CCL21 Antibody

What is the functional role of CCL21 in immune cell trafficking?

CCL21 functions as a potent chemoattractant for naïve T-cells, naïve B-cells, and immature dendritic cells. Its 37 amino acid carboxy terminal extension, which distinguishes it from other chemokines, anchors it to venule endothelium where the amino terminus can interact with its cognate receptor, CCR7. This interaction plays a crucial role in attracting naïve immune cells to sites of antigen presentation, establishing CCL21 as a key regulator of adaptive immunity by controlling dendritic cell and T cell migration . When designing experiments involving immune cell migration, it's important to consider both CCL21's immobilized and soluble forms, as both participate in gradient formation required for proper immune cell homing.

How do I select the appropriate anti-CCL21 antibody for my research?

Selection depends on your experimental purpose. For neutralization assays, choose antibodies validated for blocking CCL21-CCR7 interactions, such as those targeting the amino terminus of CCL21 . For detection applications, consider antibodies with demonstrated specificity in your sample type and technique (e.g., ICC, Western blot). Validated antibodies like the goat anti-human CCL21/6Ckine antibody have shown efficacy in multiple applications including immunocytochemistry and Simple Western assays . When selecting between monoclonal and polyclonal options, consider that monoclonals offer higher specificity while polyclonals typically provide stronger signals due to multiple epitope recognition.

What detection methods work best for visualizing CCL21 expression in tissue samples?

Immunofluorescence techniques have proven effective for detecting CCL21 in tissue samples. For example, CCL21 can be visualized in human PBMCs using antigen affinity-purified polyclonal antibodies (10 μg/mL concentration) followed by fluorescent-conjugated secondary antibodies . When studying CCL21 gradients in tissues, confocal microscopy with co-staining for lymphatic vessel markers (LYVE-1) and blood vessel markers (CD31) allows quantification of CCL21 intensity relative to these structures . The AF457 anti-CCL21 antibody has been successfully used for detecting extracellular CCL21 in skin tissues, enabling visualization of CCL21 gradients extending from lymphatic vessels .

How can I distinguish between immobilized and soluble forms of CCL21 in tissue samples?

This requires careful experimental design. For immobilized (full-length) CCL21, minimize washing steps that could remove soluble forms. Use in situ imaging with minimal tissue manipulation and antibodies that recognize the C-terminal extension (which anchors CCL21 to tissues). For soluble (cleaved) CCL21, tissue leach-out assays followed by ELISA can be effective. When analyzing both forms simultaneously, consider that extensive washing during immunostaining may bias toward detection of the immobilized form . Quantitative analysis should include measurement of CCL21 intensity at varying distances from lymphatic vessels (LVs) and CD31+ blood vessels to properly characterize gradients. For example, in wild-type skin, CCL21 intensity is greatest at the LV edge and significantly decreases with distance, while showing no significant change relative to distance from CD31+ vessels .

What are the methodological considerations for CCL21 neutralization assays?

When conducting neutralization assays, the most reliable approach uses the BaF/3 mouse pro-B cell line transfected with human CCR7, as these cells demonstrate chemotaxis in response to CCL21. The neutralization dose (ND50) for anti-CCL21 antibodies typically ranges from 0.400-4.00 μg/mL in the presence of 50 ng/mL recombinant human CCL21 . Critical controls should include: 1) cells with CCL21 alone (positive control), 2) cells with isotype-matched control antibody plus CCL21, and 3) cells without CCL21 or antibody (negative control). For accurate quantification, pre-titrate both the CCL21 concentration and antibody concentrations to establish dose-response curves before determining neutralization efficacy.

How do ACKR4-regulated CCL21 gradients impact experimental design for immune cell migration studies?

• The tissue source - ACKR4 expression varies by tissue

• The presence of both immobilized and soluble CCL21 forms

• The potential impact of ACKR4 deficiency on gradient formation

Quantitative measurements should analyze CCL21 intensity relative to distance from lymphatic vessels. For example, in wild-type skin, CCL21 intensity at the lymphatic vessel edge is significantly higher than areas 20-40μm away, while in ACKR4-deficient tissue, CCL21 intensity is 2-3 fold greater at all distances .

How are CCL21-modified dendritic cell vaccines manufactured for cancer immunotherapy?

The manufacturing process for CCL21-modified dendritic cell (CCL21-DC) vaccines involves several critical steps. Monocyte-derived dendritic cells (moDCs) are generated from patient blood or healthy donors, then transduced with a CCL21-containing adenoviral vector. This transduction significantly augments CCL21 secretion by the moDCs while minimally affecting other vaccine characteristics . The cellular composition of these vaccines is heterogeneous, containing variable proportions of passenger lymphocytes among patients. Single-cell RNA sequencing reveals further heterogeneity within the moDC compartment itself, with cells exhibiting a spectrum of DC phenotypes .

Key manufacturing considerations include:

This vaccine approach is being investigated in combination with pembrolizumab for non-small cell lung cancer treatment, potentially enhancing antitumor immune responses in patients with immunotherapy resistance .

How do I resolve inconsistent CCL21 protein detection in tissue samples?

Inconsistent detection often stems from several factors: First, consider CCL21's dual forms (immobilized and soluble). Extensive washing steps may remove soluble CCL21, biasing toward detection of the immobilized form . Second, tissue preparation techniques significantly impact detection - fresh frozen samples often preserve CCL21 better than formalin-fixed paraffin-embedded samples. For optimal results, use fresh tissues with minimal processing and validate antibody concentrations (starting with 10-20 μg/mL for immunostaining) . Finally, co-staining with lymphatic vessel markers (LYVE-1) can help identify areas of expected high CCL21 expression, as CCL21 staining in wild-type tissues is typically most intense proximal to lymphatic vessels . When troubleshooting, prepare serial dilutions of recombinant CCL21 protein as positive controls to validate antibody sensitivity.

What approaches can resolve conflicting data about CCL21 levels in different tissue compartments?

Conflicting data about CCL21 levels often result from methodological differences. To resolve these discrepancies:

• Standardize detection methods - specify whether measuring total, immobilized, or soluble CCL21

• Consider compartment-specific factors - ACKR4 expression varies by tissue, affecting CCL21 regulation

• Use multiple detection techniques - combine ELISA, immunohistochemistry, and functional assays

• Analyze gradient distributions rather than total levels alone

For example, contrary findings regarding CCL21 levels in lymph nodes of ACKR4-deficient mice were resolved by recognizing that different methods measured different CCL21 forms. Tissue leach-out assays bias toward detection of soluble CCL21, while imaging techniques better detect immobilized CCL21. When analyzing tissues with potential CCL21 gradients, quantify CCL21 intensity at defined distances from known structures (e.g., lymphatic vessels) rather than measuring global expression levels .

How can I distinguish between specific CCL21 antibody binding and potential cross-reactivity?

Cross-reactivity concerns can be addressed through several approaches. First, conduct absorption controls by pre-incubating the antibody with excess recombinant CCL21 before application to samples - this should eliminate specific staining. Second, validate results with multiple antibodies targeting different CCL21 epitopes. Third, include appropriate negative controls such as isotype-matched control antibodies and tissues known to lack CCL21 expression (e.g., normal bowel tissue has been shown to lack CCL21 expression compared to IBD tissues) . For functional validation, compare antibody effects on CCL21-mediated chemotaxis versus chemotaxis induced by other chemokines that signal through different receptors. Specificity can be quantified using neutralization dose assays, where the ND50 for anti-CCL21 antibodies should typically range from 0.400-4.00 μg/mL in the presence of 50 ng/mL recombinant human CCL21 .

How is CCL21 antibody research advancing our understanding of autoimmune disease mechanisms?

Recent research has identified altered levels of chemokine CCL21 in systemic sclerosis (SSc) associated with pulmonary arterial hypertension (PAH) . This finding connects CCL21 to vascular pathologies beyond its established role in inflammatory bowel diseases . The emerging pattern suggests CCL21 may be involved in multiple autoimmune conditions characterized by vascular inflammation and inappropriate immune cell trafficking. By targeting CCL21 with specific antibodies, researchers can dissect the contribution of this chemokine to disease progression through selective blockade of naïve immune cell recruitment while preserving memory immune responses. This approach offers advantages over broad immunosuppression by potentially allowing for targeted immunomodulation specific to disease pathology. Future research should examine CCL21 expression patterns across multiple autoimmune conditions to identify common mechanisms and potential therapeutic approaches.

What are the latest developments in multi-parametric analysis of CCL21 gradient formation?

Advanced imaging techniques combined with computational analysis now allow for more sophisticated characterization of CCL21 gradients in tissues. Researchers have quantified CCL21 intensity at defined distances from lymphatic vessels, demonstrating that in wild-type skin, CCL21 intensity is greatest at the lymphatic vessel edge and decreases significantly with distance . These gradients are maintained but amplified 2-3 fold throughout in ACKR4-deficient tissues. Future directions include:

• Integration of spatial transcriptomics with protein-level gradient analysis

• Development of live imaging techniques to visualize dynamic gradient formation

• Computational modeling of gradient formation incorporating both soluble and immobilized CCL21 forms

• Analysis of how disease states affect gradient characteristics beyond total CCL21 levels

Multi-parametric analysis approaches will help resolve longstanding questions about how functional chemokine gradients form in vivo and how they can be therapeutically manipulated in disease settings.