CCL5 Antibody

What is CCL5 and what functions make it a significant target for antibody research?

CCL5 (C-C Motif Chemokine Ligand 5), also known as RANTES (Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted), is an 8 kDa chemokine expressed by endothelial cells, platelets, smooth muscle cells, T cells, and macrophages. CCL5 exhibits the greatest affinity for CCR5, but also interacts with CCR1, CCR3, and CCR4 receptors. Due to this widespread receptor expression, CCL5 serves as a chemoattractant for multiple immune cell types, including monocytes, mast cells, dendritic cells, natural killer cells, eosinophils, basophils, CD4+ T cells, CD8+ T cells, and B cells .

In T cell biology, CCL5 regulates T-cell migration to inflammatory sites and influences T-cell differentiation through Th1 cell recruitment. Beyond immune regulation, CCL5 plays protective roles following neuronal damage, including stroke and Alzheimer's disease, by reducing oxidative stress, regulating ATP generation and synaptic complex formation in hippocampal neurons, supporting axon regeneration, and influencing brain energy metabolism . In cancer, CCL5-CCR signaling affects both tumor growth and antitumor immune responses, positioning it as a potential target for immune checkpoint blocking therapeutics .

How do CCL5 antibodies function in neutralization assays?

CCL5 neutralizing antibodies work by binding specific epitopes on the CCL5 protein, preventing its interaction with receptors and blocking downstream signaling events. A standard neutralization assay involves:

• Placing receptor-expressing cells (such as BaF3 cells transfected with human CCR5) in the top chamber of a Transwell plate

• Adding CCL5 alone or CCL5 pre-incubated with anti-CCL5 antibodies to the bottom chamber

• Measuring cell migration after incubation (typically 3 hours at 37°C)

• Calculating the neutralization dose (ND50) - the antibody concentration that reduces migration by 50%

For example, the R6G9 monoclonal antibody demonstrates dose-dependent inhibition of CCL5-induced chemotaxis of virus-specific T cells and macrophages . Similarly, commercial antibodies show neutralization with ND50 values typically between 0.1-0.5 μg/mL when tested against recombinant mouse CCL5 in chemotaxis assays using CCR5-transfected cells .

How can I validate the specificity of a CCL5 antibody?

Validating CCL5 antibody specificity requires a multi-faceted approach:

Proper validation ensures reliable interpretation of experimental results and minimizes false positives/negatives in your research system.

How do CCL5 antibodies perform in neuroinflammation models?

CCL5 antibodies have demonstrated significant efficacy in neuroinflammation models, particularly in viral-induced demyelinating disease. In mouse hepatitis virus (MHV) infection models, treatment with the R6G9 anti-CCL5 monoclonal antibody resulted in:

• Improved neurological function (clinical scores of 2.4 ± 0.1 in treated mice vs. 3.2 ± 0.1 in controls)

• Decreased T cell accumulation in the CNS (CD4+ T cell counts reduced from 1.6 × 10^5 ± 3.2 × 10^4 in controls to 1.9 × 10^4 ± 5.3 × 10^3 in treated animals by day 21 post-infection)

• Reduced demyelination in CNS tissues

The antibody works by neutralizing CCL5-mediated chemotaxis of both virus-specific T cells and macrophages. CCL5 neutralization also resulted in reduced CCL5 mRNA expression during the treatment period, suggesting a potential feedback mechanism affecting chemokine production .

When designing similar experiments, researchers should consider timing of antibody administration (prophylactic vs. therapeutic), appropriate dosing regimens, and comprehensive assessment of both clinical outcomes and cellular/molecular changes in the CNS.

What are the methodological approaches for studying CCL5 antibodies in cancer research?

CCL5 plays complex roles in cancer, affecting both tumor growth and antitumor immune responses. When using CCL5 antibodies in cancer research, consider these methodological approaches:

• Tumor microenvironment analysis:

  • Use multiplex immunohistochemistry to co-localize CCL5 with cell type-specific markers

  • Assess CCL5 expression in both tumor cells and tumor-infiltrating immune cells

  • Examine relationships between CCL5 expression and immune cell infiltration patterns

• Metabolic pathway investigation:

  • CCL5 can induce aerobic glycolysis through AMPK signaling pathway regulation

  • Compare glucose uptake, lactic acid production, and ATP levels in cancer cells with and without CCL5 neutralization

  • Assess phosphorylation of AMPK and downstream targets (ACC, c-Myc, HIF-1α, Akt)

• Epithelial-mesenchymal transition (EMT) assessment:

  • Monitor E-cadherin (epithelial marker) and vimentin (mesenchymal marker) expression

  • Determine how CCL5 antibody treatment affects EMT marker expression in cancer cells

• Therapeutic intervention studies:

  • Combine CCL5 antibodies with immune checkpoint inhibitors

  • Assess tumor growth, metastasis, and survival outcomes

  • Evaluate changes in tumor-infiltrating lymphocyte populations and activation states

Research has shown that the CCL5-CCR5 axis can promote metabolic changes in breast cancer cells co-cultured with lactate-activated macrophages, and CCL5 antibodies may interfere with these processes .

How can researchers use CCL5 antibodies to study differential receptor interactions?

CCL5 interacts with multiple receptors (primarily CCR1, CCR3, CCR4, and CCR5), each potentially mediating distinct biological effects. To study these differential interactions using antibodies:

• Receptor-specific cell models:

  • Use cell lines selectively expressing individual CCR receptors (e.g., BaF3 cells transfected with human CCR5 as used in chemotaxis assays)

  • Compare CCL5 antibody effects across these different receptor-expressing cells

  • Assess receptor-specific readouts like chemotaxis, calcium flux, or receptor internalization

• Epitope-specific antibodies:

  • Employ antibodies targeting CCL5 domains involved in specific receptor interactions

  • Compare effects with receptor-specific antagonists (e.g., Maraviroc for CCR5)

  • Correlate epitope specificity with differential inhibition of receptor-specific functions

• Signaling pathway analysis:

  • Monitor activation of receptor-specific downstream pathways

  • For example, CCL5 induces AMPK phosphorylation in a time-dependent manner in CCR5-expressing cells

  • Compare signaling patterns with and without receptor-specific inhibitors

• Combinatorial approaches:

  • Combine antibody neutralization with genetic manipulation of specific receptors

  • Use receptor knockdown/knockout models to isolate receptor-specific contributions

  • Determine whether antibody effects persist in the absence of specific receptors

These approaches help delineate which biological functions of CCL5 are mediated through which receptors, potentially informing more targeted therapeutic strategies.

What is the optimal experimental design for CCL5 antibody neutralization assays?

Based on established protocols in the literature, an optimal CCL5 neutralization assay design includes:

• Cell preparation:

  • BaF3 mouse pro-B cells transfected with human CCR5 (most commonly used)

  • Alternative: primary T cells, macrophages, or other CCR5-expressing cells

  • Cell density: typically 5 × 10^5 cells per Transwell chamber

• Stimulation conditions:

  • Recombinant CCL5 concentration: 0.01-0.025 μg/mL for human CCL5

  • Antibody titration: serial dilutions (typically 0.01-10 μg/mL)

  • Pre-incubation: 30 minutes at room temperature

• Control conditions:

  • Positive control: CCL5 without antibody

  • Negative control: media without CCL5

  • Isotype control: irrelevant antibody of same isotype

  • Specificity control: related chemokines to confirm antibody specificity

• Measurement approaches:

  • Incubation time: 3 hours at 37°C

  • Quantification methods:

    • Direct cell counting in bottom chamber (5 random high-power fields)

    • Resazurin-based quantification of migrated cells

    • Flow cytometry of collected cells

• Data analysis:

  • Generate dose-response curves

  • Calculate percent inhibition relative to positive control

  • Determine ND50 (typically 0.1-0.5 μg/mL for effective antibodies)

This standardized approach enables quantitative comparison of neutralization potency between different antibody clones and consistent evaluation across laboratories.

What are the critical parameters for optimizing immunohistochemistry with CCL5 antibodies?

Optimizing immunohistochemistry (IHC) protocols for CCL5 detection requires attention to several critical parameters:

• Tissue fixation and processing:

  • Fixative selection: neutral-buffered formalin is commonly used

  • Fixation time: minimize overfixation which can mask epitopes

  • For frozen sections: use fresh frozen tissue for optimal antigen preservation

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) is often necessary

  • Buffer selection: citrate (pH 6.0) or EDTA-based (pH 9.0) buffers

  • Example from literature: "tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic"

• Antibody optimization:

  • Concentration: 10-15 μg/mL is a typical starting point

  • Incubation conditions: overnight at 4°C or 1-3 hours at room temperature

  • Antibody diluent: typically PBS with 1-5% normal serum and 0.1-0.3% detergent

• Detection systems:

  • For brightfield: HRP-polymer systems with DAB chromogen

  • For fluorescence: appropriate secondary antibodies (e.g., NorthernLights™ 557-conjugated antibodies)

  • Signal amplification: consider tyramide signal amplification for low-abundance targets

• Tissue-specific controls:

  • Positive control tissues: human tonsil and lymphoid tissues show robust CCL5 expression

  • Negative controls: primary antibody omission and isotype controls

  • Specificity controls: pre-absorption with recombinant CCL5

CCL5 typically shows cytoplasmic localization in expressing cells, with particularly strong staining in lymphocytes as demonstrated in human tonsil sections .

How should researchers design experiments to study the impact of CCL5 antibodies on signaling pathways?

When studying how CCL5 antibodies affect signaling pathways, consider this experimental framework:

• Cell model selection:

  • Cancer cell lines: MDA-MB-231 and MCF-7 breast cancer cells (used in published studies)

  • Immune cells: THP-1 macrophages or primary immune cells

  • Transfected cell lines with controlled receptor expression

• Stimulation protocol:

  • CCL5 concentration: 50 ng/ml (standard in signaling studies)

  • Time course: multiple time points (minutes to hours)

  • Antibody pre-treatment: titration of neutralizing antibodies

• Control conditions:

  • Positive controls: direct pathway activators/inhibitors

  • Receptor controls: CCR5 antagonist Maraviroc (5μM)

  • Pathway inhibitors: compound C for AMPK inhibition (10μM)

  • siRNA knockdown of key pathway components (e.g., AMPK α1)

• Analysis methods:

  • Western blotting for phosphorylated proteins:

    • Key targets: AMPK, c-Myc, HIF-1α, Akt, and ACC

    • Include total protein controls

  • Functional readouts:

    • Glucose uptake, lactate production, ATP levels

    • Expression of downstream genes/proteins

    • Cellular phenotypes (e.g., EMT markers: E-cadherin, vimentin)

• Temporal analysis:

  • Early signaling events (minutes): receptor activation, phosphorylation cascades

  • Intermediate events (hours): transcriptional changes, protein expression

  • Late events (days): phenotypic alterations, functional outcomes

This approach allows researchers to establish cause-effect relationships between CCL5 signaling and downstream effects, and to determine how antibodies modulate these pathways.

How should researchers interpret discrepancies between CCL5 antibody detection and mRNA expression?

Discrepancies between CCL5 protein levels (detected by antibodies) and mRNA expression are common and should be interpreted considering several factors:

• Post-transcriptional regulation:

  • CCL5 mRNA may be subject to regulation by microRNAs or RNA-binding proteins

  • Translation efficiency can vary independently of transcription rates

  • mRNA stability may differ across experimental conditions

• Protein dynamics:

  • CCL5 is actively secreted, so intracellular protein levels may not reflect production

  • Proteolytic processing can affect antibody epitope recognition

  • Dimerization or aggregation of CCL5 may mask epitopes in certain assays

  • CCL5 can form dimers at low concentrations and higher-order aggregates at high concentrations

• Feedback mechanisms:

  • Treatment with anti-CCL5 antibodies can alter CCL5 mRNA expression

  • In vivo studies have shown that "Treatment with anti-CCL5 resulted in reduced mRNA expression of CCL5 during the treatment period"

  • This suggests antibody-mediated neutralization may influence transcriptional regulation

• Technical considerations:

  • Detection sensitivity differences between protein and mRNA assays

  • Antibody specificity for different CCL5 conformations or modifications

  • Sample preparation differences affecting protein recovery versus RNA isolation

• Cell source variations:

  • Different cell populations may contribute to the total CCL5 pool

  • Infiltrating T cells can express CCL5, affecting local concentrations

  • Activated T cells produce cytokines that induce CCL5 gene expression in other cells

When interpreting such discrepancies, researchers should employ multiple detection methods, examine both intracellular and secreted CCL5, and consider the temporal relationship between transcription, translation, and secretion.

What are the key considerations when comparing efficacy of different CCL5 antibody clones?

When comparing different CCL5 antibody clones, researchers should consider:

• Epitope specificity:

  • Different clones recognize distinct epitopes on CCL5

  • Clone R6G9 targets a specific epitope (KKWVQEYINYLEMS) that produces neutralizing antibodies

  • Epitope location can affect neutralization potential and receptor binding interference

• Cross-reactivity profiles:

  • Species cross-reactivity varies significantly between clones

  • Some antibodies show specificity for one species (e.g., mouse-specific)

  • Others demonstrate cross-reactivity (e.g., "detects human CCL5/RANTES and mouse CCL5/RANTES in Western blots")

  • Cross-reactivity with other chemokines should be assessed (e.g., CCL2, CCL3, CXCL9, CXCL10)

• Functional potency metrics:

  • Neutralization dose 50% (ND50): typically 0.1-0.5 μg/mL for effective antibodies

  • Binding affinity (KD values)

  • Maximum inhibition achievable

• Application-specific performance:

  • Western blot: ability to detect denatured protein

  • ELISA: sensitivity and dynamic range

  • IHC/ICC: signal-to-noise ratio in different fixation conditions

  • Functional assays: potency in chemotaxis or signaling inhibition

• Standardized comparisons:

  • Head-to-head testing under identical conditions

  • Concentration-matched comparisons (molar rather than mass-based)

  • Reference standard inclusion

By systematically evaluating these parameters, researchers can select the most appropriate antibody clone for their specific application and experimental system.

How can researchers address potential confounding factors when using CCL5 antibodies in complex disease models?

When using CCL5 antibodies in complex disease models, researchers should address these potential confounding factors:

• Chemokine network compensation:

  • CCL5 inhibition may lead to compensatory upregulation of other chemokines

  • Monitor expression of related chemokines (CCL2, CCL3, CCL4)

  • Consider "CCL5 also plays a role in reducing oxidative stress, neuroimmunology, regulating ATP generation and synaptic complex formation in hippocampal neurons"

• Receptor redundancy:

  • CCL5 signals through multiple receptors (CCR1, CCR3, CCR4, CCR5)

  • These receptors also bind other chemokines

  • Consider combined receptor blockade approaches

  • "The CCL5-CCRs signaling influences both the growth of tumors and antitumor immune responses"

• Timing-dependent effects:

  • CCL5 may have different roles at different disease stages

  • Studies have noted that "The variety of host tissues affected by viruses, added to different contexts of chronic or acute infection, makes it difficult to define a role for CCL5 in viral infections"

  • Compare prophylactic versus therapeutic antibody administration

• Cell type-specific responses:

  • Different immune cells show varying dependency on CCL5

  • CCL5 serves as chemoattractant for "monocytes, mast cells, dendritic cells, natural killer cells, eosinophils, basophils, CD4 T cells, CD8 T cells, and B cells"

  • Perform cell-specific analyses to determine differential impacts

• Concentration-dependent effects:

  • CCL5 can exhibit different activities at different concentrations

  • "Low concentrations activate GPCR-dependent pathways and high concentrations triggers CCL5 aggregation and subsequent G-protein-independent signaling"

  • Dose-response studies are essential

• Antibody penetration barriers:

  • In CNS models, blood-brain barrier penetration may be limited

  • In tumor models, abnormal vasculature affects antibody distribution

  • Consider tissue-specific pharmacokinetics and distribution

By systematically addressing these factors, researchers can more accurately interpret the specific contribution of CCL5 to disease pathogenesis and the therapeutic potential of CCL5 antibodies.

How are CCL5 antibodies being utilized in neurodegeneration research?

CCL5 antibodies are increasingly important in neurodegeneration research based on findings that CCL5 plays protective roles in several conditions:

• Neuroprotective mechanisms:

  • "Several reports documented that CCL5 plays protective roles following neuronal damage, including stroke (brain trauma) and Alzheimer's disease (AD)"

  • CCL5 antibodies can help elucidate whether these effects are direct or receptor-mediated

  • Neutralization studies can establish causality in neuroprotection models

• Neuroinflammatory modulation:

  • Anti-CCL5 treatment in viral models of demyelination "resulted in improved neurological function, decreased T cell and macrophage accumulation in the CNS, reduced demyelination"

  • Researchers can use antibodies to distinguish beneficial versus detrimental inflammation

• Metabolic regulation in neurons:

  • CCL5 is involved in "regulating ATP generation and synaptic complex formation in hippocampal neurons, axon regeneration, and brain energy metabolism"

  • Antibody neutralization studies can determine the impact of CCL5 on neuronal metabolism

• Therapeutic applications:

  • Multiple administration routes (systemic, intrathecal, intranasal)

  • Treatment timing optimization (preventive versus therapeutic)

  • Combination approaches with other neuroprotective agents

CCL5 antibodies allow researchers to target this chemokine with temporal and spatial precision, offering advantages over genetic approaches in studying its complex roles in neurodegeneration.

What methodological approaches can distinguish between different oligomeric forms of CCL5 using antibodies?

CCL5 exists in different oligomeric states with distinct biological activities. The search results indicate that "dimeric CCL5 exhibits RANTES-induced chemotaxis and inhibits HIV infection" while "aggregates cause T cell and neutrophil activation while promoting HIV infection" . To distinguish these forms:

• Conformation-specific antibodies:

  • Develop antibodies targeting epitopes unique to monomers, dimers, or higher-order aggregates

  • Validate specificity using purified oligomeric standards

  • Apply in immunoassays to quantify different forms in biological samples

• Separation techniques with antibody detection:

  • Size exclusion chromatography followed by antibody-based detection

  • Native gel electrophoresis with Western blotting

  • Analytical ultracentrifugation combined with immunoassays

• Functional characterization:

  • Compare antibody neutralization potency against different functions:

    • Chemotaxis (primarily dimeric CCL5)

    • T cell/neutrophil activation (primarily aggregated CCL5)

    • HIV infection modulation (differs between forms)

  • Correlate neutralization patterns with oligomeric composition

• In situ detection strategies:

  • Proximity ligation assays to detect oligomeric forms in tissues

  • Fluorescence resonance energy transfer (FRET) with labeled antibodies

  • Super-resolution microscopy to visualize different quaternary structures

These approaches allow researchers to determine which oligomeric forms predominate in different physiological and pathological contexts, potentially leading to more targeted therapeutic strategies addressing specific CCL5 conformations.

By employing these methodological approaches, researchers can advance our understanding of CCL5 biology and develop more targeted interventions for conditions where CCL5 plays significant roles.