CLE1 Antibody

What is CLEC1A and why is it significant in immunological research?

CLEC1A is a transmembrane receptor belonging to the C-type lectin receptor family, primarily expressed on myeloid cells, including dendritic cells and acute myeloid leukemia (AML) blasts and leukemic stem cells. The significance of CLEC1A lies in its function as a death sensor that recognizes cells undergoing programmed necrosis. CLEC1A has been identified as a negative regulator of antigen cross-presentation by conventional type-1 dendritic cells (cDC1s) to CD8+ T cells, thus playing a crucial role in modulating immune responses. Recent research has identified TRIM21 (Tripartite Motif Containing 21) as an endogenous ligand for CLEC1A, which is overexpressed in various cancers. This interaction suggests CLEC1A's potential involvement in cancer-related immune regulation, making it a promising target for immunotherapy .

What experimental approaches are used to characterize CLEC1A expression patterns?

Characterization of CLEC1A expression requires a multi-modal approach combining several techniques:

• Flow cytometry analysis: Implement multi-parameter flow cytometry using CLEC1A antibodies conjugated to fluorophores (e.g., Alexa488) in combination with lineage markers. For clinical samples, four-color staining with CLL-1/Alexa488, CD34-PE, CD45-PerCP, and CD38-APC allows identification of CLEC1A-expressing cells within specific cellular populations. Always include appropriate isotype controls (IgG-Alexa488) to account for non-specific binding .

• Immunohistochemistry (IHC): Utilize validated anti-CLEC1A antibodies for tissue section analysis, selecting appropriate retrieval buffers and detection systems based on tissue type. Both polyclonal and monoclonal antibodies are available for IHC applications, with varied specificities for human and mouse samples .

• Single-cell RNA sequencing: For comprehensive analysis of expression patterns, scRNA-seq can reveal CLEC1A expression across diverse immune populations, as demonstrated in tumor microenvironment studies that identified differential expression across myeloid cell subsets .

How should researchers select appropriate anti-CLEC1A antibodies for their experimental systems?

Selection of anti-CLEC1A antibodies requires careful consideration of multiple parameters:

When selecting antibodies, researchers should consider whether they need antibodies that recognize the native conformation (for applications like flow cytometry) or denatured epitopes (for Western blotting). For human samples, antibodies specifically validated on primary human cells are preferable. Different suppliers offer varying validation data; therefore, researchers should review supplier datasheets for target specificity, validation methodology, and potential cross-reactivities .

How can CLEC1A antibodies be optimized for studying myeloid cell function in tumor microenvironments?

Studying CLEC1A in tumor microenvironments requires specialized approaches to address the complex cellular milieu:

• Multi-parameter analysis: Combine anti-CLEC1A antibodies with markers for myeloid subsets (CD11c, CD11b, Ly6G, Ly6C for mouse; CD33, CD14, HLA-DR for human) to distinguish expression across myeloid-derived suppressor cells (MDSCs), macrophages, and dendritic cell subsets. Research has demonstrated that CLEC1A deficiency leads to reduced accumulation of immunosuppressive myeloid cells in tumors and enhanced activation of dendritic cells .

• In situ analysis: For spatial context, implement multiplexed immunofluorescence combining anti-CLEC1A antibodies with tumor and immune cell markers. This approach allows visualization of CLEC1A-expressing cells relative to tumor regions and other immune populations.

• Functional assessment: Use blocking anti-CLEC1A antibodies to evaluate their impact on myeloid cell functions including phagocytosis, cytokine production, and T cell stimulatory capacity. Studies have shown that CLEC1A blockade can invigorate dendritic cell function and enhance T cell responses in tumor models .

• Chimeric antibody development: For therapeutic evaluation, generate chimeric antibodies by cloning the variable regions of anti-CLEC1A monoclonal antibodies into expression vectors containing human IgG1 constant regions. This approach allows testing of human-compatible antibodies in preclinical models using techniques such as RACE/RT-PCR followed by stable transfection into Chinese hamster ovary cells .

What methodologies enable effective investigation of CLEC1A internalization dynamics?

CLEC1A internalization represents a crucial parameter for developing antibody-drug conjugates and understanding receptor regulation. Investigation requires:

• Immunofluorescence microscopy protocol:

  • Seed CLEC1A-expressing cells on poly-L-lysine coated coverslips

  • Preincubate cells in PBS-10% human serum (15 minutes)

  • Incubate with fluorophore-conjugated anti-CLEC1A antibody (10 μg/mL) at 4°C for 1 hour

  • Wash with ice-cold buffer and transfer to 37°C to allow internalization

  • Fix with 4% paraformaldehyde and mount with appropriate media containing nuclear counterstain

  • Analyze using confocal or fluorescence microscopy at different time points

• Flow cytometry-based internalization assay:

  • Incubate cells with fluorescently-labeled anti-CLEC1A antibodies at 4°C

  • Transfer to 37°C for various time intervals

  • Measure reduction in surface fluorescence over time as indicator of internalization

  • Include surface quenching steps to distinguish between internalized and surface-bound antibodies

• Biochemical fractionation:

  • Biotinylate cell surface proteins before internalization

  • After antibody incubation and temperature shift, separate plasma membrane from endosomal fractions

  • Detect CLEC1A using immunoblotting to track movement between compartments

These methods have revealed that anti-CLEC1A antibodies can be efficiently internalized upon receptor binding, supporting their potential use as vehicles for targeted drug delivery .

How can researchers investigate CLEC1A's interaction with its ligand TRIM21?

Investigation of CLEC1A-TRIM21 interactions requires specialized biochemical and cellular approaches:

• Affinity purification coupled with mass spectrometry:

  • Perform co-immunoprecipitation using recombinant CLEC1A (Fc-tagged or His-tagged)

  • Incubate with protein extracts from necrotic cells

  • Analyze eluted proteins by SDS-PAGE followed by LC-MS identification

  • Validate specific interactions through Western blotting with anti-TRIM21 antibodies

This approach successfully identified TRIM21 as an endogenous ligand for CLEC1A with approximately 50% coverage in mass spectrometry analysis and a corresponding molecular weight of 52 kDa .

• Validation through multiple tagging strategies:

  • Use different tagging approaches (Fc-tag, His-tag) to confirm specificity

  • Include appropriate controls (e.g., irrelevant C-type lectin receptors like CLEC7A)

  • Perform reciprocal co-immunoprecipitation with anti-TRIM21 antibodies followed by detection with anti-CLEC1A antibodies

• Knockdown verification:

  • Implement shRNA-mediated knockdown of TRIM21

  • Demonstrate reduced binding of CLEC1A to cells with diminished TRIM21 expression

  • Confirm using both Western blot and flow cytometry approaches

How can researchers evaluate the impact of CLEC1A on antigen cross-presentation?

Evaluating CLEC1A's role in regulating antigen cross-presentation by dendritic cells requires specialized functional assays:

• In vitro cross-presentation assay:

  • Generate bone marrow-derived dendritic cells from wild-type and CLEC1A-deficient animals

  • Expose dendritic cells to necrotic tumor cells containing model antigens (e.g., OVA)

  • Co-culture with antigen-specific CD8+ T cells (e.g., OT-I T cells for OVA)

  • Measure T cell activation via proliferation, cytokine production, or activation marker expression

  • Compare cross-presentation efficiency between CLEC1A-sufficient and CLEC1A-deficient dendritic cells

• Antibody blocking studies:

  • Isolate human dendritic cells, particularly the cDC1 subset

  • Pretreat with anti-CLEC1A blocking antibodies or isotype controls

  • Assess cross-presentation capacity using tumor-associated antigens

  • Measure resultant T cell activation

• In vivo tumor models with CLEC1A manipulation:

  • Establish tumor models in wild-type and CLEC1A-knockout mice

  • Monitor tumor growth, survival, and T cell responses

  • Deplete CD8+ T cells to confirm mechanism of action

  • Analyze memory response through tumor rechallenge experiments

Research has shown that CLEC1A deficiency enhances cross-presentation of dead cell-associated antigens by cDC1s to CD8+ T cells, resulting in more robust antitumor immunity. Additionally, CLEC1A-deficient mice demonstrated increased survival in hepatocellular carcinoma models, with effects partially dependent on CD8+ T cells .

What methodological approaches can assess the therapeutic potential of anti-CLEC1A antibodies?

Evaluation of anti-CLEC1A antibodies for therapeutic applications requires comprehensive preclinical assessment:

• Antibody humanization/chimerization:

  • Clone variable regions of anti-CLEC1A monoclonal antibodies using RACE/RT-PCR

  • Fuse with human IgG1 constant regions in expression vectors

  • Co-transfect into Chinese hamster ovary cells

  • Select stable cell lines secreting full-length chimeric antibodies

• In vitro cytotoxicity assessment:

  • Test antibody-dependent cellular cytotoxicity (ADCC) against CLEC1A-expressing cell lines and primary AML blasts

  • Evaluate complement-dependent cytotoxicity (CDC)

  • Assess direct killing mechanisms and internalization capacity

• In vivo efficacy models:

  • Establish xenograft models using CLEC1A-expressing tumor cells

  • Treat with anti-CLEC1A antibodies alone or in combination with standard therapies

  • Monitor tumor growth, survival, and immune changes

  • Analyze tumor microenvironment by flow cytometry and single-cell RNA sequencing

• Combination therapy assessment:

  • Investigate synergy between anti-CLEC1A antibodies and chemotherapy

  • Evaluate combinations with immune checkpoint inhibitors

  • Analyze mechanistic basis for observed synergies

Research has demonstrated that targeting CLEC1A with antagonist antibodies can enhance antitumor immunity in preclinical models, particularly when combined with other therapeutic modalities .

How can researchers address variability in CLEC1A detection across different sample types?

Variability in CLEC1A detection can arise from multiple sources, requiring specific troubleshooting approaches:

• Tissue fixation optimization for IHC:

  • Test multiple fixation protocols (formalin, paraformaldehyde, methanol)

  • Optimize antigen retrieval methods (heat-induced vs. enzymatic)

  • Compare antibody clones that recognize different epitopes

  • Implement positive controls (known CLEC1A-expressing tissues)

• Flow cytometry optimization:

  • Use fresh rather than frozen samples when possible

  • Implement blocking steps with human/mouse serum to reduce non-specific binding

  • Compare direct conjugated antibodies vs. primary-secondary detection systems

  • Validate with knockout/knockdown controls

• Heterogeneity management in clinical samples:

  • Stratify samples based on disease characteristics

  • Implement multi-parameter analysis to identify specific cell subsets

  • Consider single-cell approaches for highly heterogeneous samples

• Quantification standardization:

  • Develop standardized protocols with defined positive thresholds

  • Use calibration beads for flow cytometry

  • Include internal reference populations

What controls are essential for validating experimental findings with CLEC1A antibodies?

Proper experimental controls are crucial for ensuring reliable results with CLEC1A antibodies:

Implementation of these controls enables researchers to distinguish genuine CLEC1A detection from technical artifacts, ensuring robust and reproducible experimental findings .

How might researchers advance understanding of CLEC1A function in disease contexts?

Future research on CLEC1A should focus on several promising directions:

• Comprehensive ligand identification: Beyond TRIM21, additional CLEC1A ligands may exist in different pathological contexts. Systematic approaches combining proximity labeling, mass spectrometry, and focused screening of candidate molecules will help complete the CLEC1A ligandome.

• Signaling pathway characterization: Detailed analysis of signaling events downstream of CLEC1A activation, particularly in different myeloid cell subsets, will provide mechanistic insights into its immune regulatory functions.

• Single-cell multi-omics: Integration of scRNA-seq with additional single-cell modalities (ATAC-seq, proteomics) will reveal how CLEC1A expression and function varies across myeloid cell states in health and disease.

• Therapeutic antibody optimization: Development of next-generation anti-CLEC1A antibodies with enhanced properties (higher affinity, optimized effector functions, reduced immunogenicity) could improve their clinical potential.

• Biomarker potential: Investigation of soluble CLEC1A or CLEC1A-expressing circulating cells as potential biomarkers for disease progression or treatment response in cancer and inflammatory conditions.

These approaches will expand our understanding of CLEC1A biology and may reveal novel therapeutic applications for targeting this receptor in various disease contexts .