CLEC2D Human

What is CLEC2D and what are its basic structural characteristics?

CLEC2D is a C-type lectin-like receptor that forms both homodimers and heterodimers with other receptors, notably TLR2 (Toll-like receptor 2). Structurally, CLEC2D belongs to the C-type lectin family and contains characteristic domains that facilitate protein-protein interactions. Crystal structure analyses have revealed that NKR-P1 (which interacts with CLEC2D/LLT1) forms homodimers in an arrangement that enables LLT1 binding in two distinct modes, allowing the bridging of two LLT1 molecules . This specific structural arrangement is critical for understanding CLEC2D's functional capabilities in immune regulation.

To study CLEC2D structure, researchers commonly employ:

• X-ray crystallography for detailed structural analysis

• SEC-SAXS (Size Exclusion Chromatography-Small Angle X-ray Scattering) for analyzing complex formations in solution

• dSTORM super-resolution microscopy for visualizing receptor arrangements on cell surfaces

What cellular types express CLEC2D in humans and how is expression regulated?

CLEC2D/LLT1 is primarily expressed on activated monocytes and B cells . Additionally, research has demonstrated elevated expression of CLEC2D in various cancer types, including clear cell renal cell carcinoma (ccRCC) , colorectal cancer, breast cancer, prostate cancer, lung cancer, and liver cancer .

Expression analysis methods include:

• Flow cytometry using anti-human LLT1-PE antibodies for cell surface detection

• Immunohistochemistry (IHC) for tissue expression analysis

• RT-qPCR for mRNA expression quantification

• Bioinformatic analysis of expression using databases such as TCGA and GEPIA

Regulatory factors influencing CLEC2D expression remain an active area of research, with evidence suggesting that tumor microenvironment conditions may upregulate its expression in cancer cells.

How can researchers accurately detect and measure CLEC2D expression?

Methodological approaches for CLEC2D detection include:

Flow Cytometry Protocol:

• Prepare single-cell suspensions from target tissues or cell lines

• Block cells with human Fc fragment to prevent nonspecific binding

• Stain with anti-human LLT1-PE antibodies (or isotype control mouse IgG1-PE antibodies)

• Analyze using flow cytometry (e.g., Beckman Coulter Cytomics FC500)

• Process data with analysis software (e.g., FlowJo v10)

Immunofluorescence Confocal Microscopy:

• Culture cells on coverslips overnight and fix with 2% paraformaldehyde

• Block with solution containing human Fc fragment

• Stain with mouse anti-human LLT1-PE antibody overnight at 4°C

• Mount coverslips and image using confocal microscopy (e.g., with 488 nm wavelength)

Expression Analysis in Tumor Tissues:
Researchers can assess CLEC2D expression in patient samples using immunohistochemistry and correlate with clinicopathological features as demonstrated in ccRCC studies .

What is the relationship between CLEC2D and natural killer (NK) cell function?

CLEC2D/LLT1 plays a critical role in regulating NK cell activity. It serves as a ligand for NKR-P1 (CD161), a C-type lectin-like receptor expressed on NK cells . This interaction is primarily inhibitory, helping to maintain NK cell self-tolerance when CLEC2D is expressed on normal activated immune cells .

Research findings indicate:

• CLEC2D-NKR-P1 interaction forms an inhibitory immune synapse

• This interaction suppresses NK cell cytotoxicity against CLEC2D-expressing cells

• Blocking this interaction with antibodies enhances NK cell-mediated lysis of target cells

• The interaction involves receptor clustering that overcomes weak ligand-receptor affinity to trigger signal transduction

For functional studies of this interaction, researchers commonly employ:

• 51Cr release cytotoxicity assays with primary NK cells against CLEC2D-expressing targets

• Blocking studies using anti-CLEC2D antibodies

• siRNA knockdown of CLEC2D to assess NK cell cytotoxicity changes

How does CLEC2D regulate antifungal immunity?

CLEC2D forms homodimers or heterodimers with TLR2 that negatively regulate antifungal immunity through suppression of IRF5-mediated IL-12 production . Mechanistically:

• Both CLEC2D homodimers and CLEC2D/TLR2 heterodimers show higher binding affinity to fungi-derived β-glucans than TLR2 homodimers alone

• Upon β-glucan binding, these dimeric forms mediate ubiquitination and degradation of MyD88

• This inhibits activation of transcription factor IRF5 and subsequent IL-12 production

• Reduced IL-12 leads to decreased IFN-γ-producing NK cells, which are important for antifungal defense

Clec2d-deficient female mice demonstrate resistance to Candida albicans infection due to increased IL-12 production and enhanced generation of IFN-γ-producing NK cells .

What experimental approaches are effective for studying CLEC2D-mediated signaling?

To investigate CLEC2D signaling, researchers can employ several methodological approaches:

Genetic Manipulation:

• siRNA knockdown using SMARTpool: ON-TARGETplus CLEC2D siRNAs (25 nM final concentration)

• CRISPR-Cas9 gene editing for complete knockout models

• Validation of knockdown by flow cytometry with anti-LLT1 antibodies

Protein Interaction Studies:

• Co-immunoprecipitation assays to detect CLEC2D-TLR2 heterodimer formation

• Proximity ligation assays to visualize protein interactions in situ

• Quantitative ligand binding assays to measure β-glucan binding capacity

Signaling Pathway Analysis:

• Western blotting for MyD88 ubiquitination and degradation

• Luciferase reporter assays for IRF5 activation

• Cytokine ELISAs for IL-12 production measurement

• Phospho-flow cytometry for intracellular signaling events

How is CLEC2D expression altered in human cancers and what is its prognostic significance?

CLEC2D shows significant expression alterations across multiple cancer types. Comprehensive analysis reveals:

In clear cell renal cell carcinoma (ccRCC):

*p < 0.05

What mechanisms underlie CLEC2D's role in tumor progression?

CLEC2D appears to promote tumor progression through several potential mechanisms:

• Immune Evasion: CLEC2D expression on tumor cells engages with NKR-P1A (CD161) on NK cells, inhibiting NK cell-mediated cytotoxicity against tumor cells

• Cytokine Modulation: CLEC2D may affect IFN-gamma production through ERK pathway activation in NK cells

• Tumor Growth Promotion: Though the exact mechanisms require further investigation, CLEC2D appears to promote tumor growth in animal models

The research confirms that blocking LLT1 (CLEC2D) on triple-negative breast cancer cells with antibodies disrupts the interaction with NKRP1A and enhances NK cell-mediated lysis of these cancer cells , suggesting therapeutic potential.

How can CLEC2D be targeted therapeutically in cancer research?

Experimental approaches for targeting CLEC2D in cancer research include:

Antibody-Based Approaches:

• Use of unconjugated goat anti-human CLEC2D antibodies to block interaction with NKRP1A

• Development of monoclonal antibodies with optimized blocking capacity

• Investigation of antibody-drug conjugates targeting CLEC2D-expressing tumor cells

Genetic Silencing:

• siRNA transfection (25 nM) for transient knockdown of CLEC2D in cancer cell lines

• Stable shRNA or CRISPR-Cas9 approaches for long-term studies

Functional Assays to Evaluate Targeting Efficacy:

• 51Cr release assays to measure NK cell cytotoxicity against target cells

• Co-culture systems with primary NK cells and cancer cells

• In vivo tumor models to assess tumor growth and immune infiltration

Research suggests that docetaxel can suppress immunotherapy efficacy of natural killer cells toward castration-resistant prostate cancer cells through targeting CLEC2D (LLT1) , highlighting the importance of considering drug interactions when developing CLEC2D-targeted therapies.

What are the molecular dynamics of CLEC2D-NKR-P1 interaction clusters in the immune synapse?

The CLEC2D-NKR-P1 interaction forms complex clusters that serve as the foundation of an inhibitory immune synapse. Research has revealed:

• NKR-P1 forms homodimers in an unexpected arrangement that enables binding to LLT1 in two distinct modes, bridging two LLT1 molecules

• These interaction clusters help overcome the weak affinity between individual receptors and ligands

• Formation of these clusters has been observed through multiple methodological approaches:

  • SEC-SAXS analysis in solution

  • dSTORM super-resolution microscopy on cell surfaces

  • Functional signaling studies with freshly isolated NK cells

• Effective NKR-P1 inhibitory signaling requires ligation of both LLT1 binding interfaces

Understanding these molecular interactions requires sophisticated biophysical and imaging techniques, including crystallography, super-resolution microscopy, and advanced biochemical assays.

What contradictions exist in the current understanding of CLEC2D function?

Several areas of contradiction or uncertainty persist in CLEC2D research:

• Context-Dependent Effects: While CLEC2D generally inhibits NK cell activity, context-specific effects may exist, particularly in different disease states

• Species-Specific Differences: Discrepancies between mouse and human CLEC2D signaling pathways complicate translational research

• Cancer-Specific Functions: The mechanisms by which CLEC2D promotes tumor progression in different cancer types may vary substantially

• Relationship to Other Immune Checkpoints: How CLEC2D-NKR-P1 interactions complement or counteract other immune checkpoint pathways remains incompletely understood

As noted in research on ccRCC, "the molecular mechanism of CLEC2D's influence on the progression of ccRCC, failed to find downstream key proteins and signaling pathways, and failed to elucidate the cause of the influence on immune infiltration" requires "multidimensional omics to screen downstream key proteins and pathways" .

What novel technologies are advancing CLEC2D research?

Emerging technologies enhancing CLEC2D research include:

Single-Cell Analysis:

• Single-cell RNA sequencing to identify heterogeneous expression patterns

• Single-cell proteomics to characterize receptor interactions at individual cell level

Advanced Imaging:

• Super-resolution microscopy such as dSTORM to visualize receptor clustering

• Live-cell imaging to track receptor dynamics during immune synapse formation

Multidimensional Omics:

• Integration of genomics, transcriptomics, and proteomics data

• Systems biology approaches to model CLEC2D signaling networks

Humanized Mouse Models:

• Development of models that better recapitulate human CLEC2D-NKR-P1 interactions

• Patient-derived xenografts to study CLEC2D in personalized cancer models

What are the optimal conditions for studying CLEC2D dimerization and ligand binding?

For robust dimerization and ligand binding studies, researchers should consider:

Protein Expression and Purification:

• Use of mammalian expression systems for proper glycosylation

• Careful buffer optimization to maintain physiological dimeric states

• Addition of stabilizing agents to preserve protein-protein interactions

Binding Assays:

• Quantitative ligand binding assays with purified proteins

• Surface plasmon resonance for measuring binding kinetics

• Microscale thermophoresis for detecting interactions in solution

Structural Studies:

• X-ray crystallography for atomic-level details

• Cryo-electron microscopy for complex assemblies

• Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

Experimental conditions should mimic physiological environments whenever possible, including appropriate pH, salt concentration, and calcium levels, given CLEC2D's nature as a C-type lectin receptor.

How should researchers design experiments to study CLEC2D in primary human samples?

When working with primary human samples:

Sample Collection and Processing:

• Fresh samples should be processed rapidly to maintain cell viability

• Appropriate anticoagulants for blood samples (e.g., EDTA or heparin)

• Cryopreservation protocols optimized for maintaining receptor expression

Analysis of CLEC2D Expression:

• Multiparameter flow cytometry to correlate with other immune markers

• Immunohistochemistry with validated antibodies for tissue samples

• RT-qPCR with appropriate reference genes for mRNA quantification

Functional Studies:

• Isolation of primary NK cells using negative selection to avoid receptor activation

• Use of autologous systems when possible

• Inclusion of appropriate controls for donor variability

Bioinformatic integration across multiple platforms (e.g., GEPIA, TCGA, cBioPortal) can provide valuable insights when analyzing clinical relevance .

What are the most promising areas for future CLEC2D research?

Several high-priority research directions include:

• Targeted Therapeutics: Development of blocking antibodies or small molecules targeting CLEC2D-NKR-P1 interactions for cancer immunotherapy

• Combination Approaches: Investigating CLEC2D blockade in combination with other immune checkpoint inhibitors

• Biomarker Development: Validation of CLEC2D as a prognostic or predictive biomarker in multiple cancer types

• Structural Biology: Resolving additional structural details of receptor-ligand complexes and signaling assemblies

• Signaling Networks: Comprehensive mapping of CLEC2D signaling networks in different cellular contexts

• Precision Medicine: Exploring patient-specific variation in CLEC2D expression and function for personalized treatment approaches

As noted in recent research, "multidimensional omics to screen downstream key proteins and pathways" will be essential for elucidating the complete mechanisms of CLEC2D function .