CLEC10A Antibody

What is CLEC10A and why is it significant in immunological research?

CLEC10A (CD301, macrophage galactose-type C-type lectin) is a C-type lectin receptor that has been identified as a specific marker for human CD1c+ dendritic cells (DCs). Its significance in immunological research stems from its highly specific expression pattern, making it valuable for distinguishing CD1c+ DCs (also known as conventional DCs type 2 or cDC2) from other immune cell populations .

While other markers like CLEC9A have been established for CD141+ DCs (cDC1), CLEC10A fills an important gap by providing a specific marker for CD1c+ DCs. This specificity is crucial for accurately identifying and isolating these cells from various lymphohematopoietic tissues, enabling more precise investigation of their functions in both normal physiology and disease states .

Additionally, CLEC10A demonstrates high endocytic potential, rapidly internalizing upon ligand binding, which makes it a promising candidate for antigen-targeting approaches in research and potential therapeutic applications .

Which immune cell populations express CLEC10A?

CLEC10A exhibits a predominantly CD1c+ DC-specific expression pattern across multiple human lymphohematopoietic tissues. According to flow cytometric analyses, CLEC10A is expressed on approximately 80% of CD1c+ DCs in blood, thymus, and spleen, demonstrating a consistent expression profile across these tissues .

Limited expression is observed in other immune cell populations:

• Approximately 5% of blood monocytes

• About 20% of thymic B cells

• Roughly 20% of splenic monocytes/macrophages

Notably, CLEC10A expression is absent on CD141+ DCs, plasmacytoid DCs (pDCs), T cells, and NK cells . This restricted expression pattern makes CLEC10A particularly valuable as a specific marker for CD1c+ DCs across different human tissues.

What are the standard methods for detecting CLEC10A expression?

Detection of CLEC10A expression can be accomplished through several methodological approaches:

Transcriptomic Analysis:
Transcriptional analysis of CLEC10A mRNA expression in sorted cell populations has demonstrated strong expression in CD1c+ DCs across different tissues, with low expression in intermediate monocytes (CD14++CD16+) .

Flow Cytometry:
For protein-level detection, flow cytometry is the predominant methodology. Standard protocols involve:

• Single cell suspensions stained with anti-CLEC10A antibodies (e.g., clone H037G3 from BioLegend)

• Common fluorochromes include PE or APC conjugates

• Appropriate isotype controls (mouse IgG2a, clone MOPC-173) should be included

• Multi-parameter analysis with additional markers for distinguishing DC subsets (CD1c, CD141, CD303) and other lineages (CD3, CD14, CD19, CD20, CD56, NKp46)

Ligand-Based Detection:
CLEC10A can also be detected using its natural ligands:

• FITC-coupled MUC-1 peptide glycosylated with N-acetylgalactosamine (Tn antigen)

• Typically used at 10 μg/ml concentration

• Non-glycosylated MUC-1 peptide serves as a negative control

What is the role of CLEC10A in dendritic cell function?

CLEC10A plays several important roles in dendritic cell function, particularly in CD1c+ DCs:

Endocytic Activity:
CLEC10A demonstrates rapid internalization upon binding of specific antibodies or natural ligands, suggesting its involvement in antigen uptake and processing. This endocytic capacity makes it potentially valuable for targeted delivery of antigens to CD1c+ DCs .

Cytokine Production Modulation:
When CD1c+ DCs are exposed to a CLEC10A-specific bivalent ligand (MUC-1 peptide glycosylated with N-acetylgalactosamine) in combination with TLR7/8 stimulation (R848), there is enhanced secretion of several cytokines:

• TNFα (pro-inflammatory)

• IL-8 (neutrophil chemotactic factor)

• IL-10 (anti-inflammatory)

This suggests CLEC10A signaling can modulate the inflammatory response of CD1c+ DCs when co-stimulated with TLR ligands.

Immunoregulatory Function:
In murine models, the homolog of CLEC10A (Clec10a/MGL1/CD301a) plays an important role in skin homeostasis against house dust mite (HDM)-induced dermatitis by inhibiting TLR4-mediated inflammatory cytokine production through its inhibitory immunoreceptor tyrosine activating motif .

What are the best practices for using anti-CLEC10A antibodies in flow cytometry?

When using anti-CLEC10A antibodies in flow cytometric applications, researchers should consider the following best practices:

Antibody Selection and Validation:

• The clone H037G3 (available from BioLegend) has been validated for specific detection of CLEC10A on human CD1c+ DCs

• PE or APC conjugates are commonly used fluorochromes

• Always include appropriate isotype controls (mouse IgG2a, clone MOPC-173) to assess non-specific binding

Staining Protocol:

• Prepare single cell suspensions from blood or tissues (6-8 × 10^6 cells recommended)

• Stain cells with anti-CLEC10A antibody along with a comprehensive antibody panel to identify DC subsets and exclude other lineages

• Incubate for 15 minutes at 4°C

• Wash cells and analyze by flow cytometry

Gating Strategy:

• First exclude lineage-positive cells (CD3, CD14, CD19, CD20, CD56, NKp46)

• Gate on HLA-DR+ cells to identify antigen-presenting cells

• Further discriminate DC subsets using CD1c, CD141, and CD303

• Assess CLEC10A expression on these subpopulations

Analysis Considerations:

• Account for tissue-specific variations in CLEC10A expression

• Consider that approximately 80% of CD1c+ DCs across tissues express CLEC10A, while a small fraction remains CLEC10A-negative

• Recognize that low expression can be detected on some non-DC populations

How can CLEC10A antibodies be used to isolate CD1c+ dendritic cells?

CLEC10A antibodies offer a promising approach for isolating highly purified CD1c+ DCs from human samples:

Sequential Magnetic Separation Strategy:

• Initial enrichment of total dendritic cells through depletion of lineage-positive cells

• Positive selection using anti-CLEC10A antibodies

Flow Cytometry-Based Cell Sorting:

• Stain cells with anti-CLEC10A and other DC markers

• Gate on lineage-negative, HLA-DR+, CD1c+, CLEC10A+ cells

• Sort using a high-speed cell sorter

Advantages Over CD1c-Only Selection:

• Higher specificity for conventional CD1c+ DCs

• Reduces contamination with CD1c+ monocytes or B cells

• Allows isolation of a functionally distinct subset of CD1c+ DCs

Purity Assessment:
After isolation, confirm purity by re-analysis of a small aliquot for:

• CLEC10A expression (>95% should be positive)

• Absence of lineage markers

• Positive expression of CD1c and HLA-DR

What are the known ligands for CLEC10A and their effects on dendritic cell function?

CLEC10A recognizes specific glycan structures and has several identified ligands that affect dendritic cell function:

N-acetylgalactosamine (GalNAc/Tn antigen):

• Primary carbohydrate ligand recognized by CLEC10A

• Often found on mucin-type O-linked glycoproteins

MUC-1 Peptide with GalNAc Modifications:

• Glycosylated MUC-1 peptide (βAla-GVTSAPDTRPAPGSTAPPAHGVT-NH₂) with N-acetylgalactosamine at Serine 4 and Threonine 15

• Binds specifically to CLEC10A on CD1c+ DCs

• When combined with TLR7/8 ligand R848, enhances secretion of:

  • TNFα (pro-inflammatory)

  • IL-8 (neutrophil chemoattractant)

  • IL-10 (immunoregulatory)

Functional Effects of Ligand Binding:

• Receptor Internalization: Ligand binding triggers rapid internalization of CLEC10A

• Modulation of TLR Responses: Can enhance cytokine production when combined with TLR stimulation

• Potential Immunoregulatory Role: May contribute to balancing inflammatory responses through IL-10 production

In mouse models, a mucin-like molecule in house dust mites has been identified as a ligand for mouse Clec10a and its human homolog Asgr1, with application of this ligand showing amelioration of TLR4 ligand-induced dermatitis .

How does CLEC10A expression differ across human tissues?

CLEC10A demonstrates a remarkably consistent expression pattern on CD1c+ DCs across different human lymphohematopoietic tissues, while showing some variation in its expression on other immune cell populations:

This consistent expression profile across different tissues demonstrates that CLEC10A is a reliable marker for CD1c+ DCs regardless of tissue origin. The data indicate an "organ-irrespective expression profile" for CLEC10A on CD1c+ DCs, making it a valuable marker for identifying this cell population in diverse anatomical locations .

What are the technical considerations when designing CLEC10A internalization assays?

When designing internalization assays for CLEC10A, researchers should consider several technical aspects to ensure reliable and reproducible results:

Protocol Design:

• Antibody Labeling: Use PE-coupled anti-CLEC10A antibody (clone H037G3) or appropriate isotype control (mouse IgG2a, MOPC-173)

• Initial Surface Labeling: Stain cells on ice (15 minutes) to prevent premature internalization

• Internalization Period: Incubate cells at 37°C for various time points (5, 15, 30, and 60 minutes)

• Control Condition: Maintain a sample on ice for 60 minutes to establish baseline (non-internalized) state

• Detection of Remaining Surface Receptors: Label with anti-PE primary antibody followed by fluorescently-tagged secondary antibody (e.g., anti-goat-A647)

Critical Parameters:

• Cell Viability: Ensure >90% viability for accurate assessment

• Temperature Control: Strict maintenance of 4°C during initial labeling and 37°C during internalization phase

• Antibody Concentration: Titrate to ensure saturation without excess

• Time Points: Include multiple time points to establish internalization kinetics

Data Analysis Approach:

• Calculate internalization rates as percentage of initial surface expression

• Account for natural receptor turnover in control samples

• Consider analyzing both median fluorescence intensity and percentage of positive cells

Alternative Methodologies:

• pH-sensitive Fluorophores: Allow direct visualization of internalization into acidic endosomal compartments

• Confocal Microscopy: Provides spatial information about receptor trafficking

• Biochemical Approaches: Surface biotinylation followed by stripping and Western blotting can quantify internalization

How can CLEC10A be targeted for antigen delivery in immunotherapy research?

CLEC10A offers significant potential as a target for antigen delivery in immunotherapy research due to its specific expression on CD1c+ DCs and rapid internalization properties:

Targeting Strategies:

• Antibody-Antigen Conjugates:

  • Direct conjugation of antigens to anti-CLEC10A antibodies

  • May utilize chemical crosslinking or recombinant fusion protein approaches

  • Clone H037G3 has demonstrated efficient internalization properties

• Glycan-Modified Antigens:

  • Incorporation of N-acetylgalactosamine (GalNAc/Tn antigen) into antigenic peptides or proteins

  • Example: MUC-1 peptide glycosylated with N-acetylgalactosamine at specific serine/threonine residues

  • Natural ligand approach may offer physiological targeting mechanism

• Nanoparticle-Based Delivery:

  • Development of nanoparticles decorated with CLEC10A ligands

  • Allows co-delivery of antigens and adjuvants

  • Potential for controlled release of cargo

Experimental Considerations:

• Adjuvant Selection: Combining CLEC10A targeting with TLR7/8 agonists (e.g., R848) has shown enhanced cytokine production

• Antigen Processing Efficiency: Monitor intracellular trafficking to endosomal/lysosomal compartments

• Cross-Presentation Capacity: Assess ability of targeted CD1c+ DCs to present antigens to CD8+ T cells

• In Vivo Models: Consider using humanized mouse models expressing human CLEC10A or assessing the human homolog Asgr1

Potential Applications:

• Cancer Immunotherapy: Delivery of tumor antigens to CD1c+ DCs

• Vaccination: Enhanced humoral and cellular responses to vaccine antigens

• Autoimmunity: Delivery of tolerogenic signals to modulate immune responses

• Allergic Conditions: Targeting immunoregulatory pathways based on Clec10a's role in house dust mite-induced dermatitis

What are the differences between mouse and human CLEC10A expression and function?

Understanding the differences between mouse and human CLEC10A is crucial for translational research and proper experimental design:

Genetic and Structural Differences:

• Gene Duplication: Mice possess two homologs of human CLEC10A:

  • CD301a/MGL1: More closely related to human CLEC10A

  • CD301b/MGL2: Distinct expression pattern and binding specificity

• Human Homolog: In humans, asialoglycoprotein receptor 1 (Asgr1) has been identified as a functional homolog of mouse Clec10a in some contexts, particularly in house dust mite-induced skin inflammation

Expression Pattern Disparities:

Functional Differences:

• Carbohydrate Recognition:

  • Human CLEC10A: Primarily recognizes N-acetylgalactosamine (Tn antigen)

  • Mouse CD301a/MGL1: Preferentially binds Lewis X and Lewis A structures

  • Mouse CD301b/MGL2: Recognizes N-acetylgalactosamine similar to human CLEC10A

• Immunoregulatory Function:

  • Mouse Clec10a plays a role in skin homeostasis against house dust mite-induced dermatitis

  • Mouse Clec10a inhibits TLR4-mediated inflammatory cytokine production through its inhibitory immunoreceptor tyrosine activating motif

  • Similar immunoregulatory functions are likely present in human CLEC10A but with potential differences in specific pathways

Experimental Implications:

• Data from mouse models using CD301a/MGL1 cannot be directly translated to human CLEC10A function

• Humanized mouse models or consideration of Asgr1 may be more appropriate for translational studies

• Experimental design should account for these species-specific differences when targeting CLEC10A/MGL receptors

How does CLEC10A signaling interact with TLR pathways in dendritic cells?

The interaction between CLEC10A signaling and Toll-like receptor (TLR) pathways represents an important area of research for understanding dendritic cell function:

Signaling Crosstalk:

• Enhanced Cytokine Production: When CD1c+ DCs are stimulated with both CLEC10A ligands (MUC-1 peptide glycosylated with N-acetylgalactosamine) and TLR7/8 agonist R848, there is enhanced secretion of:

  • TNFα: Pro-inflammatory cytokine

  • IL-8: Neutrophil chemotactic factor

  • IL-10: Anti-inflammatory/regulatory cytokine

• Balanced Immune Response: The concurrent production of both pro-inflammatory (TNFα, IL-8) and anti-inflammatory (IL-10) cytokines suggests CLEC10A may help balance inflammatory responses initiated by TLR signaling

Mechanistic Insights:

• Inhibitory Signaling: In mouse models, Clec10a has been shown to inhibit TLR4-mediated inflammatory responses through its inhibitory immunoreceptor tyrosine activating motif in its cytoplasmic portion

• Skin Homeostasis: This inhibitory effect contributes to skin homeostasis against house dust mite (HDM)-induced dermatitis

Therapeutic Implications:

• Targeting CLEC10A in Inflammatory Disorders: The inhibitory effect on TLR-mediated inflammation suggests potential applications in treating inflammatory skin conditions

• Balancing Vaccine Adjuvanticity: CLEC10A targeting could be combined with TLR agonists to achieve balanced immune activation with both effector and regulatory components

• Personalized Approaches: Understanding individual variations in CLEC10A/TLR crosstalk may inform personalized treatment strategies

Future Research Directions:

• Further elucidation of the molecular mechanisms underlying CLEC10A-TLR crosstalk

• Investigation of cell type-specific differences in signaling interactions

• Development of targeted therapeutics that exploit this signaling interaction for immunomodulation