CLEC4C Recombinant Monoclonal Antibody

What is CLEC4C and why is it significant for immunological research?

CLEC4C (C-type lectin domain family 4 member C), also known as BDCA-2, CD303, DLEC, or HECL, is a type II membrane protein containing one C-type lectin domain. It serves as a specific surface marker of human plasmacytoid dendritic cells (pDCs) and plays central roles in antigen presentation and immune tolerance . CLEC4C functions as a lectin-type cell surface receptor involved in antigen capturing by dendritic cells, with particular importance in regulating antiviral immunity . Its significance stems from its ability to inhibit type I interferon (IFN) production, particularly IFNα, making it a critical regulator of immune responses during viral infections and potentially in autoimmune conditions .

For researchers, CLEC4C represents an important target for studying pDC biology, antiviral immunity, and immunoregulatory mechanisms. Its specific expression pattern allows for precise identification and isolation of pDCs from mixed cell populations, while its functional properties offer insights into immune modulation pathways.

How do CLEC4C recombinant monoclonal antibodies differ from polyclonal alternatives?

CLEC4C recombinant monoclonal antibodies offer several methodological advantages over polyclonal alternatives:

For instance, Abcam's anti-CLEC4C recombinant monoclonal antibody [EPR21985] can be obtained conjugated to APC for flow cytometry applications , while polyclonal alternatives like Proteintech's 17941-1-AP are primarily validated for ELISA applications . The choice between formats should be guided by specific experimental requirements, with recombinant monoclonals offering superior reproducibility for longitudinal studies.

What are the optimal experimental conditions for using CLEC4C recombinant monoclonal antibodies in flow cytometry?

When designing flow cytometry experiments with CLEC4C recombinant monoclonal antibodies, researchers should consider the following methodological approach:

• Sample preparation: Fresh isolation of peripheral blood mononuclear cells (PBMCs) is recommended, as cryopreservation can alter surface marker expression. Process samples within 24 hours of collection to maintain pDC viability.

• Antibody titration: Despite manufacturer recommendations, perform titration experiments to determine optimal antibody concentration. Research indicates that approximately 5 μg of CLEC4C-CRD tetramers per 250,000 cells provides adequate staining intensity .

• Blocking protocol: Include a Fc receptor blocking step (10-15 minutes at room temperature) prior to antibody staining to prevent non-specific binding, especially when working with FcR-expressing cells.

• Staining buffer optimization: Use a buffer containing 1-2% protein (BSA or FBS) and 0.1% sodium azide to maintain antibody stability and prevent internalization of surface markers.

• Gating strategy: When identifying pDCs, use a sequential gating approach combining CLEC4C/CD303 with other markers such as HLA-DR, CD123, and lineage markers (CD3, CD14, CD19, CD56) for accurate identification.

• Controls: Include both isotype controls and fluorescence-minus-one (FMO) controls to establish gating boundaries. Blocking experiments with unlabeled anti-CLEC4C can be used to confirm specificity, as demonstrated in studies where anti-CLEC4C mAb strongly inhibited CLEC4C-CRD-PE binding to monocytes while isotype-matched antibodies had no effect .

For APC-conjugated CLEC4C antibodies, minimize exposure to light during staining procedures and analysis to prevent fluorophore photobleaching.

How can CLEC4C antibodies be utilized to study plasmacytoid dendritic cell function in vitro?

CLEC4C antibodies serve as valuable tools for studying pDC function through several methodological approaches:

• Isolation and purification of pDCs: CLEC4C antibodies can be used for positive selection of pDCs from PBMCs using magnetic bead separation or flow cytometry-based sorting. While positive selection provides high purity, researchers should be aware that antibody binding to CLEC4C may induce signaling and potentially alter cellular functions.

• Modulation of type I IFN production: Experimental evidence shows that CLEC4C engagement inhibits TLR9-induced type I IFN production. Researchers can leverage this property by pre-incubating pDCs with CLEC4C antibodies before TLR stimulation to assess downstream effects on IFN production and signaling pathways . This approach has revealed that CLEC4C transmits intracellular signals through an associated transmembrane adaptor, the FcεRγ, which recruits protein tyrosine kinase Syk, inducing tyrosine phosphorylation and calcium mobilization .

• Co-culture systems: Studies demonstrate that CLEC4C ligand-expressing cells can modulate pDC function when co-cultured. For example, research has shown that the presence of autologous monocytes significantly reduces the production of IFNα mRNA and protein in CpG-activated pDCs . Researchers can use CLEC4C antibodies to block these interactions and determine specificity.

• Glycan-mediated interactions: To study CLEC4C binding to its natural ligands, researchers can manipulate terminal galactose expression using exoglycosidases. Data indicates that hydrolysis of the β1–4-glycosidic bond with β-(1–4)-galactosidase markedly reduces CLEC4C binding to target cells (from 74% to 25%) , providing a method to investigate the importance of specific glycan structures in CLEC4C-mediated regulation.

• Signaling pathway analysis: Using CLEC4C antibodies to trigger receptor signaling, researchers can study downstream molecular events including calcium flux, protein phosphorylation, and gene expression changes associated with pDC regulation.

How can CLEC4C recombinant monoclonal antibodies be employed to investigate the intersection of viral infection and autoimmunity?

Investigating the dual role of CLEC4C in viral infections and autoimmunity requires sophisticated experimental approaches:

• Ex vivo analysis of patient samples: CLEC4C recombinant monoclonal antibodies can be used to assess pDC activation states in patients with viral infections versus autoimmune conditions. Flow cytometric analysis comparing CLEC4C expression levels and correlation with IFN-α production can provide insights into disease-specific pDC dysregulation. Research indicates that CLEC4C can block IFNα production by binding to oligosaccharides with terminal residues of β1–4- or β1–3-galactose , suggesting potential mechanisms for viral immune evasion.

• Glycan modulation experiments: Advanced glycomics approaches can be combined with CLEC4C antibody studies to investigate how viral infection alters the glycosylation patterns of cell surface proteins. Researchers can employ neuraminidase treatment to unmask CLEC4C binding sites, which has been shown to completely block IFNα production by pDCs , mimicking potential viral strategies for immune suppression.

• Single-cell analysis: Coupling CLEC4C antibody staining with single-cell RNA sequencing or mass cytometry allows researchers to identify distinct pDC subpopulations and their functional states across disease contexts. This approach can reveal heterogeneity in pDC responses that may contribute to either effective viral clearance or pathological autoimmunity.

• Receptor engagement dynamics: Using CLEC4C antibodies in combination with live-cell imaging techniques enables real-time visualization of receptor clustering, internalization, and trafficking following ligand binding. These dynamics may differ between viral and autoimmune settings, providing insights into context-specific pDC regulation.

• Therapeutic intervention models: In preclinical models, CLEC4C-targeting strategies can be tested for their ability to modulate immune responses in both viral infection and autoimmunity contexts. These studies could establish the therapeutic potential of targeting this pathway to enhance antiviral immunity or suppress autoimmune inflammation.

What are the critical considerations when using CLEC4C antibodies to study cross-talk between plasmacytoid dendritic cells and other immune cell populations?

Studying immune cell cross-talk involving pDCs and CLEC4C requires careful methodological considerations:

• Multi-parameter experimental design: When investigating cellular interactions, combine CLEC4C antibodies with markers for other cell populations (T cells, B cells, conventional DCs, etc.) and functional readouts. Research has demonstrated that CLEC4C specifically binds monocytes and monocyte-derived dendritic cells but not CD14− peripheral blood mononuclear cells (which mostly include T and B lymphocytes), natural killer cells, or EBV-transformed B cell lines , indicating selective interaction patterns.

• Trans-well vs. direct co-culture systems: To distinguish between contact-dependent and soluble factor-mediated interactions, implement parallel experimental setups. Evidence suggests that direct cellular contact with CLEC4C ligand-expressing cells significantly impacts pDC function, as demonstrated by the reduction of IFNα production when pDCs are co-cultured with monocytes but not with B-EBV cells (CLEC4C-CRD-PE-negative) .

• Temporal dynamics: Establish time-course experiments to capture the kinetics of cellular interactions, as CLEC4C-mediated inhibition of type I IFN production may have different temporal requirements than positive regulatory pathways.

• Microenvironmental factors: Consider tissue-specific conditions when studying CLEC4C-mediated interactions. The glycosylation patterns that determine CLEC4C ligand availability may vary across tissues and disease states, affecting the functional outcomes of pDC interactions with other cell types.

• Receptor competition analysis: When multiple C-type lectin receptors are present, competitive binding assays using labeled CLEC4C antibodies can determine receptor hierarchy and functional dominance. Research comparing CLEC4C to other C-type lectins (like DC-SIGN, DC-SIGN receptor, and langerin) reveals distinct binding specificities, with CLEC4C recognizing terminal galactose residues similar to the hepatic asialo-glycoprotein receptor (ASPG-R) .

• Integrating proteomic approaches: Coupling CLEC4C antibody-based cell sorting with proteomics can identify novel interaction partners and signaling complexes formed during pDC cross-talk with other immune cells.

What are the common pitfalls when working with CLEC4C recombinant monoclonal antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with CLEC4C recombinant monoclonal antibodies:

• Epitope masking by glycosylation: CLEC4C binds to terminal galactose residues on glycans, but these can be masked by sialic acid residues in many cell types. Experimental evidence shows that neuraminidase treatment to remove sialic acids can dramatically reveal CLEC4C binding sites . When unexpected negative results occur, consider enzymatic treatment of samples with neuraminidase before antibody staining.

• Low frequency target cells: pDCs typically represent <0.5% of peripheral blood leukocytes, making detection challenging. Implement enrichment steps (like density gradient separation followed by negative selection) before antibody staining to increase the probability of detecting rare pDC populations.

• Antibody internalization: C-type lectin receptors including CLEC4C can internalize upon ligand binding. To minimize this effect, perform staining at 4°C rather than 37°C and include sodium azide in staining buffers to inhibit cellular metabolism and receptor trafficking.

• Cross-reactivity with other C-type lectins: Validate antibody specificity using knockout controls or blocking experiments. Research comparing CLEC4C to other C-type lectins has shown that it has distinctive binding properties, specifically recognizing complex type sugars with terminal galactose .

• Batch-to-batch variability: Despite the improved consistency of recombinant antibodies, perform quality control with each new lot. Flow cytometry standardization using calibration beads can help normalize data across experiments.

• Storage and handling: Antibody functionality can be compromised by improper storage. Follow manufacturer recommendations for storage conditions, which typically include keeping antibodies at -20°C and avoiding repeated freeze-thaw cycles. Products like Proteintech's antibodies are stable for one year after shipment when stored properly .

How can researchers optimize CLEC4C antibody-based assays for detecting low-abundance ligands or interactions?

To enhance detection sensitivity when studying low-abundance CLEC4C interactions:

• Signal amplification strategies: Implement biotin-streptavidin systems or tyramide signal amplification to enhance detection sensitivity. Studies have successfully used tetrameric forms of CLEC4C-CRD-PE for improved avidity in binding assays , demonstrating the effectiveness of multivalent formats.

• Proximity ligation assays (PLA): This technique can detect protein-protein interactions with single-molecule sensitivity. By combining CLEC4C antibodies with antibodies against potential interaction partners, researchers can visualize rare or transient binding events that might be missed by conventional co-immunoprecipitation.

• Super-resolution microscopy: Techniques like STORM or PALM coupled with fluorophore-conjugated CLEC4C antibodies enable visualization of receptor nanoclustering and co-localization with signaling components below the diffraction limit.

• Enzymatic pre-treatments: Optimize sample preparation by systematically testing different glycosidases to unmask potential CLEC4C binding sites. Research has demonstrated that β-(1–4)-galactosidase treatment significantly reduces CLEC4C binding , suggesting that careful enzymatic manipulation can reveal physiologically relevant interactions.

• Recombinant protein engineering: Generate high-avidity reagents by creating multimeric forms of CLEC4C extracellular domains. Researchers have successfully produced soluble forms of the CLEC4C ectodomain that can be used to probe glycan arrays and identify specific binding partners .

• Quantitative binding assays: Employ surface plasmon resonance (SPR) or bio-layer interferometry (BLI) with immobilized CLEC4C recombinant proteins to measure binding kinetics with potential ligands. Data shows that immobilized human CLEC4C can bind anti-CLEC4C recombinant antibody with EC50 values of 7.658-12.99 ng/mL in functional ELISA assays , providing a reference point for assay sensitivity.

How can CLEC4C recombinant monoclonal antibodies be utilized to explore the therapeutic potential of targeting plasmacytoid dendritic cells in inflammatory diseases?

The therapeutic targeting of pDCs via CLEC4C represents an emerging research frontier:

• Selective depletion models: CLEC4C antibodies conjugated to toxins or used in CAR-T cell designs can selectively eliminate pDCs in preclinical models to evaluate the impact on disease progression. By tracking outcomes in inflammatory disease models following pDC depletion, researchers can establish causality between pDC activity and pathology.

• Functional modulation without depletion: Since CLEC4C engagement inhibits IFNα production, antibodies can be designed to modify pDC function rather than deplete these cells. Studies have demonstrated that CLEC4C-mediated suppression of IFNα production is regulated by the masking/unmasking of galactose moieties , suggesting that modulating this pathway could provide therapeutic benefit in conditions characterized by excessive type I IFN production.

• Biomarker development: CLEC4C antibodies can be used to monitor pDC numbers, activation status, and receptor expression patterns during disease progression and therapeutic intervention. Quantitative flow cytometry using standardized CLEC4C antibodies may reveal correlations between pDC phenotypes and disease activity or treatment response.

• Combination therapy approaches: Evaluate how CLEC4C-targeting strategies interact with established immunotherapies, such as checkpoint inhibitors or cytokine antagonists. The intersection of CLEC4C signaling with other immune pathways may reveal synergistic treatment opportunities.

• Targeted drug delivery: Exploit CLEC4C's endocytic properties to deliver therapeutic payloads specifically to pDCs. Research shows that CLEC4C efficiently targets ligands into antigen-processing and peptide-loading compartments , indicating potential for delivering immunomodulatory compounds directly to these cells.

What are the methodological considerations for studying CLEC4C glycan binding specificity in different tissue microenvironments?

Investigating tissue-specific CLEC4C-glycan interactions requires specialized approaches:

• Tissue-specific glycomic profiling: Compare glycan structures across different tissues using mass spectrometry and lectin arrays to identify potential CLEC4C binding partners. Research has established that CLEC4C specifically recognizes complex type sugars with terminal galactose, particularly non-sialylated galactose-terminated biantennary glycans containing the trisaccharide epitope Gal(β1-3/4)GlcNAc(β1-2)Man .

• In situ proximity labeling: Develop tissue-specific CLEC4C proximity labeling systems to identify glycoprotein ligands in their native context. This approach can reveal tissue-specific interaction partners that may not be detected in reductionist in vitro systems.

• Ex vivo tissue slice models: Apply fluorescently labeled CLEC4C recombinant proteins to tissue slices with and without glycosidase treatments to map the distribution of binding sites across different anatomical compartments. This spatial information can provide insights into potential in vivo interaction sites.

• Competitive binding assays: Design experiments to compare CLEC4C binding in the presence of tissue-specific glycan mixtures or other C-type lectins. This approach can reveal how the tissue microenvironment may influence CLEC4C function through competitive or cooperative interactions.

• Glycan array modifications: Customize glycan arrays to include tissue-specific oligosaccharide structures. Research using glycan arrays has already identified that CLEC4C recognizes with high specificity terminal residues of β1–4- or β1–3-galactose at the end of biantennary complex sugars , but this may be expanded to include tri- and tetra-antennary complex type oligosaccharides that weren't present in available arrays.

• Recombinant glycoprotein engineering: Generate tissue-specific glycoprotein variants with defined glycan structures to systematically evaluate CLEC4C binding preferences under controlled conditions.