CCL21 Antibody, FITC conjugated

Basic Research Questions

• What is CCL21 and what biological roles does it play in immune function?

CCL21 (Chemokine C-C Motif Ligand 21) is a chemokine that plays crucial roles in immune cell migration and function. It serves as a key mediator of dendritic cell (DC) and T cell trafficking, particularly for homing to lymphoid tissues. CCL21 primarily signals through the CCR7 receptor, though recent research suggests differential downstream effects compared to other CCR7 ligands .

Biologically, CCL21 functions include:

• Facilitating DC migration from peripheral tissues to lymph nodes

• Guiding naive T cell trafficking within lymphoid organs

• Mediating long-distance chemotaxis of naive T lymphocytes

• Contributing to immune response in various pathological conditions including rheumatoid arthritis and cancer

Recent findings indicate that CCL21 can trigger stable polarization and support long-range chemotaxis of naive T cells, even in soluble form, contrary to earlier beliefs that it required substrate adsorption to be functional .

• How should researchers prepare samples for optimal CCL21 detection using FITC-conjugated antibodies?

For optimal detection of CCL21 using FITC-conjugated antibodies, sample preparation depends on the experimental context:

For tissue sections:

• Fix tissues appropriately (typically 4% paraformaldehyde)

• For paraffin sections: Perform antigen retrieval using citrate buffer (pH 6.0)

• Block with appropriate serum (5-10% normal serum) for 1 hour at room temperature

• Incubate with anti-CCL21-FITC antibody at manufacturer-recommended dilution (typically 1:50-1:200) overnight at 4°C

• Wash thoroughly with PBS (3-5 times, 5 minutes each)

• Mount with anti-fade mounting medium

For flow cytometry:

• Prepare single-cell suspensions from relevant tissues

• For intracellular staining: Fix and permeabilize cells using commercial kits

• Block Fc receptors to reduce non-specific binding

• Incubate with anti-CCL21-FITC at recommended concentration (typically 5-10 μg/ml)

• Wash cells 2-3 times before analysis

Note that CCL21 can be both produced by cells and captured from the environment, which may affect detection strategies . When examining CCL21 expression in T cells, be aware that detection might represent captured rather than synthesized protein .

• What are the most appropriate positive and negative controls for CCL21 antibody validation?

Positive controls:

• High endothelial venules (HEVs) in lymph nodes, which express high levels of CCL21

• Stromal cells in T cell zones of lymphoid tissues

• Lymphatic endothelial cells (positive for LYVE-1)

• Fibroblasts and epithelial cells in liver cancer tissues

Negative controls:

• Isotype-matched control antibody (rabbit IgG for rabbit polyclonal antibodies)

• Tissues known to lack CCL21 expression (e.g., muscle tissue)

• Pre-absorption control: pre-incubate the antibody with recombinant CCL21 protein before staining

• For knockout validation: tissues from CCL21-deficient mice (though complete knockouts may not be viable)

For comprehensive validation, researchers should perform parallel staining with another validated anti-CCL21 antibody targeting a different epitope, and correlate protein detection with mRNA expression through RT-PCR or RNA-seq data .

• What are the typical flow cytometry gating strategies for CCL21-positive cells?

When analyzing CCL21-positive cells by flow cytometry, consider these gating strategies:

• Initial gating:

  • FSC/SSC to identify cellular populations and exclude debris

  • Viability dye to exclude dead cells

  • Single-cell gating using FSC-H vs FSC-A to exclude doublets

• CCL21-specific gating:

  • Set FITC threshold based on isotype control or fluorescence-minus-one (FMO) controls

  • For T cells: Gate on CD3+ cells first, then analyze CCL21-FITC signal intensity

  • For dendritic cells: Gate on CD11c+ cells, then analyze CCL21-FITC signal

• Special considerations:

  • For identifying cells that capture vs. produce CCL21: Compare ex vivo staining with in vitro cultured cells (CCL21 captured by T cells disappears after 5 hours of culture)

  • T cells constitute a major population of CCL21+ cells in the spleen, likely binding CCL21 via CCR7

  • The intensity of detectable surface CCL21 varies by tissue type (lymph nodes > spleen > blood)

A comprehensive analysis should include measurement of both percentage of positive cells and mean fluorescence intensity (MFI) to quantify expression levels.

Intermediate Research Questions

• How do researchers differentiate between cell-produced and cell-captured CCL21 in experimental settings?

Differentiating between produced and captured CCL21 is methodologically challenging but critical for accurate interpretation. Research strategies include:

Methodological approach:

• Temporal analysis: CCL21 detected ex vivo on CD11b- cells disappears after 5 hours of in vitro culture, suggesting capture rather than production

• RNA-protein correlation:

  • Perform flow cytometry for CCL21 protein detection

  • In parallel, conduct RT-PCR or RNA-seq on sorted cell populations

  • Cells with protein but no mRNA likely represent CCL21 capture rather than production

  • Single-cell RNA sequencing has shown that in liver cancer, CCL21 is mainly derived from stromal cells like fibroblasts and epithelial cells, not immune cells

• Culture experiments:

  • Culture cells in CCL21-free conditions for extended periods

  • Cells that maintain CCL21 expression likely produce it endogenously

  • Cells that lose expression over time likely captured it

• Receptor blocking:

  • Treat cells with CCR7 antagonists prior to staining

  • Reduction in CCL21 staining after receptor blocking suggests capture via CCR7

• What are the key technical considerations when using CCL21 antibodies in migration and chemotaxis assays?

When studying CCL21-mediated migration using antibodies, researchers should consider these technical aspects:

Experimental design:

• Gradient establishment:

  • For Boyden chamber assays: Use 0.001-100 ng/ml concentration range of CCL21, with VEGF (10 ng/ml) as positive control

  • For microfluidic assays: Create soluble gradients based on diffusion to study long-range chemotaxis

  • Include controls without ICAM-1 or other adhesion ligands to distinguish between adhesion-dependent and independent migration

• Specificity controls:

  • Block cells with anti-CCR7 antibody (10 μg/ml for 1 hour at 37°C) before migration assays

  • Include appropriate IgG control antibodies

  • Compare CCL21-induced migration with other chemokines like CCL19 that share receptors

• Readouts:

  • Count migrating cells (average of three high-power fields/well, in triplicate)

  • Analyze cell polarization as an early indicator of response

  • Track individual cells to distinguish directed chemotaxis from random chemokinesis

• Antibody applications:

  • Use anti-CCL21 antibodies to neutralize activity in functional blocking studies

  • Employ FITC-conjugated CCL21 antibodies to visualize gradient formation

  • Consider immobilized versus soluble CCL21 scenarios, as recent research shows CCL21 can trigger naive T lymphocyte chemotaxis while in bulk solution

Recent findings demonstrate that CCL21 gradients trigger both hapotaxis (migration along surface-bound gradients) and chemotaxis (migration in soluble gradients) in naive T lymphocytes .

• How does CCL21 expression correlate with immunotherapy response in cancer research?

Recent studies have revealed significant correlations between CCL21 expression and immunotherapy response, particularly in cancer research:

Key findings:

• Hepatocellular carcinoma (HCC):

  • Transcriptome analysis showed significantly higher CCL21 levels in HCC patients responding to immune checkpoint inhibitors (ICIs)

  • HCC patients with high serum CCL21 levels showed better sensitivity to immunotherapy

  • The area under the ROC curve (AUC) for serum CCL21 in predicting immunotherapy response was 0.73-0.74

• Non-small cell lung cancer (NSCLC):

  • CCL21 gene-modified dendritic cell vaccines enhance antitumor immune responses

  • In situ vaccination with CCL21-DC sensitized immune-resistant murine NSCLC to immune checkpoint inhibitors

  • CCL21-DC obliterated tumor-promoting neutrophils and promoted sustained infiltration of CD8+ cytolytic and CD4+ Th1 lymphocytes

• Predictive modeling:

  • A nomogram incorporating CCL21 with tumor size, gamma-glutamyl transferase, and neutrophil-to-lymphocyte ratio showed excellent predictive performance (AUC: 0.863)

  • CCL21 serves as an independent predictive biomarker for immunotherapy response regardless of tumor stage

• Mechanism:

  • High CCL21 expression correlates with greater infiltration of immune cells in tumors

  • CCL21 inhibits N2 neutrophil polarization by suppressing NF-κB pathway activation

  • Combination of CCL21 with ICIs potentially offers a novel therapeutic strategy

These correlations provide valuable insights for patient selection and treatment strategies in cancer immunotherapy.

• What methodological approaches can detect differences in CCL21 distribution across tissue microenvironments?

Detecting CCL21 distribution across tissue microenvironments requires specialized techniques:

Methodological approaches:

• Dual immunofluorescence staining:

  • Co-stain with FITC-conjugated anti-CCL21 antibody and markers for specific structures

  • For vascular association: Pair with anti-VWF (von Willebrand Factor) antibody

  • For lymphatic vessels: Combine with anti-LYVE-1 antibody

  • Visualize with appropriate secondary antibodies (e.g., Texas red-labeled anti-goat for CCL21, FITC-conjugated anti-rabbit for VWF)

• Gradient visualization:

  • Use confocal microscopy with z-stack imaging to create 3D reconstructions

  • Employ fluorescence intensity profiling across tissue sections

  • ACKR4 (atypical chemokine receptor 4) shapes CCL21 distribution in barrier tissues and prevents leakage

• Quantification methods:

  • For soluble CCL21: Quantify from tissue homogenates using ELISA

  • For tissue-bound CCL21: Use ImageJ or similar software to measure fluorescence intensity relative to distance from structures of interest

  • Hemoglobin measurement in matrigel plugs can assess associated vascularization

• Functional assessment:

  • Create matrigel plugs containing CCL21 (4 μg) to assess in vivo effects

  • Compare with negative controls (PBS) and positive controls (bFGF, 100 ng)

  • Analyze after appropriate time period (e.g., 10 days) for vascularity and cellular infiltration

Recent research has shown that without ACKR4, extracellular CCL21 gradients in barrier sites become saturated and nonfunctional, preventing dendritic cells from homing directly to lymphatic vessels .

Advanced Research Questions

• How can researchers optimize CCL21-DC vaccine manufacturing processes for maximum efficacy?

Optimizing CCL21-DC vaccine manufacturing involves multiple critical considerations:

Key optimization parameters:

• Cellular composition analysis:

  • The cellular makeup of CCL21-DC vaccines is heterogeneous due to variable proportions of passenger lymphocytes

  • Single-cell RNA sequencing reveals heterogeneity within the monocyte-derived DC (moDC) compartment, with cells spanning a spectrum of DC phenotypes

  • Monitor and potentially standardize passenger lymphocyte content

• Transduction optimization:

  • CCL21-containing adenoviral vector transduction augments CCL21 secretion by moDCs

  • Transduction has minimal effect on other vaccine characteristics

  • Optimize vector-to-cell ratios and transduction protocols

• Storage considerations:

  • A single freeze-thaw cycle for stored vaccines is associated with minor alterations to DC phenotype

  • Compare fresh vs. frozen vaccine characteristics and efficacy

  • Develop optimal cryopreservation protocols

• Source material impact:

  • Use of healthy donors rather than patient autologous blood shows comparable but not identical results

  • Consider standardizing source material when possible

  • Validate differences between autologous and allogeneic approaches

• Functional validation:

  • Assess CCL21 secretion levels post-manufacturing

  • Evaluate DC activation markers (CD80, CD86, MHC-II)

  • Test migration capability and T cell stimulatory capacity

Recent clinical trials have used CCL21-DC vaccines in combination with pembrolizumab for non-small cell lung cancer, highlighting the translational relevance of these optimization strategies .

• What techniques are most effective for studying the interplay between CCL21-mediated immune cell recruitment and immunotherapy response?

Studying the interplay between CCL21-mediated immune cell recruitment and immunotherapy response requires sophisticated experimental approaches:

Effective techniques:

• In situ vaccination models:

  • Murine NSCLC models with distinct driver mutations (e.g., KrasG12D/P53+/-/Lkb1-/-, KrasG12D/P53+/-, Kras) provide systems with varying tumor mutational burden

  • Combination therapy with CCL21-DC in situ vaccination plus immune checkpoint inhibitors

  • Longitudinal sample collection for comprehensive analyses

• Cellular analysis methods:

  • Flow cytometry to track changes in immune cell populations over time

  • Single-cell RNA sequencing to characterize cellular heterogeneity and functional states

  • Whole-exome sequencing to assess immunoediting of tumor subclones

• Tumor microenvironment evaluation:

  • Single-cell genomic sequencing with GSEA (Gene Set Enrichment Analysis) to identify pathway alterations

  • CIBERSORT algorithm to determine correlations between CCL21 levels and immune cell types

  • ssGSEA to assess relative abundance of immune cells in tumor tissues with different CCL21 expression levels

• Functional immunological assays:

  • Assessment of neutrophil polarization states (N1 vs. N2) and their impact on immunotherapy response

  • Evaluation of CD8+ T cell infiltration, expansion, and effector function both locally and systemically

  • Characterization of progenitor T cell enrichment in the tumor microenvironment

Research demonstrates that CCL21-DC obliterates tumor-promoting neutrophils, promotes sustained infiltration of CD8+ cytolytic and CD4+ Th1 lymphocytes, and enriches progenitor T cells in the tumor microenvironment .

• How can researchers distinguish between soluble and immobilized CCL21 effects on immune cell behavior?

Distinguishing between soluble and immobilized CCL21 effects requires specialized experimental design:

Methodological approaches:

• Microfluidic systems:

  • Create soluble gradients based on diffusion to study long-range chemotaxis

  • Design non-adherent single channels without ICAM-1 or other adhesion ligands to isolate effects of soluble CCL21

  • Analyze cell polarization as a first indicator of response to soluble CCL21

• Surface immobilization techniques:

  • Immobilize CCL21 on surfaces at controlled densities

  • Compare migration on CCL21-coated vs. uncoated surfaces

  • Use specialized migration chambers that allow simultaneous assessment of responses to soluble and immobilized chemokine

• Functional readouts:

  • Track individual cells to distinguish directed chemotaxis from random chemokinesis

  • Measure cell polarization in response to different presentations of CCL21

  • Assess downstream signaling pathways activated by different forms of CCL21

• Scavenging mechanisms:

  • Study the role of ACKR4 in scavenging both soluble and immobilized CCL21

  • Examine how ACKR4 shapes CCL21 distribution in barrier tissues and prevents leakage

  • Investigate how extracellular CCL21 gradients can become saturated and nonfunctional without proper regulation

Recent research has challenged previous claims that CCL21 does not stimulate naive T lymphocytes unless adsorbed on a substrate, demonstrating that CCL21 can trigger stable polarization and long-range chemotaxis of cells in soluble form .

• What are the technical challenges in developing predictive models using CCL21 as a biomarker for immunotherapy response?

Developing predictive models using CCL21 as a biomarker for immunotherapy response faces several technical challenges:

Technical considerations and solutions:

• Sample collection and processing:

  • Standardization of blood collection timing relative to treatment

  • Consideration of tissue vs. serum CCL21 levels (tumor tissues showed differential CCL21 expression in responding vs. non-responding patients)

  • Development of robust protocols for sample processing to maintain CCL21 stability

• Assay selection and validation:

  • Optimization of detection thresholds for ELISA or other quantitative methods

  • Cross-validation across different detection platforms

  • Establishment of reference ranges for different cancer types

• Integration with other biomarkers:

  • LASSO regression analysis can identify the most useful predictors to combine with CCL21

  • A nomogram incorporating CCL21 with tumor size, gamma-glutamyl transferase (γ-GT), and neutrophil to lymphocyte ratio (NLR) showed excellent predictive performance (AUC: 0.863)

  • Calibration curves should demonstrate good consistency between actual observation and nomogram prediction

• Biological variability factors:

  • CCL21 expression independence from tumor stage requires stratified analysis

  • Source variability: CCL21 in liver cancer is mainly derived from stromal cells like fibroblasts and epithelial cells

  • Receptor expression (CCR7) may not correlate with immunotherapy response despite being the principal receptor for CCL21

• Clinical validation requirements:

  • Prospective validation in diverse patient cohorts

  • Evaluation of predictive value in different cancer types

  • Assessment of temporal dynamics during treatment course