Clec4e Antibody

What is CLEC4E and why is it important for immunological research?

CLEC4E (C-type lectin domain family 4 member E), also known as Mincle, is a pattern recognition receptor (PRR) of the innate immune system that recognizes damage-associated molecular patterns (DAMPs) of abnormal self and pathogen-associated molecular patterns (PAMPs) of bacteria and fungi. The protein consists of a 19 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 179 amino acid extracellular domain containing the C-type lectin domain .

CLEC4E is particularly important for research because:

• It recognizes mycobacterial trehalose 6,6'-dimycolate (TDM), a cell wall glycolipid with potent adjuvant immunomodulatory functions

• It plays a critical role in host defense against Mycobacterium tuberculosis (Mtb)

• It contributes to inflammatory responses through recognizing DAMPs released during non-homeostatic cell death

• It has emerged as a potential therapeutic target for host-directed therapies against drug-resistant pathogens

Which cell types express CLEC4E and how can this be detected?

CLEC4E is primarily expressed on:

• Monocytes

• Macrophages (including bone marrow-derived macrophages)

• Immature dendritic cells

• Some B cells (upregulated following anti-thymocyte globulin treatment in transplantation models)

Detection methods include:

• Flow cytometry using specific anti-CLEC4E antibodies (recommended application for most commercial antibodies)

• Western blotting for protein expression analysis

• Immunohistochemistry/immunofluorescence for tissue section analysis

• qRT-PCR for transcript analysis

For flow cytometry applications, optimal antibody concentration is typically 0.25 μg per 10^6 cells, though this should be optimized for specific experimental conditions .

What are the most reliable applications for CLEC4E antibodies in basic research?

Based on the validated applications reported in the search results, the most reliable applications include:

• Flow cytometry: Most commercial antibodies are validated for flow cytometric analysis of CLEC4E expression, particularly useful for examining expression on different immune cell populations

• Western blotting: For detection of CLEC4E protein in cell or tissue lysates, with expected molecular weight of approximately 30 kDa

• Immunohistochemistry/immunofluorescence: For visualization of CLEC4E expression in tissue sections or fixed cells

• CyTOF (mass cytometry): Some antibodies are validated as "CyTOF-ready," allowing for highly multiplexed analysis of CLEC4E alongside other markers

When selecting antibodies for these applications, researchers should verify specificity using appropriate controls, such as CLEC4E-transfected versus control-transfected cells as demonstrated in the validation data .

How can CLEC4E antibodies be used to investigate the receptor's role in autophagy induction against Mycobacterium tuberculosis?

CLEC4E has been demonstrated to play a novel role in inducing autophagy against Mtb infection. To investigate this function:

• Experimental design approach:

  • Use anti-CLEC4E antibodies to block receptor function in macrophage cultures infected with Mtb

  • Compare with isotype controls to assess specificity

  • Alternatively, use anti-CLEC4E antibodies to immunoprecipitate the receptor and identify interacting proteins involved in autophagy signaling

• Key markers to assess:

  • LC3-I to LC3-II conversion (autophagy marker)

  • p62/SQSTM1 degradation

  • Formation of autophagosomes (by microscopy)

  • Co-localization of Mtb with autophagic vesicles

• Relevant findings to build upon:

  • CLEC4E signaling activates MYD88, PtdIns3K, STAT1, and RELA/NFKB pathways

  • It increases lysosome biogenesis and enhances macroautophagy

  • Macrophages from autophagy-deficient (atg5 knockout or Becn1 knockdown) mice showed elevated survival of Mtb despite CLEC4E activation

  • Combined stimulation of CLEC4E and TLR4 (denoted as C4.T4) significantly enhanced bactericidal activity against Mtb

What technical considerations are important when using CLEC4E antibodies to study receptor signaling complexes?

When investigating CLEC4E signaling complexes, several technical considerations are crucial:

• Preserving protein-protein interactions:

  • Use mild detergents (e.g., 1% NP-40 or 1% digitonin) for cell lysis to maintain associated proteins

  • Include protease and phosphatase inhibitors to prevent degradation of signaling components

  • Consider crosslinking approaches for transient interactions

• Important binding partners to assess:

  • CLEC4D/MCL (forms heteromeric complexes with CLEC4E)

  • FcRγ chain (FCER1G) - associates through an Arg residue in the CLEC4E transmembrane segment

  • SYK kinase - critical for downstream signaling

  • CARD9 complex components

• Validation approaches:

  • Use SYK inhibitors (e.g., piceatannol) to confirm specificity of signaling

  • Include controls for non-specific binding in immunoprecipitation experiments

  • Consider CLEC4E knockout or knockdown models as negative controls

• Species-specific considerations:

  • Human and mouse CLEC4E have different ligand specificity - mouse CLEC4E does not appear to interact with TDB, GroMM, or cholesterol crystals

How can researchers effectively use CLEC4E antibodies in models of host-directed therapy against tuberculosis?

Based on research showing CLEC4E's role in host defense against Mtb, researchers can use CLEC4E antibodies in host-directed therapy models as follows:

• Experimental approaches:

  • Use anti-CLEC4E antibodies to track receptor expression in animal models receiving C4.T4 agonist therapy

  • Employ flow cytometry to assess changes in CLEC4E expression on macrophage populations following treatment

  • Conduct immunohistochemistry on lung sections to visualize CLEC4E expression in granulomatous lesions

• Key experimental findings to consider:

  • Administration of C4.T4 (CLEC4E and TLR4 agonists) to Mtb-challenged mice protects them, as indicated by significantly reduced bacterial burden in lungs, liver, and spleen

  • C4.T4 treatment substantially decreases the number of granulomas in the lung

  • Treatment with C4.T4 in combination with rifampicin (RIF) considerably boosts the killing efficacy of the drug compared to drug alone

  • 1 mg/kg body weight of RIF in combination with C4.T4 decreased Mtb burden equivalent to 10 mg/kg of RIF alone

  • C4.T4 treatment expanded the pool of Mtb-specific Th1 and Th17 cells

• Methodological considerations:

  • Include appropriate isotype controls to rule out non-specific effects

  • Assess CLEC4E expression before and after treatment to correlate with treatment efficacy

  • Consider using CLEC4E knockout animal models as negative controls

What are the optimal fixation and staining protocols for detecting CLEC4E in flow cytometry experiments?

For optimal detection of CLEC4E by flow cytometry, the following protocol is recommended based on available data:

• Cell preparation:

  • Harvest cells (peripheral blood monocytes, macrophages, or dendritic cells)

  • Wash cells in cold PBS containing 1% BSA or FBS

  • Adjust to 1×10^6 cells per 100 μl staining buffer

• Fixation options:

  • For surface staining only: use fresh cells without fixation

  • For combined surface/intracellular staining: fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

• Staining procedure:

  • Block Fc receptors with human FcR blocking reagent (5-15 minutes)

  • Incubate with primary anti-CLEC4E antibody (0.25 μg per 10^6 cells is recommended starting concentration)

  • For unconjugated antibodies, follow with appropriate fluorophore-conjugated secondary antibody

  • For direct staining, use conjugated antibodies such as Alexa Fluor 488 or Alexa Fluor 700-conjugated anti-CLEC4E

• Controls to include:

  • Isotype control antibody at the same concentration

  • FMO (fluorescence minus one) controls

  • Positive control: CLEC4E-transfected cell line (if available)

  • Negative control: untransfected cell line or CLEC4E-negative cell population

Based on validation data, this approach has successfully detected CLEC4E expression in transfected HEK293 cells versus control cells .

What factors affect CLEC4E expression and how should these be controlled for in antibody-based experiments?

Several factors influence CLEC4E expression, which researchers should account for when designing experiments:

• Activation status of myeloid cells:

  • CLEC4E expression is upregulated on Mtb-infected macrophages compared to uninfected cells

  • Expression levels change during macrophage differentiation and activation

• Inflammatory stimuli:

  • Microbial products (e.g., TLR ligands) can alter CLEC4E expression

  • Inflammatory cytokines may influence receptor levels

  • Cell death in surrounding tissue can impact expression through DAMP release

• Experimental manipulations that affect expression:

  • Anti-thymocyte globulin (ATG) treatment upregulates CLEC4E expression on B cells in transplantation models

  • CD4 T cell depletion affects CLEC4E expression in a CD4-dependent manner

• Control measures:

  • Include time-matched controls for all experimental conditions

  • Standardize cell isolation procedures to minimize activation

  • Consider kinetic analyses to capture expression changes over time

  • Include both transcript (qRT-PCR) and protein-level analyses to distinguish between transcriptional and post-transcriptional regulation

How can researchers validate CLEC4E antibody specificity and avoid cross-reactivity with other C-type lectin receptors?

To ensure antibody specificity and avoid cross-reactivity with related C-type lectin receptors, researchers should implement these validation steps:

• Genetic validation approaches:

  • Test antibodies on CLEC4E knockout or knockdown cells/tissues

  • Use CLEC4E-transfected cell lines as positive controls compared to empty vector-transfected controls

  • Consider testing on cells from CLEC4E knockout mice if working with mouse models

• Cross-reactivity assessment:

  • Test against cells expressing related C-type lectins, especially CLEC4D/MCL which associates with CLEC4E

  • Perform Western blots to confirm single band of expected molecular weight

  • Consider peptide blocking experiments using the immunizing peptide

• Functional validation:

  • Use CLEC4E-specific inhibitors (e.g., piceatannol for SYK inhibition) to confirm specificity of signaling

  • Validate with alternative antibody clones targeting different epitopes

  • Correlate protein detection with transcript levels by parallel qRT-PCR analysis

• Species considerations:

  • Human CLEC4E shares 65% and 68% amino acid sequence identity with mouse and rat CLEC4E, respectively, within the extracellular domain

  • Confirm species specificity when working with cross-species models

How should researchers interpret conflicting data between CLEC4E expression and function across different experimental systems?

When encountering conflicting data regarding CLEC4E expression or function across experimental systems:

• Species-specific differences:

  • Human and mouse CLEC4E have distinct ligand recognition patterns:

    • Human CLEC4E binds TDB, GroMM, and cholesterol crystals

    • Mouse CLEC4E does not appear to interact with these ligands

  • Consider these differences when translating findings between animal models and human systems

• Cell type-specific functions:

  • CLEC4E functions differently in various cell types:

    • In macrophages: primarily involved in pathogen recognition and inflammatory responses

    • In B cells: contributes to T cell recovery after lymphoablation

  • Validate findings across relevant cell types for your research question

• Contextual signaling:

  • CLEC4E signaling outcomes depend on co-receptors and cellular context:

    • Combined signaling with TLR4 (C4.T4) enhances bactericidal activity

    • Different downstream effects occur depending on specific ligands engaged

  • Examine co-receptor expression and activation in your experimental system

• Technical reconciliation approaches:

  • Use multiple antibody clones targeting different epitopes

  • Employ complementary detection methods (flow cytometry, Western blotting, immunofluorescence)

  • Include genetic approaches (knockdown/knockout) alongside antibody-based detection

  • Consider post-translational modifications that might affect antibody recognition

What emerging research directions are utilizing CLEC4E antibodies beyond traditional immunological applications?

Several innovative research directions are utilizing CLEC4E antibodies beyond classical immunology:

• Transplantation biology:

  • CLEC4E expression is upregulated in B cells from heart allograft recipients treated with anti-thymocyte globulin

  • Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution

  • This presents new opportunities for modulating transplant rejection through CLEC4E-targeted approaches

• Host-directed therapies:

  • Combination of CLEC4E activation with conventional antibiotics shows promise for enhancing treatment efficacy

  • Intracellular killing of Mtb was achieved with a 10-fold lower dose of isoniazid or rifampicin in conjunction with C4.T4 agonists

  • CLEC4E antibodies are valuable tools for monitoring receptor expression during such therapeutic approaches

• Autophagy regulation:

  • The novel role of CLEC4E in inducing autophagy through MYD88 represents a potential therapeutic target

  • Antibodies allow tracking of this process in various disease models

  • This mechanism could be exploited beyond infectious diseases to conditions where autophagy modulation is beneficial

• Cardiovascular research:

  • CLEC4E binds cholesterol crystals deposited in atherosclerotic plaques

  • This interaction may contribute to inflammatory processes in atherosclerosis

  • Antibodies against CLEC4E can help elucidate its role in cardiovascular pathologies

How can researchers design experiments to distinguish between CLEC4E-dependent and -independent effects in complex immune responses?

To differentiate CLEC4E-dependent from -independent effects in complex immune responses, researchers should consider these experimental designs:

• Genetic approaches:

  • Use CLEC4E knockout models or CRISPR/Cas9-mediated deletion

  • Create bone marrow chimeras with selective deletion in specific cell types (as demonstrated in studies with mixed chimeras lacking Mincle only in B lymphocytes)

  • Apply conditional knockout systems if constitutive knockout has developmental effects

• Antibody-based interventions:

  • Use blocking antibodies against CLEC4E to inhibit receptor function

  • Include appropriate isotype controls

  • Titrate antibody concentration to establish dose-dependency

  • Consider timing of antibody administration (prophylactic vs. therapeutic)

• Pharmacological tools:

  • Apply specific inhibitors of downstream signaling components:

    • SYK inhibitors (e.g., piceatannol)

    • TLR4 inhibitor (e.g., CLI-095) as a comparison for specificity

  • Use pathway-specific inhibitors to dissect signaling events

• Combinatorial approach:

  • Implement simultaneous analysis of multiple receptors

  • Design experiments comparing:

    • CLEC4E agonist alone

    • TLR4 agonist alone

    • Combined stimulation (C4.T4)

    • Controls for each condition

  • Measure multiple outcome parameters (bacterial killing, cytokine production, autophagy, etc.)

What is the optimal experimental design for studying CLEC4E's role in B cell-mediated T cell recovery after lymphoablation?

Based on findings that CLEC4E expression is upregulated in B cells following anti-thymocyte globulin treatment and contributes to T cell recovery, an optimal experimental design would include:

• Animal model setup:

  • Heart allograft transplantation in mice (wild-type vs. CLEC4E-deficient)

  • Treatment with murine anti-thymocyte globulin (mATG)

  • Generation of mixed bone marrow chimeras lacking CLEC4E only in B lymphocytes (to distinguish B cell-specific effects)

• Treatment conditions:

  • mATG (0.5 mg i.p.) on days 0 and 4 post-transplant

  • Control groups: non-transplanted mice with mATG, transplanted mice without mATG

  • Additional conditions: anti-CD154 mAb MR1 or agonistic anti-CD40 mAb FGK4.5 treatment

• Analysis timepoints:

  • Day 8 after transplantation (when B cell numbers recover to pre-depletion levels)

  • Multiple timepoints to track T cell reconstitution kinetics

• Key readouts:

  • Flow cytometry analysis of CLEC4E expression on B cells

  • Assessment of B cell production of pro-inflammatory cytokines

  • Measurement of T lymphocyte reconstitution

  • Allograft survival and rejection parameters

How can researchers design CLEC4E antibody-based imaging approaches to study granuloma formation in tuberculosis models?

To study granuloma formation in tuberculosis models using CLEC4E antibody-based imaging:

• Sample preparation methodologies:

  • Prepare lung sections from Mtb-infected animal models (mice or guinea pigs)

  • Process tissues with minimal fixation to preserve epitope accessibility

  • Consider dual immunofluorescence to simultaneously visualize CLEC4E and bacterial markers or cell type-specific markers

• Staining protocol:

  • Apply validated anti-CLEC4E antibodies suitable for immunohistochemistry/immunofluorescence

  • Include appropriate isotype controls

  • Use secondary antibodies with minimal background in lung tissue

  • Counterstain with DAPI for nuclear visualization

• Analysis parameters:

  • Compare CLEC4E expression in:

    • Granulomatous lesions vs. normal lung tissue

    • Center vs. periphery of granulomas

    • Different treatment conditions (untreated, C4.T4, antibiotics, combination)

  • Quantify colocalization with macrophage markers, bacterial burden, and autophagy markers

• Advanced imaging techniques:

  • Consider intravital imaging using fluorescently labeled anti-CLEC4E antibodies in accessible TB models

  • Apply tissue clearing methods for whole-organ 3D visualization

  • Use super-resolution microscopy for subcellular localization of CLEC4E in relation to Mtb

What methodological approaches can differentiate between CLEC4E's role in autophagy versus direct antimicrobial responses?

To distinguish CLEC4E's contributions to autophagy from its direct antimicrobial effects:

• Cellular models with autophagy manipulation:

  • Use macrophages from autophagy-deficient models:

    • atg5 knockout mice

    • Becn1 knockdown mice

  • Apply autophagy inhibitors (e.g., 3-methyladenine, bafilomycin A1)

  • Compare with autophagy inducers (e.g., rapamycin) as positive controls

• Key assays to perform:

  • Bacterial survival assays (CFU determination) with and without autophagy inhibition

  • LC3 puncta formation and p62 degradation analysis

  • ROS production measurement

  • Phagosome-lysosome fusion assessment

  • Cytokine production analysis

• Temporal analysis strategy:

  • Track early (0-6h) vs. late (24-48h) responses

  • Correlate timing of autophagy induction with bacterial killing

  • Analyze sequential activation of signaling pathways

• Molecular dissection approach:

  • Target specific components of CLEC4E signaling:

    • MYD88 (involved in CLEC4E-induced autophagy)

    • PtdIns3K (required for autophagosome formation)

    • STAT1 and RELA/NFKB (involved in inflammatory responses)

  • Use siRNA or inhibitors to block individual pathways while maintaining CLEC4E activation