of Th1-, Th2-, and Th17-type cytokines

• Upregulation of Toll-like receptors

• Potential recognition of pathogen-associated molecular patterns

LILRA6 activation in the context of infections could enhance inflammatory responses and antimicrobial effector functions. This suggests potential therapeutic applications:

• Development of LILRA6 agonists to enhance immunity against specific pathogens

• Targeted modulation of LILRA6 signaling to boost vaccine responses

• Integration of LILRA6 polymorphism analysis in personalized infection risk assessment

The receptor may be particularly relevant in the context of pathogens like Mycoplasma hyorhinis, Streptococcus pneumonia, Candida albicans, and Legionella pneumophila, where LILR family members have been implicated in host defense mechanisms .

What are the optimal methods for producing and validating recombinant LILRA6 for research applications?

For producing functional recombinant LILRA6:

• Expression Systems:

  • HEK293 cells for mammalian glycosylation patterns

  • CHO cells for stable high-yield production

  • E. coli for non-glycosylated fragments (e.g., single domains)

• Construct Design:

  • Full extracellular domain (aa 1-424) for most applications

  • Fc-fusion proteins for enhanced stability and detection

  • Inclusion of polyhistidine or other purification tags

• Validation Methods:

  • ELISA binding assays with known ligands (e.g., Angiopoietin-like 7)

  • Flow cytometry to confirm binding to cellular targets

  • Reporter cell assays to verify functional activity

  • Western blotting under reducing and non-reducing conditions

When studying LILRA6, typical ED50 values for functional binding assays range from 0.1-0.6 μg/mL when coated at 2 μg/mL concentration, providing a benchmark for experimental design .

How can researchers effectively design experiments to distinguish LILRA6 functions from other LILR family members?

To distinguish LILRA6 functions from other LILR family members:

• Use gene-specific knockdown/knockout approaches:

  • CRISPR-Cas9 targeting unique LILRA6 sequences

  • siRNA/shRNA designed against divergent regions

  • Validation with allele-specific PCR

• Employ blocking antibodies with verified specificity:

  • Pre-validation against recombinant proteins

  • Cross-adsorption to remove antibodies recognizing shared epitopes

  • Isotype-matched controls

• Utilize receptor chimeras:

  • Swap extracellular domains with cytoplasmic domains

  • Create LILRA6/LILRB3 chimeras to isolate signaling effects

  • Compare with wild-type receptors

• Implement allele-specific functional assays:

  • Express distinct LILRA6 alleles in reporter systems

  • Compare binding and activation profiles

  • Correlate with genetic variations

When interpreting results, researchers should account for the inherent challenges in distinguishing LILRA6 from LILRB3 due to their high sequence similarity and presence on the same cell populations .

What advanced analytical approaches can help resolve the complex relationship between LILRA6 CNV, expression, and function?

To address the complex relationship between LILRA6 CNV, expression, and function:

• Integrated Genomic and Transcriptomic Analysis:

  • Digital PCR for absolute copy number determination

  • RNA-seq with allele-specific analysis

  • Long-read sequencing to resolve structural variations

• Single-Cell Approaches:

  • scRNA-seq to correlate expression with cell states

  • Mass cytometry for protein-level analysis

  • Imaging mass cytometry for spatial context

• Systems Biology Integration:

  • Pathway enrichment analysis based on CNV status

  • Network modeling of signaling differences

  • Machine learning to identify functional patterns

• Longitudinal Studies:

  • Track expression changes during immune responses

  • Correlate with disease progression

  • Monitor in response to therapeutic interventions

• Functional Genomics:

  • CRISPR-based screens in relevant cell types

  • Isogenic cell lines with defined LILRA6 copy numbers

  • Synthetic biology approaches to control expression levels

These advanced approaches can help resolve how LILRA6 copy number influences receptor density on the cell surface, potentially affecting signaling thresholds upon ligand engagement and ultimately modulating immune cell function in both health and disease contexts .

What are the most promising areas for future LILRA6 research and therapeutic development?

The most promising areas for LILRA6 research include:

• Precision medicine applications based on LILRA6 genotyping:

  • Prediction of autoimmune disease susceptibility

  • Cancer risk stratification

  • Personalized immunotherapy approaches

• Development of LILRA6-targeted therapeutics:

  • Agonistic or antagonistic antibodies

  • Small molecule modulators of signaling

  • Engineered ligands with controlled receptor activation properties

• Understanding the evolutionary significance of LILRA6 CNV:

  • Population genetics across diverse ethnic groups

  • Selection pressures maintaining polymorphism

  • Comparative immunology in non-human primates

• Elucidation of the complete "ligandome":

  • Systematic screening for additional binding partners

  • Structural biology of receptor-ligand complexes

  • Context-dependent ligand recognition