CCL22 Antibody

What is CCL22 and why is it significant in immunological research?

CCL22 is a 10.6 kilodalton chemokine that binds to the CCR4 receptor present on multiple cell types. It has significant immunological importance as it is secreted by M2 macrophages and contributes to Th2 responses including phagocytosis, tissue repair, and wound healing . CCL22 has been implicated in various biological processes including regulatory T cell communication, inflammatory regulation, and pathogenesis of certain diseases. Research significance stems from its dual role in both maintaining normal immune homeostasis and contributing to pathological conditions such as atopic diseases and tumor immunosuppression .

What are the primary applications of anti-CCL22 antibodies in research?

Anti-CCL22 antibodies serve multiple critical research functions, most commonly employed in:

• Western blotting (WB) for protein expression quantification

• Enzyme-linked immunosorbent assays (ELISA) for CCL22 detection in biological samples

• Flow cytometry (FCM) for cellular analysis

• Immunohistochemistry on paraffin-embedded tissues (IHC-p) for localization studies

• Neutralization assays to block CCL22 function

These applications allow researchers to study CCL22's role in cytokine signaling networks and characterize its expression across different experimental conditions . Anti-CCL22 antibodies have been particularly valuable in studies examining immune regulation, cancer biology, and inflammatory diseases.

How do CCL22 antibodies differ in their species reactivity and what should researchers consider when selecting them?

When selecting anti-CCL22 antibodies, species reactivity is a critical consideration. Available antibodies show varying degrees of cross-reactivity:

• Human-specific antibodies: Most commonly available and well-characterized

• Cross-reactive antibodies: Some recognize human, mouse, and guinea pig CCL22

Researchers should consider:

• The experimental model system (human tissues, mouse models, etc.)

• The species homology of the CCL22 epitope targeted by the antibody

• Validation data demonstrating specificity in the species of interest

• Whether the research requires detecting specific motifs or regions unique to CCL22 in particular species

For studies involving orthologous comparisons, selecting antibodies validated across multiple species becomes crucial to ensure consistent detection methodologies .

How can CCL22 antibodies be utilized to study the tumor microenvironment and immune escape mechanisms?

CCL22 antibodies provide powerful tools for investigating tumor-associated macrophage (TAM) interactions and immune evasion. Research has demonstrated that TAM-derived CCL22 contributes to immunosuppressive tumor microenvironments by:

• Recruiting regulatory T cells (Tregs) through the CCL22:CCR4 axis

• Inducing FAK signaling pathways in tumor cells

• Suppressing anti-cancer immune responses

Methodologically, researchers can employ CCL22 antibodies in multiplex immunohistochemistry to map the spatial relationship between CCL22-expressing cells and Tregs within tumor sections. Flow cytometric analysis with CCL22 antibodies can quantify the proportion of CCL22-producing cells in tumor digests. In experimental models, neutralizing CCL22 antibodies can be used to block the CCL22:CCR4 axis, potentially revealing therapeutic avenues and mechanistic insights into immune escape .

What role does CCL22 play in autoimmune and inflammatory conditions, and how can antibodies help elucidate these mechanisms?

CCL22 has been implicated in the pathogenesis of multiple inflammatory and autoimmune conditions. Studies indicate its role in:

• Atopic diseases such as asthma and atopic dermatitis via CCR4 binding

• Abdominal aortic aneurysm (AAA), where both tissue and circulating CCL22 concentrations show significant association

• Dextran sodium sulfate (DSS)-induced colitis models, where CCL22 appears to regulate inflammatory processes

Researchers can employ CCL22 antibodies in several advanced applications:

• Quantitative tissue analysis comparing diseased versus healthy tissues (illustrated by median relative density measurements in AAA vs. atherothrombosis: 2.57 vs. 1.40, p=0.01)

• Co-localization studies to identify CCL22-producing cells in inflammatory lesions

• Tracking dynamic changes in CCL22 expression throughout disease progression

• Mechanistic studies using neutralizing antibodies to block CCL22 function in experimental disease models

For AAA specifically, immunohistochemistry with anti-CCL22 antibodies has demonstrated localization adjacent to macrophage markers, with concomitant staining for the CCL22 receptor (CCR4), suggesting autocrine/paracrine signaling mechanisms in disease pathogenesis .

What experimental approaches can reveal the impact of CCL22 deficiency on immune responses?

Studies examining CCL22 deficiency have yielded important insights into its immunoregulatory functions. Researchers have employed several strategic approaches:

• Genetic knockout models: Ccl22^−/−^ mice display enhanced T cell responses upon OVA vaccination, with OVA-specific cytotoxic T cells more than doubled compared to wild-type, and 2.7-fold more IFN-γ–positive CTLs upon restimulation .

• Cell transfer experiments: Vaccination with dendritic cells from Ccl22^−/−^ mice induces substantially stronger T cell immune responses compared to wild-type DCs, with more than double the frequency of antigen-specific and IFN-γ–positive T cells .

• Ex vivo functional assays: Isolating T cells from CCL22-deficient environments to assess their function through proliferation, cytokine production, and cytotoxicity assays.

• Reconstitution studies: Determining whether reintroduction of recombinant CCL22 can restore immunoregulatory function in deficient models.

These approaches collectively demonstrate that CCL22 deficiency reduces regulatory T cell suppression and enhances effector T cell responses, highlighting CCL22's role as a critical immune checkpoint molecule .

What are the optimal protocols for quantifying CCL22 in cell supernatants and biological fluids?

For precise CCL22 quantification in biological samples, researchers should consider:

HTRF Detection Method:

• Sample volume: 16 μL

• Assay format: 384-well low-volume white plate or 96-well plate

• Protocol: Sample dispensed directly into assay plate, followed by pre-mixed HTRF® antibodies added in a single step

• Scalability: Can be adapted to 1536-well format by proportional volume adjustment

• Analysis: 4 Parameter Logistic (4PL) curve fitting recommended over linear analysis

Enhanced Sensitivity for Serum/Plasma:

• For low abundance detection in serum or plasma, AlphaLISA assays may provide sufficient sensitivity

• Avoid highly hemolyzed samples

• Consider sample pre-clearing through centrifugation to remove particulates

Normalization Approaches:

• For tissue samples, normalize measurements to total protein concentration (relative density units/μg of protein)

• For comparative studies, paired statistical tests (e.g., Wilcoxon sign rank tests) may be appropriate for analyzing body vs. neck AAA biopsies

Median values with interquartile ranges should be reported for non-parametric data distributions, as demonstrated in comparative tissue analysis studies (e.g., CCL22 in AAA: 2.57 (2.01–3.03) vs. atherothrombosis: 1.40 (1.15–1.45)) .

What considerations are important when designing immunohistochemistry experiments with CCL22 antibodies?

Effective immunohistochemistry (IHC) with CCL22 antibodies requires careful methodological planning:

• Tissue Preparation and Fixation:

  • Paraffin-embedded tissues require appropriate antigen retrieval methods

  • Consider tissue-specific fixation protocols that preserve CCL22 epitopes

• Antibody Selection and Validation:

  • Choose antibodies validated specifically for IHC applications

  • Confirm specificity using appropriate positive and negative controls

  • Consider using multiple antibodies targeting different epitopes for confirmation

• Co-localization Studies:

  • Design multi-color staining panels to identify CCL22-producing cells

  • In tumor and inflammatory contexts, consider co-staining with:

    • Macrophage markers (to identify TAMs)

    • CCR4 (to visualize receptor-ligand interactions)

    • T cell markers (to correlate with Treg infiltration)

• Quantification Methods:

  • Develop consistent scoring systems for CCL22 expression

  • Consider digital pathology approaches for unbiased quantification

  • Correlate staining patterns with clinical outcomes or experimental endpoints

Previous studies have successfully employed IHC to demonstrate co-localization of CCL22 staining adjacent to macrophage markers, with corresponding CCR4 expression, suggesting autocrine/paracrine signaling networks in pathological tissues .

How can researchers effectively use neutralizing CCL22 antibodies in functional studies?

Neutralizing antibodies against CCL22 provide valuable tools for mechanistic studies. Effective implementation requires:

• Dose Optimization:

  • Perform dose-response experiments to determine minimal effective concentration

  • Assess target engagement through residual free CCL22 measurement

• Controls:

  • Include isotype control antibodies to account for nonspecific effects

  • Consider comparing results with genetic approaches (e.g., CRISPR knockout, siRNA)

• Timing Considerations:

  • For acute effects, determine appropriate pre-treatment window

  • For chronic studies, establish dosing schedule based on antibody half-life

• Readout Systems:

  • Chemotaxis assays to assess inhibition of CCL22-mediated cell migration

  • T reg recruitment assays in tumor models

  • Signaling pathway analysis (e.g., FAK/AKT activation) in target cells

• In Vivo Applications:

  • Consider route of administration (intravenous, intraperitoneal, intratumoral)

  • Assess tissue penetration and target engagement

  • Monitor for potential immune responses against the neutralizing antibody

Studies have demonstrated that neutralizing CCL22 function can significantly impact tumor growth and inflammatory processes, making these antibodies valuable tools for both mechanistic studies and therapeutic development .

What are common challenges in detecting CCL22 in tissue samples and how can they be overcome?

Researchers frequently encounter several challenges when detecting CCL22 in tissues:

• Low Signal Intensity:

  • Enhance detection through signal amplification systems (e.g., tyramide signal amplification)

  • Optimize antigen retrieval methods for specific tissue types

  • Use high-affinity antibodies with demonstrated tissue reactivity

  • Increase antibody concentration or incubation time (with appropriate controls)

• High Background:

  • Implement rigorous blocking steps using appropriate blocking agents

  • Reduce primary antibody concentration

  • Perform additional washing steps

  • Consider tissue-specific autofluorescence quenching for fluorescent detection

• Epitope Masking:

  • Test multiple fixation protocols to preserve epitope accessibility

  • Try antibodies targeting different CCL22 epitopes

  • Use enzymatic or heat-based antigen retrieval methods

• Quantification Challenges:

  • Develop consistent region-of-interest selection criteria

  • Use digital image analysis to reduce subjective interpretation

  • Normalize to appropriate reference markers

  • Report data as shown in validated studies (e.g., median relative density units/μg with interquartile ranges)

• Cross-Reactivity:

  • Validate antibody specificity using CCL22-knockout tissues or blocking peptides

  • Compare staining patterns from multiple independent antibodies

Researchers successfully addressing these challenges have demonstrated significant differences in CCL22 expression between diseased and healthy tissues, as evidenced by the 1.84-fold difference observed between AAA and atherothrombosis samples (p=0.01) .

How can researchers address variability in CCL22 detection across different experimental systems?

Addressing variability in CCL22 detection requires systematic standardization:

• Assay Standardization:

  • Implement consistent sample collection, processing, and storage protocols

  • Use internal controls and reference standards across experiments

  • Calibrate detection using recombinant CCL22 standard curves

  • Maintain consistent lot numbers for critical reagents when possible

• Biological Variability Management:

  • Increase biological replicates to account for natural variation

  • Consider time-of-day effects on chemokine expression

  • Account for stimulus-dependent variation in CCL22 production

  • Control for cell density and passage number in in vitro systems

• Technical Approach Harmonization:

  • Employ statistical methods appropriate for the data distribution

  • Consider normalization to housekeeping proteins or total protein

  • Use the 4 Parameter Logistic (4PL) curve fitting for ELISA-based detection

  • Report clear interquartile ranges for non-parametric data

• Cross-Platform Validation:

  • Confirm key findings using orthogonal detection methods

  • Consider protein-mRNA correlation studies

  • Validate antibody performance across different application methods

In comparative studies, consistent methodology has revealed significant biological differences, such as the 2.18-fold higher CCL22 levels observed in AAA body versus neck tissue (37.56 vs. 17.24 relative density units/μg, p=0.01) , highlighting the importance of methodological consistency.

How are CCL22 peptide vaccines being developed as immunotherapeutic strategies in cancer?

Recent advances in CCL22-targeted immunotherapy show promising directions for cancer treatment:

• Mechanism of Action:

  • CCL22 peptide vaccines induce CCL22-specific T-cell responses (measured by interferon-γ secretion ex vivo)

  • These vaccines modulate the tumor microenvironment by:

    • Increasing CD8+ T cell infiltration

    • Enhancing M1 macrophage presence

    • Improving CD8/Treg and M1/M2 macrophage ratios

  • The net effect is augmentation of anti-tumor immune responses

• Preclinical Evidence:

  • Vaccination with CCL22-derived peptides has been shown to:

    • Reduce tumor growth

    • Prolong survival in mouse models

    • Generate measurable CCL22-specific T-cell responses in both BALB/c and C57BL/6 mice

• Immune Monitoring Approaches:

  • Multi-color flow cytometry to assess changes in tumor-infiltrating immune cells

  • Ex vivo interferon-γ secretion assays to measure CCL22-specific T-cell responses

  • Immunohistochemistry to evaluate spatial distribution of immune populations

• Combination Therapy Potential:

  • CCL22-targeting approaches may complement existing immunotherapies

  • By altering the immunosuppressive tumor microenvironment, CCL22 vaccines could potentially enhance efficacy of checkpoint inhibitors

This approach represents a novel immunotherapeutic modality that focuses on modulating the tumor microenvironment rather than directly targeting tumor cells .

What role does CCL22 play in the focal adhesion kinase (FAK) activation pathway, and how can this be studied?

Emerging research has revealed an unexpected connection between CCL22 and FAK signaling in cancer:

• Signaling Relationship:

  • Tumor-associated macrophages (TAMs) produce abundant CCL22

  • TAM-derived CCL22 induces phosphorylation of focal adhesion kinase (pFAK Tyr)

  • CCL22 levels in tumor stroma positively correlate with intratumoral pFAK levels

• Experimental Approaches:

  • In vivo models: Intravenous CCL22 injection (0.1 μg/kg; twice/week) increased tumor size and enhanced AKT activation

  • Combination therapy studies: FAK inhibitors (e.g., VS-6063) more effectively inhibited tumor malignancy in CCL22-treated groups

  • Quantification methods: ELISAs for measuring Ki67, CD31, and LYVE-1 expression

• Clinical Correlations:

  • Expression of CCL22 in tumor stroma appears to correlate with pFAK levels

  • This correlation suggests potential prognostic significance

  • The relationship provides a mechanistic understanding of microenvironment-mediated oncogenic signaling

• Therapeutic Implications:

  • Dual targeting of CCL22 and FAK pathways may offer synergistic anti-tumor effects

  • FAK inhibitors may be particularly effective in tumors with high CCL22 expression

These findings highlight a novel mechanism of "microenvironment-mediated cellular addiction" to specific oncogenic signaling pathways, suggesting new approaches for strategic therapeutic interventions .