CLEC1A Antibody

What is CLEC1A and where is it expressed in different species?

CLEC1A is a C-type lectin receptor with a carbohydrate recognition domain in its extracellular region but no known signaling motif in its cytoplasmic domain . This receptor demonstrates high evolutionary conservation between species, with approximately 70% homology between human and mouse variants . Expression studies have shown that CLEC1A is primarily found on myeloid cells including dendritic cells (DCs), macrophages, monocytes, and neutrophils . In humans specifically, CLEC1A expression is restricted to CD16− myeloid DCs in blood and monocyte-derived DCs (moDCs) . Additionally, CLEC1A is highly expressed in endothelial cells, as demonstrated in both human and rodent models .

The expression pattern varies in response to cytokines, with transforming growth factor-β (TGFβ) enhancing CLEC1A expression on moDCs, while inflammatory stimuli downregulate its expression . This context-dependent expression suggests an important immunoregulatory role in different physiological and pathological conditions.

How do CLEC1A antibodies perform in different experimental applications?

CLEC1A antibodies demonstrate variable performance across different experimental applications. Based on current validation data:

When choosing a CLEC1A antibody, researchers should consider the host species (rabbit antibodies like the one described in search result offer good reactivity to both human and mouse CLEC1A), clonality (polyclonal antibodies provide multiple epitope recognition), and the specific experimental application required .

What are the known functions of CLEC1A in the immune system?

CLEC1A functions as an immune checkpoint in myeloid cells, particularly in dendritic cells, where it plays an inhibitory role in regulating T cell responses . Several key functions have been identified:

• Dead cell sensing: CLEC1A recognizes cells killed by programmed necrosis, with its ligands appearing at late stages of programmed necrosis rather than during early apoptosis .

• Dampening dendritic cell activation: Evidence from both human and rat studies demonstrates that CLEC1A acts as an inhibitory receptor that restrains dendritic cell activation and subsequent T helper 17 (Th17) responses .

• Regulation of antigen cross-presentation: In CLEC1A-deficient models, there is enhanced cross-presentation of dead cell-associated antigens by conventional type-1 DCs, suggesting CLEC1A normally limits this process .

• Protection against excessive inflammation: CLEC1A-deficient rats exhibit exacerbated CD4+ Th1 and Th17 responses both in vitro and in vivo, indicating that CLEC1A prevents excessive T-cell priming and effector responses .

• Host defense role: CLEC1A has been implicated in host defense against Aspergillus fumigatus by recognizing 1,8-dihydroxynaphthalene-melanin on the fungal surface .

What methodological approaches are recommended for validating CLEC1A antibody specificity?

To ensure reliable research outcomes, validation of CLEC1A antibody specificity should follow these methodological steps:

• Positive and negative control samples: Use cell lines with known CLEC1A expression (positive controls) and CLEC1A knockout models or CLEC1A-negative cell types (negative controls) . The generation of CLEC1A knockout mice or rats provides excellent negative controls .

• Blocking peptide controls: Utilize the synthesized peptide used as immunogen to confirm binding specificity in competitive binding assays .

• Cross-species reactivity testing: Confirm reactivity across target species (e.g., human and mouse) if using the antibody for comparative studies .

• Western blot analysis: Verify detection of the expected molecular weight band (~32 kDa) for CLEC1A .

• Multi-technique confirmation: Compare results across different detection methods (e.g., flow cytometry, immunohistochemistry, and Western blot) to ensure consistent staining patterns.

• siRNA knockdown verification: In cell culture systems, compare antibody signal in cells treated with CLEC1A-specific siRNA versus control siRNA to confirm specificity.

What experimental approaches best reveal CLEC1A's role in cancer immunotherapy?

CLEC1A has emerged as a promising target for cancer immunotherapy based on its function as a myeloid immune checkpoint. Researchers exploring this area should consider these methodological approaches:

• Combinatorial therapy studies: Design experiments that combine CLEC1A blockade with conventional chemotherapies. Research has shown that blocking CLEC1A in combination with chemotherapeutic agents like gemcitabine (GEM) or cyclophosphamide (CPA) significantly reduced tumor growth in mouse models, with complete tumor elimination in 37-75% of CLEC1A knockout mice compared to 10-36% in wild-type mice .

• Tumor microenvironment profiling: Analyze changes in tumor-infiltrating immune cells using flow cytometry and single-cell RNA sequencing. In CLEC1A-deficient models, researchers observed reduced accumulation of immunosuppressive myeloid cells in tumors and enhanced activation state of dendritic cells, leading to increased T cell responses .

• Mechanistic investigation using bone marrow chimeras: Generate bone marrow chimeric mice with CLEC1A deficiency only in the hematopoietic compartment to confirm the specific role of myeloid-expressed CLEC1A in antitumor immunity .

• CD8+ T cell depletion studies: Perform antibody-mediated CD8+ T cell depletion in CLEC1A-deficient tumor models to determine the contribution of CD8+ T cells to the observed antitumor effects .

• Memory response assessment: Challenge cured CLEC1A-deficient mice with tumor cells to evaluate development of memory immune responses .

How can researchers effectively study CLEC1A-TRIM21 interactions?

The identification of TRIM21 as an endogenous ligand for CLEC1A represents a significant advance in understanding this receptor's biology . To study this interaction:

• Affinity purification with mass spectrometry: Use co-immunoprecipitation with Fc-CLEC1A fusion proteins and protein extracts from necrotic cells, followed by liquid chromatography-mass spectrometry (LC-MS) to identify interaction partners. This approach successfully identified TRIM21 as a CLEC1A ligand with 50% mean coverage .

• Confirmation by Western blot: Validate specific interactions using Western blot analysis of co-immunoprecipitates, with appropriate controls (such as irrelevant C-type lectin receptors like CLEC7A) .

• Direct interaction assays: Implement surface plasmon resonance (Biacore) and enzyme-linked immunosorbent assay (ELISA) to confirm direct interaction between recombinant TRIM21 and CLEC1A .

• Expression correlation studies: Analyze expression of TRIM21 in various tumor cell lines treated with chemotherapies like cisplatin or staurosporine, and correlate with CLEC1A binding .

• Functional studies with TRIM21 knockdown/knockout: Investigate how modulating TRIM21 affects CLEC1A-dependent functions in dendritic cells and antitumor immunity.

What considerations are important when studying CLEC1A in different species models?

When working with CLEC1A across different species models, researchers should account for several important factors:

• Conservation and differences: While CLEC1A shows high evolutionary conservation (70% homology between human and mouse), species-specific differences may exist in expression patterns, regulation, and function .

• Cross-reactivity of tools: Select antibodies that have been validated for cross-reactivity between species of interest. The polyclonal antibody described in result shows reactivity to both human and mouse CLEC1A .

• Ligand conservation: Evidence suggests CLEC1A ligands are conserved between species. Human Fc-CLEC1A can bind to dead murine splenocytes and vice versa .

• Generation of species-specific knockout models: Different approaches have been used to generate CLEC1A-deficient animals, including homologous recombination techniques for mice and rats . These models are crucial for studying in vivo functions.

• Disease model selection: CLEC1A has been studied in different disease models across species, including cancer models in mice and experimental autoimmune encephalomyelitis in rats . The appropriate model should be selected based on the specific research question.

How can CLEC1A expression be accurately quantified in clinical samples?

For clinical research applications, accurate quantification of CLEC1A expression requires robust methodology:

• Quantitative PCR (qPCR): For measuring CLEC1A mRNA levels in tissues or sorted cell populations. This approach was used to demonstrate that decreased expression of CLEC1A in human lung transplants predicts the development of chronic rejection .

• Flow cytometry of tissue suspensions: For quantifying CLEC1A protein expression on specific cell subsets within clinical samples. This approach is particularly useful for identifying CLEC1A expression on CD16− myeloid DCs in human blood .

• Immunohistochemistry with digital pathology: For spatial analysis of CLEC1A expression in tissue sections with precise quantification using digital image analysis.

• Single-cell RNA sequencing: For comprehensive analysis of CLEC1A expression across the diversity of cell types in complex tissues. This approach has been used to analyze the transcriptional changes in immune cell populations infiltrating hepatocellular carcinoma tumors in CLEC1A-deficient versus wild-type mice .

• Standardization with reference samples: Include standardized positive and negative control samples with each batch of clinical specimens to account for technical variability.

What factors may lead to inconsistent CLEC1A antibody staining results?

Several factors can contribute to inconsistent CLEC1A antibody staining:

• Context-dependent expression: CLEC1A expression is regulated by cytokines, with TGFβ enhancing and inflammatory stimuli suppressing its expression . If samples are collected under different inflammatory conditions, staining intensity may vary significantly.

• Cell type specificity: CLEC1A expression is restricted to specific cell types such as CD16− myeloid DCs in human blood . Staining on mixed cell populations may appear heterogeneous, which could be misinterpreted as inconsistent results.

• Fixation sensitivity: The epitope recognized by the antibody may be sensitive to particular fixation methods. Optimization of fixation protocols (duration, fixative concentration) is crucial for consistent results.

• Detection method limitations: Different secondary detection systems (fluorescent vs. enzymatic) may have varying sensitivity thresholds.

• Antibody batch variation: Particularly with polyclonal antibodies, lot-to-lot variation can occur. Researchers should record lot numbers and standardize their protocols accordingly.

• Sample preparation techniques: Variations in tissue processing, antigen retrieval methods, or cell permeabilization protocols can significantly impact staining quality and consistency.

How can researchers reconcile contradictory data regarding CLEC1A function?

When faced with contradictory data regarding CLEC1A function, researchers should consider:

• Context-dependent roles: CLEC1A may have different functions depending on the disease model or experimental system. For example, it plays a protective role in experimental autoimmune encephalomyelitis but an immunosuppressive role in cancer models .

• Cell type-specific effects: CLEC1A is expressed on multiple cell types including different DC subsets, macrophages, neutrophils, and endothelial cells . Its function may vary depending on the cell type being studied.

• Compensatory mechanisms in knockout models: Long-term deficiency of CLEC1A may lead to compensatory upregulation of other CLRs or immune pathways, potentially confounding interpretations of knockout phenotypes.

• Species differences: Despite high conservation, functional differences may exist between human and rodent CLEC1A. Researchers should specify the species being studied and avoid overgeneralizing findings.

• Methodological variations: Different blocking antibodies, knockout strategies, or experimental readouts may lead to apparently contradictory results. Careful evaluation of methodological details is essential for reconciling discrepancies.

• Integrated approach: When possible, use multiple complementary techniques (genetic models, blocking antibodies, in vitro and in vivo systems) to build a more robust understanding of CLEC1A function.

What controls should be included when validating CLEC1A knockout models?

Proper validation of CLEC1A knockout models requires rigorous controls:

• Genotyping confirmation: Verify the knockout at the DNA level through PCR-based genotyping strategies that can distinguish between wild-type, heterozygous, and homozygous knockout animals .

• mRNA expression analysis: Confirm absence of CLEC1A mRNA using RT-PCR or qPCR with primers specific to different exons of the CLEC1A gene.

• Protein expression verification: Use validated antibodies to confirm absence of CLEC1A protein by Western blot, flow cytometry, or immunohistochemistry in relevant tissues.

• Off-target effects assessment: Evaluate expression of closely related C-type lectin receptors to ensure the knockout strategy did not affect their expression.

• Immune cell composition analysis: As demonstrated in search result , conduct thorough immune cell phenotyping to detect any developmental abnormalities or baseline changes in immune cell populations (as shown in table S1) .

• Functional rescue experiments: When possible, reintroduce CLEC1A expression in knockout cells or animals to confirm that observed phenotypes are directly attributable to CLEC1A deficiency.

What challenges exist in detecting CLEC1A ligands beyond TRIM21?

While TRIM21 has been identified as an endogenous ligand for CLEC1A , detecting additional ligands presents several challenges:

How might CLEC1A function differently in cancer versus autoimmune disease contexts?

Research suggests context-dependent roles for CLEC1A across disease models:

• Cancer context: In tumor models, CLEC1A functions as an immune checkpoint that limits antitumor immunity . CLEC1A deficiency reduces accumulation of immunosuppressive myeloid cells in tumors, increases dendritic cell activation, and enhances T cell responses . These effects translate to improved survival in multiple cancer models when CLEC1A is absent or blocked.

• Autoimmune context: In contrast, CLEC1A-deficient mice develop milder symptoms in experimental autoimmune encephalomyelitis, a model for multiple sclerosis . This suggests that in autoimmune conditions, CLEC1A may play a protective role against excessive inflammation.

• Transplantation setting: In human lung transplants, decreased expression of CLEC1A predicts the development of chronic rejection and associates with higher levels of IL-17A . This indicates a potential protective role in preventing allograft rejection.

These seemingly contradictory functions might be explained by CLEC1A's role in regulating the balance between protective immunity and immunopathology in a context-dependent manner. Future research should investigate the molecular mechanisms underlying these differential effects, potentially focusing on tissue-specific microenvironments and interaction with other immunoregulatory pathways.

What novel therapeutic approaches could target the CLEC1A pathway?

Several promising therapeutic approaches targeting CLEC1A are emerging:

• Antagonistic antibodies: Anti-human CLEC1A antagonist antibodies have been identified that enhance antitumor immunity in CLEC1A-humanized mice . These could be developed as cancer immunotherapy agents, particularly in combination with chemotherapy.

• Combination therapy strategies: Given the synergistic effects observed when combining CLEC1A blockade with chemotherapies like gemcitabine or cyclophosphamide , rational combination therapies targeting multiple pathways could maximize therapeutic efficacy.

• TRIM21-targeted approaches: Since TRIM21 has been identified as an endogenous ligand for CLEC1A , therapeutic strategies disrupting this interaction might offer an alternative to direct CLEC1A targeting.

• Cell-based therapies: Engineering dendritic cells with CLEC1A knockdown or knockout could enhance their immunostimulatory capacity for cancer immunotherapy applications.

• Small molecule inhibitors: Developing small molecules that block CLEC1A signaling might offer advantages over antibody-based approaches in terms of tissue penetration and dosing flexibility.

• Agonistic approaches for autoimmunity: In contrast to antagonism for cancer therapy, CLEC1A agonists might be beneficial in autoimmune disease contexts where CLEC1A appears to play a protective role .

How does CLEC1A interact with other immune checkpoint molecules?

Understanding CLEC1A's position within the broader immune checkpoint landscape remains an important research direction:

• Pathway integration: Investigation is needed to determine how CLEC1A signaling integrates with other inhibitory pathways in myeloid cells, such as PD-L1/PD-1, VISTA, or LILRB family members.

• Complementary mechanisms: Unlike many immune checkpoints that directly modulate T cell activation, CLEC1A primarily acts on myeloid cells to indirectly influence T cell responses . This distinct mechanism may offer opportunities for complementary targeting alongside T cell-directed checkpoint inhibitors.

• Compensatory regulation: Studies should assess whether blocking one checkpoint (e.g., PD-1) leads to compensatory upregulation of CLEC1A or vice versa, which would influence combination therapy design.

• Shared ligand interactions: The identification of TRIM21 as a CLEC1A ligand raises questions about whether this or other ligands might also interact with additional immune regulatory receptors.

• Biomarker potential: Expression patterns of CLEC1A alongside other immune checkpoints might serve as biomarkers for predicting response to immunotherapy, particularly in identifying patients who might benefit from combination approaches.