LILRB2 Human

What is the molecular structure of LILRB2 and how does it compare to other LILR family members?

LILRB2 contains four immunoglobulin (Ig) domains (D1-D4), a transmembrane domain, and 3-4 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail . These ITIMs interact with tyrosine phosphatases, inhibiting activating signals in immune cells . Unlike LILRB1, which is widely expressed in monocytes, dendritic cells, B cells, and subsets of NK and T cells, LILRB2 expression is restricted primarily to the myelomonocytic lineage . LILRB2 binds to HLA molecules through its two N-terminal extracellular domains (D1 and D2), with D3 and D4 appearing to function as scaffolds for the D1-D2 binding . A distinctive feature of LILRB2 compared to LILRB1 is that it does not require the presence of β2m when binding to HLA molecules .

What methodologies are most effective for characterizing LILRB2 genetic diversity?

For comprehensive characterization of LILRB2 genetic diversity, next-generation sequencing (NGS) combined with specialized bioinformatics approaches is most effective . The bioinformatics strategy using tools like hla-mapper software is crucial for achieving reliable read mapping of LILRB2, particularly due to its high sequence similarity with other LILR family members . This approach is especially important since LILRB2 arose from intergenic recombination and gene duplication, with identity levels among the LILR D1-D4 domains ranging from 63% to 84% when aligned to LILRB1 . Full exon sequencing is necessary to capture all potential variants, as demonstrated in studies that identified numerous SNVs arranged into multiple haplotypes .

How do researchers differentiate between LILRB2 isoforms in experimental settings?

Researchers can differentiate between LILRB2 isoforms using a combination of techniques. Alternative mRNA processing gives rise to eight different protein isoforms from LILRB2, each with distinct cell expression profiles . Methodologically, this requires:

• RT-PCR with isoform-specific primers to distinguish between alternative splice variants

• Western blotting with antibodies targeting unique epitopes in different isoforms

• Flow cytometry to detect surface expression patterns on different cell types

• Mass spectrometry for precise protein identification and characterization

• Reporter systems with isoform-specific constructs to monitor functional differences

These approaches can help distinguish between the various LILRB2 isoforms that may have distinct biological functions in different cellular contexts.

What patterns of selection have been observed in LILRB2 coding regions, and what methodologies best detect them?

Both positive and purifying selection patterns have been detected in LILRB2 coding regions . Methodologically, several approaches have proven effective:

• FUBAR (Fast Unconstrained Bayesian AppRoximation) analysis has identified residues under positive selection, suggesting amino acid replacements resulting in beneficial functional changes .

• FoldX4 stability analysis has classified specific SNPs (like rs1128646 and rs7247538) as stabilizing mutations, while others (such as rs386056*G) cause slight destabilizing effects .

• Population genetics approaches examining haplotype structure and frequency in diverse populations reveal evidence of selection .

• Comparative genomics showing that LILRB2, like other LIR family members, is rapidly evolving with larger interspecies differences compared to the genome average .

These findings collectively provide strong evidence supporting the directional selection regime hypothesis for LILRB2 evolution.

How do linkage disequilibrium patterns in LILRB2 differ across populations, and what implications does this have for research?

Linkage disequilibrium (LD) patterns in LILRB2 are highly population-specific, being affected by factors such as non-random mating, selection, mutation, migration, admixture, genetic drift, and effective population size . Research in admixed populations like Brazilians has revealed that:

• Variants differentiating specific LILRB2 haplogroups (a, c, d) are part of the same LD block and are in high or complete LD with each other (r² > 0.5) .

• These population-specific LD patterns may not be directly extrapolated to worldwide populations without comprehensive evaluation .

The implications for research include:

• Need for population-specific genetic association studies

• Potential for uncovering novel variants in admixed populations

• Importance of considering ancestral background when interpreting LILRB2 genetic data

• Necessity for tailored approaches to identify disease associations in different populations

What is the relationship between LILRB2 polymorphisms and protein stability/function?

LILRB2 polymorphisms can significantly affect protein stability and function, with both structural and evolutionary implications . Research has shown:

Some polymorphisms directly affect the interaction with ligands. For example, non-conserved residues in the binding region on D1D2 (such as Q76, R80, and R84 in LILRB2 compared to Y76, D80, and R84 in LILRB1) contribute to the higher affinity of LILRB2 for HLA-G compared to LILRB1 . Additionally, residues T36 and A38 in LILRB2 recognize the 195–197 loop of HLA-G, demonstrating how specific polymorphisms can affect ligand recognition .

What is the molecular mechanism of Angptl2 binding to LILRB2, and how can researchers experimentally validate it?

The molecular mechanism of Angptl2 binding to LILRB2 involves:

• Angptl2 expressed in mammalian cells forms high-molecular-weight (HMW) species, which is required for activation of LILRB2 .

• A specific motif in the first and fourth Ig domains of LILRB2 is critical for Angptl2 binding and signaling activation .

• While individual Ig domains don't bind Angptl2, Ig1 and Ig2 in combination display about 50% of the maximal binding, while Ig3 and Ig4 in combination display about 10%, indicating the major binding site resides in Ig1 and Ig2 .

Researchers can experimentally validate this interaction using:

• Chimeric receptor reporter systems that fuse the LILRB2 extracellular domain with transmembrane/intracellular domains containing immunoreceptor tyrosine-based activation motifs linked to reporter genes .

• Flow cytometry-based cell-surface ligand binding assays with domain- and site-specific mutations to map critical binding regions .

• Confocal microscopy to visualize receptor clustering upon ligand binding, as evidenced by the change from ring-like to spot-like distribution of LILRB2 on cell membranes .

How does multimerization of ligands affect LILRB2 activation, and what methodological approaches can assess this?

Ligand multimerization is crucial for LILRB2 activation and subsequent signaling . Key findings include:

• Angptl2 expressed in mammalian cells exists as high-molecular-weight species necessary for LILRB2 activation .

• Immobilized anti-LILRB2 antibodies induce more potent activation than soluble antibodies or Angptl2, suggesting that ligand presentation in a multimerized form is critical .

• Receptor clustering appears to be the mechanism of activation, as demonstrated by the change in LILRB2-ECD distribution from a ring-like to a spot-like pattern after treatment with immobilized antibodies .

Methodological approaches to assess this include:

• Gel filtration chromatography to characterize the molecular weight of ligands

• Chemical cross-linking studies to analyze ligand multimerization

• Surface plasmon resonance to measure binding kinetics of monomeric versus multimeric ligands

• Confocal microscopy with fluorescently labeled receptor to visualize clustering

• FRET (Förster Resonance Energy Transfer) analysis to measure receptor proximity upon activation

How does LILRB2 signaling differ between Angptl2 and HLA-G binding, and what techniques best measure these differences?

LILRB2 signaling differs significantly between Angptl2 and HLA-G binding:

• Angptl2 binding to LILRB2 is more potent than and not completely overlapped with HLA-G binding .

• In reporter cell assays, Angptl2-treated LILRB2 reporter cells induced significantly greater GFP expression (18.95% ± 0.95%) than control cells (5.34% ± 1.19%), while even high concentrations of HLA-G (130 μg/mL) failed to activate GFP expression .

• Angptl2 binding to LILRB2 induces activation of SHP-2 and Ca²⁺/calmodulin-dependent kinase, which are critical for supporting HSC activity .

Techniques to measure these differences include:

• Chimeric receptor reporter systems with quantifiable outputs (e.g., GFP expression)

• Phosphorylation assays for downstream signaling molecules (SHP-1, SHP-2)

• Calcium flux assays to measure intracellular calcium mobilization

• Competitive binding assays to determine relative affinities

• Functional assays specific to cell types of interest (e.g., HSC expansion assays)

What mechanisms underlie LILRB2's role in hematopoietic stem cell maintenance and expansion?

LILRB2 plays crucial roles in hematopoietic stem cell (HSC) maintenance and expansion through several mechanisms:

• LILRB2 is expressed on human HSCs and its activation supports ex vivo expansion of these cells .

• Binding of Angptls to LILRB2 induces activation of SHP-2 and Ca²⁺/calmodulin-dependent kinase, types of factors known to be critical for supporting HSC activity .

• LILRB2 signaling inhibits differentiation and promotes self-renewal, helping to maintain the stem cell state .

• The interaction with Angptls leads to specific downstream signaling events that support HSC expansion while preserving stemness properties .

These findings have led to the development of specialized culture systems containing defined cytokines and immobilized anti-LILRB2 antibodies that support stable and reproducible ex vivo expansion of repopulating human cord blood HSCs .

How can researchers optimize serum-free culture conditions for LILRB2-mediated HSC expansion?

Optimization of serum-free culture conditions for LILRB2-mediated HSC expansion requires attention to several key factors:

• Ligand presentation: Immobilized anti-LILRB2 antibodies induce more potent activation than soluble antibodies or Angptl2 . The immobilization approach is critical as it supports receptor clustering for optimal signaling activation .

• Cytokine combination: A defined mixture of cytokines must be combined with LILRB2 activation to support HSC expansion while maintaining stemness .

• Culture duration: Determining the optimal culture period to balance expansion with preservation of repopulating potential.

• Antibody concentration: Titration of immobilized anti-LILRB2 concentration to achieve optimal receptor activation without inducing negative effects.

• Substrate selection: The surface used for antibody immobilization can affect both antibody orientation and HSC interaction.

• Medium formulation: Beyond cytokines, other medium components must be optimized to support HSC metabolism and prevent differentiation.

What are the contradictions in data regarding LILRB2 function in normal versus leukemic stem cells?

Research has revealed both similarities and contradictions regarding LILRB2 function in normal versus leukemic stem cells:

Similarities:

• LILRB2 supports self-renewal in both normal HSCs and leukemic stem cells .

• Both cell types utilize LILRB2 signaling for maintenance and expansion .

• Similar downstream signaling pathways (SHP-2 and Ca²⁺/calmodulin-dependent kinase) are activated in both contexts .

Contradictions:

• While LILRB2 activity is regulated in normal HSCs, it appears to be dysregulated in leukemic cells .

• The balance between self-renewal and differentiation mediated by LILRB2 differs between normal and leukemic contexts.

• The requirement for LILRB2 in leukemia development suggests potential therapeutic targeting approaches that must spare normal HSCs.

These contradictions present both challenges and opportunities for developing targeted therapies that disrupt LILRB2 function in leukemic cells while preserving normal HSC activity.

What bioinformatic pipelines are most effective for analyzing LILRB2 sequence data from diverse populations?

Effective bioinformatic pipelines for analyzing LILRB2 sequence data must address several challenges, particularly for diverse or admixed populations:

• Specialized mapping tools: Software like hla-mapper is essential for reliable read mapping of LILRB2 due to its high sequence similarity with other LILR family members (63-84% identity) .

• Pipeline components:

  • Quality control and read trimming

  • Specialized alignment algorithms for genes with homologs

  • Variant calling optimized for polymorphic regions

  • Haplotype phasing

  • Selection analysis (FUBAR, FoldX4)

  • Population structure analysis

  • Linkage disequilibrium assessment

• Population-specific considerations:

  • For admixed populations, ancestry-informed analysis is crucial

  • Local ancestry inference may be needed for regions surrounding LILRB2

  • Population-specific LD patterns must be considered when interpreting results

• Validation strategies:

  • Cross-platform validation of identified variants

  • Functional prediction of missense variants

  • Conservation analysis across species

How can researchers effectively design domain-swapping experiments to study LILRB2 binding specificity?

Domain-swapping experiments are valuable for studying LILRB2 binding specificity. Based on research findings, an effective experimental design should:

• Target key domains: Focus on D1 and D2 domains as they display about 50% of maximal binding to Angptl2, while D3 and D4 in combination display only about 10% .

• Create chimeric constructs:

  • LILRB2 D1-D2 + LILRB1 D3-D4

  • LILRB1 D1-D2 + LILRB2 D3-D4

  • LILRB2 D1 + LILRB1 D2-D4

  • LILRB1 D1 + LILRB2 D2-D4

• Include site-directed mutagenesis:

  • Target residues identified in positive selection analyses

  • Focus on non-conserved residues between LILRB1 and LILRB2 (e.g., Y76, D80, R84 in LILRB1 vs. Q76, R80, R84 in LILRB2)

  • Mutate residues involved in HLA-G recognition (T36, A38)

• Utilize appropriate assay systems:

  • Chimeric receptor reporter systems

  • Flow cytometry-based cell-surface binding assays

  • Protein stability assessments

• Include both ligands:

  • Test Angptl2 binding and activation

  • Compare with HLA-G binding patterns

  • Evaluate differences in downstream signaling

What experimental systems best recapitulate LILRB2 function in primary human cells versus cell lines?

To accurately study LILRB2 function, researchers must carefully consider experimental systems that recapitulate physiological conditions:

Primary Human Cell Systems:

• Isolated CD34+ hematopoietic stem/progenitor cells from cord blood or bone marrow

• Primary monocytes and dendritic cells

• Ex vivo expanded HSCs using defined conditions with immobilized anti-LILRB2

• Patient-derived leukemia samples

Cell Line Models:

• Chimeric receptor reporter systems using appropriate cell backgrounds

• CRISPR-engineered cell lines with modified LILRB2 loci

• Inducible expression systems to control LILRB2 levels

Considerations for Experimental Design:

• Ligand presentation: Immobilized versus soluble ligands produce different outcomes

• Multimerization status: Ensure ligands form appropriate high-molecular-weight species

• Physiological expression levels: Avoid overexpression artifacts

• Signaling readouts: Include multiple downstream pathways (SHP-1/2, calcium flux)

• Functional endpoints: Measure effects on differentiation, proliferation, and cellular function

Technical Recommendations:

• Use primary cells for final validation of mechanisms identified in cell lines

• Implement single-cell approaches to account for heterogeneity in primary populations

• Consider 3D culture systems to better mimic in vivo microenvironments

• Employ multiple ligands to compare signaling outcomes

• Include genetic diversity analysis when using primary cells from different donors

How might LILRB2 genetic diversity influence susceptibility to hematological malignancies?

LILRB2 genetic diversity likely influences susceptibility to hematological malignancies through several mechanisms:

• Functional polymorphisms in LILRB2 may influence immune response diversity among individuals and consequently, their susceptibility to diseases .

• Specific haplotypes and SNPs may affect LILRB2's ability to regulate HSC self-renewal versus differentiation, potentially predisposing to leukemia in certain genetic backgrounds .

• LILRB2 is required for leukemia development as it inhibits differentiation and promotes self-renewal of leukemic progenitors . Genetic variants that enhance or diminish these functions could modify leukemia risk.

• Selection signatures detected in LILRB2 coding regions suggest functional importance of certain polymorphisms that may influence disease susceptibility.

• Population-specific patterns of LILRB2 genetic diversity may contribute to differences in hematological malignancy incidence across ethnic groups.

Future research should focus on large-scale association studies correlating LILRB2 genotypes with disease incidence and outcomes across diverse populations.

What are the implications of LILRB2's role in HSC expansion for clinical cell therapy applications?

LILRB2's role in HSC expansion has significant implications for clinical cell therapy applications:

• Development of improved ex vivo expansion protocols: Immobilized anti-LILRB2 antibodies induce more potent activation than Angptl2, leading to better HSC expansion . This approach can help overcome limited cell numbers in cord blood transplantation.

• Serum-free culture systems: The development of defined, serum-free cultures containing immobilized anti-LILRB2 antibodies supports stable expansion of repopulating human cord blood HSCs , reducing variability and regulatory concerns for clinical applications.

• Preservation of stemness: LILRB2 activation supports expansion while maintaining repopulating potential , addressing a key challenge in HSC expansion protocols.

• Targeted approaches: Understanding the specific motif in LILRB2's Ig domains critical for activation enables the development of more specific agonists for clinical use.

• Quality control: Knowledge of LILRB2 signaling provides molecular markers to assess the quality of expanded HSCs before clinical use.

How might therapeutic targeting of LILRB2 differ between autoimmune conditions and cancer contexts?

Therapeutic targeting of LILRB2 would require fundamentally different approaches in autoimmune conditions versus cancer contexts:

Autoimmune Conditions:

• Goal: Enhance LILRB2 inhibitory function to suppress overactive immune responses

• Approach: Develop agonistic antibodies or multimeric ligands that activate LILRB2 signaling

• Target cells: Myeloid cells, dendritic cells

• Considerations: Avoid excessive immunosuppression that might increase infection risk

• Biomarkers: Monitor SHP-1/2 activation in myeloid cells

Cancer Context:

• Goal: Block LILRB2 inhibitory signals to enhance anti-tumor immunity and/or inhibit leukemic stem cell self-renewal

• Approach: Develop antagonistic antibodies or small molecules that prevent LILRB2 signaling

• Target cells: Tumor-associated macrophages, leukemic stem cells

• Considerations: Potential effects on normal HSCs must be carefully evaluated

• Biomarkers: Assess differentiation markers in leukemic cells, immune activation in tumor microenvironment

These divergent approaches highlight the context-dependent nature of LILRB2 function and the need for careful therapeutic design based on disease mechanism.