CLEC2B Human

What is CLEC2B and how is it classified in the protein family system?

CLEC2B (Activation-induced C-type lectin) is a 30-35 kDa variably glycosylated type-2 transmembrane protein belonging to the C-type lectin-like receptor (CTLR) family. It specifically belongs to the subgroup of CLEC2 proteins that also includes CLEC2A/KACL, CLEC2D/LLT, and CD69/CLEC2C, all encoded by the natural killer gene complex (NKC) . Human CLEC2B contains a single C-type lectin domain in its extracellular region and a notably short cytoplasmic tail of just 7 amino acids .

What is the cellular expression pattern of CLEC2B in humans?

CLEC2B is predominantly expressed on cells of myeloid lineage, specifically monocytes, macrophages, and granulocytes . Its expression is dynamically regulated, with significant upregulation observed on TLR-activated monocytes. Interestingly, IL-12 + IL-18 activated NK cells also show increased CLEC2B expression, suggesting a broader role in immune cell communication than initially thought .

What cell culture systems are optimal for studying CLEC2B function?

For in vitro studies of CLEC2B, researchers should consider using:

• Human monocytic cell lines (e.g., U937 cells have been validated for CLEC2B expression as demonstrated in flow cytometry experiments)

• Primary human monocytes isolated from peripheral blood

• Human NK cell cultures for studying CLEC2B-NKp80 interactions

Cell culture conditions should typically include:

• DMEM medium supplemented with 10% fetal bovine serum

• Incubation at 37°C in a 5% CO2 atmosphere

• For activation studies, appropriate TLR ligands or cytokine combinations (IL-12+IL-18) should be considered to upregulate CLEC2B expression

What are validated detection methods for CLEC2B in research applications?

Several validated detection methods for CLEC2B include:

Flow Cytometry:
U937 cells have been successfully stained using Mouse Anti-Human AICL/CLEC-2B Monoclonal Antibody (MAB9059), with Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody . Specificity can be confirmed using isotype control antibodies (MAB002).

Antibody-Based Detection:

• Monoclonal Mouse IgG1 (Clone #1064935) has been validated for human CLEC2B detection

• Antibodies have been validated for specificity using transduced cell lines expressing HA-tagged CLEC2B compared to vector-only controls

Protein Interaction Studies:
Recombinant Human AICL/CLEC-2B Fc Chimera Protein can be used for binding studies. When coated at 2 μg/mL, it has been shown to bind Recombinant Human Galectin-1 with a typical ED50 of 0.6-3 μg/mL .

How can researchers effectively study CLEC2B-mediated signaling pathways?

To investigate CLEC2B-mediated signaling:

• Receptor Engagement Studies:

  • Use plate-bound or soluble anti-CLEC2B antibodies to trigger signaling

  • Measure TNF production by ELISA as a functional readout of CLEC2B activation in monocytes

• Downstream Signaling Analysis:

  • Western blotting to detect phosphorylated signaling proteins

  • Transcriptomic analysis to identify genes regulated following CLEC2B engagement

  • Inhibitor studies using specific pathway blockers to delineate signaling cascades

• Statistical Analysis:

  • Data should be recorded in Excel and analyzed using software like GraphPad Prism

  • Results should be analyzed by a two-tailed t-test with p < 0.05 considered significant

  • Data should be expressed as mean ± standard error

What approaches can be used to study the CLEC2B-NKp80 interaction axis?

The CLEC2B-NKp80 interaction represents a crucial immune regulatory axis. To study this:

• Co-culture Systems:

  • Establish co-cultures of monocytes (CLEC2B+) and NK cells (NKp80+)

  • Use blocking antibodies against either CLEC2B or NKp80 to confirm specificity

• Functional Assays:

  • Cytotoxicity assays to measure NK cell-mediated lysis of CLEC2B-expressing monocytes

  • Cytokine profiling to assess reciprocal activation

• Imaging Approaches:

  • Confocal microscopy to visualize receptor clustering at the immune synapse

  • Live-cell imaging to track dynamic interactions between these cell types

How does human CLEC2B compare structurally and functionally to other CLEC2 family proteins?

The CLEC2 family shows a pattern of genetically coupled receptor/ligand pairs of CTLRs. This raises important questions about why genes of receptors and ligands are tightly coupled despite no apparent polymorphism . The family members share structural features of CTLDs (C-type lectin-like domains), including six conserved cysteines and the WIGL motif .

How does murine CLEC-2 differ from human CLEC2B?

Murine CLEC-2 shows some notable differences from human CLEC2B:

• Expression Pattern:

  • Murine CLEC-2 is strongly expressed on peripheral blood neutrophils

  • Expression is weaker on bone-marrow or elicited inflammatory neutrophils

  • Also expressed on platelets (CD61+ cells)

• Detection Methods:

  • Specific antibodies have been developed to detect murine CLEC-2, including both polyclonal and monoclonal antibodies

  • Expression can be confirmed using CLEC-2 transduced cell lines versus vector controls

• Strain Differences:

  • Similar levels of expression are detected across different mouse strains including BALB/c, C57BL/6, and 129/Sv mice

What are common technical challenges in studying CLEC2B and how can they be addressed?

• Protein Stability Issues:

  • CLEC2B is a glycosylated protein that can be sensitive to storage conditions

  • Recommendation: For recombinant proteins, use carrier-free versions for applications where BSA might interfere, otherwise use BSA-containing formulations for enhanced stability

  • Avoid repeated freeze-thaw cycles and use manual defrost freezers

• Expression Variability:

  • CLEC2B expression levels can vary based on activation state

  • Recommendation: Include appropriate positive controls and standardize activation protocols

• Antibody Specificity:

  • Ensure antibody specificity through careful validation

  • Recommendation: Test antibodies on known positive and negative cell lines, and always include isotype controls

How should researchers approach experimental design for CLEC2B studies?

• Sample Size and Statistical Power:

  • Ensure adequate biological replicates (n ≥ 3)

  • Perform power calculations before beginning experiments

  • Use appropriate statistical tests (two-tailed t-tests for simple comparisons)

• Controls:

  • Include isotype controls for antibody experiments

  • Use vector-only controls for recombinant expression systems

  • Include unstimulated cells as baseline controls for activation studies

• Reproducibility Considerations:

  • Standardize protocols across experiments

  • Document lot numbers of critical reagents

  • Consider validation across different cell types where appropriate

What are key unanswered questions about CLEC2B that merit further investigation?

• Signaling Complexity:

  • How does the short (7 amino acid) cytoplasmic tail of CLEC2B mediate its signaling functions?

  • What adapter proteins interact with CLEC2B?

• Disease Relevance:

  • How is CLEC2B expression altered in inflammatory or infectious diseases?

  • Does CLEC2B play a role in cancer immunosurveillance?

• Evolutionary Conservation:

  • To what extent are CLEC2B functions conserved across species?

  • Are there functional homologues serving similar roles across species despite limited sequence homology?

• Therapeutic Potential:

  • Could modulation of the CLEC2B-NKp80 axis have therapeutic applications?

  • What would be the consequences of CLEC2B targeting in inflammatory conditions?

What emerging methodologies might advance CLEC2B research?

• Single-cell Technologies:

  • Single-cell RNA sequencing to identify heterogeneity in CLEC2B expression

  • Mass cytometry for high-dimensional phenotyping of CLEC2B+ cells

• CRISPR/Cas9 Applications:

  • Genome editing to create CLEC2B knockout models

  • Knock-in reporter systems for live tracking of CLEC2B expression

• Advanced Imaging:

  • Super-resolution microscopy to study receptor clustering

  • Intravital imaging to track CLEC2B+ cells in vivo