CCR8 Recombinant Monoclonal Antibody

What is CCR8 and why is it a significant research target?

CCR8 (C-C motif chemokine receptor 8) is a G protein-coupled receptor predominantly expressed in regulatory T (Treg) cells and T helper 2 cells. The receptor's significance stems from its increased expression in Tregs within cancer microenvironments, its role in enhancing Treg migration activity, and its anti-apoptotic properties in T cell leukemia and lymphoma. These characteristics suggest CCR8 plays a critical role in cancer development and progression, making it both a valuable diagnostic marker and potential therapeutic target . CCR8 may also be referred to in literature by alternative designations including C-C CKR-8, CC-CKR-8, CCR-8, CDw198, CKRL1, C-C chemokine receptor type 8, and CC chemokine receptor 8, with a reported molecular mass of approximately 40.8 kilodaltons .

What are the principal applications of CCR8 monoclonal antibodies in research?

CCR8 monoclonal antibodies serve multiple research purposes:

• Flow cytometry: Detection of endogenous and exogenous CCR8 expression on cell surfaces

• Immunocytochemistry: Visualization of CCR8 distribution in cellular contexts

• Neutralization assays: Blocking CCR8-ligand interactions to study functional significance

• Cancer immunotherapy development: CCR8-targeting approaches for cancer treatment

• Basic mechanistic studies: Elucidation of CCR8-dependent processes in normal physiology and disease

In flow cytometry applications, CCR8 antibodies have demonstrated efficacy in detecting the receptor on both transfected cells overexpressing CCR8 and in cell lines with endogenous expression, such as P388 (mouse lymphocyte-like cells) and J774-1 (mouse macrophage-like cells) .

How should researchers validate a new CCR8 monoclonal antibody for specific applications?

Proper validation of CCR8 monoclonal antibodies requires a systematic approach:

• Positive and negative control testing: Test antibody binding to:

  • CCR8-overexpressing transfected cell lines (positive control)

  • Parental non-transfected cell lines (negative control)

• Species cross-reactivity assessment: Determine if the antibody recognizes CCR8 from multiple species or is species-specific (human, mouse, etc.)

• Application-specific validation:

  • For flow cytometry: Compare staining patterns with isotype controls

  • For immunocytochemistry: Perform blocking experiments with recombinant CCR8 protein

  • For neutralization assays: Test dose-dependent inhibition of chemotaxis or signaling

• Endogenous expression verification: Confirm detection of native CCR8 in cells known to express the receptor (e.g., Tregs, specific cell lines)

A thorough validation study should include quantitative analysis of antibody performance metrics including signal-to-noise ratio, specificity parameters, and reproducibility across multiple experimental conditions.

What experimental controls are essential when working with CCR8 antibodies?

Essential controls for CCR8 antibody experiments include:

The incorporation of these controls is critical as demonstrated in studies where rat anti-human CCR8 monoclonal antibody (MAB1429) effectively neutralized chemotaxis induced by CCL1/I-309 in a dose-dependent manner, with typical neutralization doses (ND50) of 0.01-0.05 μg/mL .

How can researchers optimize CCR8 antibody usage for flow cytometry applications?

Optimization of CCR8 antibody usage for flow cytometry requires attention to several technical parameters:

• Titration determination: Instead of using manufacturer-recommended concentrations without verification, researchers should perform antibody titration experiments to determine optimal concentration for:

  • Maximum specific signal

  • Minimal background

  • Optimal signal-to-noise ratio

• Sample preparation considerations:

  • Fresh vs. fixed cells: Some epitopes may be sensitive to fixation

  • Membrane permeabilization: Necessary only if targeting intracellular domains

  • Buffer composition: Test multiple staining buffers to reduce non-specific binding

• Multi-parameter panel design:

  • When combining CCR8 detection with other markers, perform fluorochrome compensation controls

  • Consider marker co-expression patterns for proper gating strategy

  • Test for antibody interference when using multiple antibodies

• Data analysis refinements:

  • Use fluorescence minus one (FMO) controls for accurate gating

  • Compare median fluorescence intensity (MFI) shifts rather than percent positive alone

  • Establish consistent gating strategies across experiments

Published protocols have demonstrated successful CCR8 detection in human peripheral blood cells using flow cytometry with monoclonal antibody MAB1429, followed by an anti-Rat IgG APC-conjugated secondary antibody in combination with lineage markers such as CD14 .

What approaches can resolve discrepancies in CCR8 detection between different antibody clones?

When faced with discrepant results between different CCR8 antibody clones, researchers should implement the following troubleshooting strategy:

• Epitope mapping comparison:

  • Different antibody clones recognize distinct epitopes that may be differentially accessible

  • Some epitopes may be masked by protein-protein interactions in certain cell types

  • Post-translational modifications might affect epitope recognition

• Side-by-side validation using multiple techniques:

  • Compare flow cytometry, immunocytochemistry, and western blot results

  • Correlate protein detection with mRNA expression (RT-PCR or RNA-seq)

  • Use gene editing (CRISPR/Cas9) to create CCR8 knockout controls

• Reconciliation through experimental design:

  • Design experiments that account for known limitations of each antibody

  • Use multiple antibody clones targeting different epitopes

  • Implement functional assays to complement binding studies

The literature indicates potential discrepancies in antibody performance, as evidenced by a review reporting that one anti-human CCR8 antibody failed to bind specifically to CCR8-transfected CHO and HEK293 cells despite proper controls functioning correctly .

How can researchers troubleshoot common issues with CCR8 antibody applications?

Common technical challenges with CCR8 antibodies can be addressed through systematic troubleshooting:

For example, when using C8Mab-3 for immunocytochemistry, researchers successfully detected both exogenous and endogenous mouse CCR8 by optimizing staining protocols for specific cell types .

What factors should researchers consider when selecting between different CCR8 antibody clones?

Selection between different CCR8 antibody clones should be guided by:

• Application compatibility:

  • Some clones perform better in specific applications (flow cytometry vs. immunohistochemistry)

  • Review validation data for each clone in your intended application

  • Consider whether native or denatured protein detection is required

• Species reactivity:

  • Determine which species is relevant to your research (human, mouse, etc.)

  • Consider whether cross-reactivity with other species is beneficial or problematic

  • Check sequence homology between target species and immunogen used for antibody generation

• Clone-specific characteristics:

  • Binding affinity and avidity can vary significantly between clones

  • Epitope location may affect ability to detect CCR8 in different contexts

  • Some clones may have neutralizing activity while others only bind without functional effects

• Experimental validation history:

  • Review publications using specific clones for similar experiments

  • Consider clones with demonstrated reliability in peer-reviewed research

  • Evaluate manufacturer validation data comprehensively

For example, C8Mab-2 (rat IgG2b, kappa) and C8Mab-3 (rat IgG1, kappa) are both effective for detecting mouse CCR8, but may have different performance characteristics based on their isotype and development method .

How is CCR8 antibody technology advancing cancer immunotherapy approaches?

CCR8 antibody technology is driving significant advances in cancer immunotherapy through several mechanisms:

• Targeting immunosuppressive Tregs:

  • CCR8 is highly expressed on tumor-infiltrating Tregs

  • Anti-CCR8 antibodies can selectively deplete these immunosuppressive cells

  • This approach may enhance anti-tumor immune responses by reducing immunosuppression in the tumor microenvironment

• Combination therapy strategies:

  • CCR8 antibodies are being investigated in combination with:

    • Immune checkpoint inhibitors (anti-PD-1/PD-L1)

    • Chemotherapy regimens

    • Other immunomodulatory agents

  • These combinations aim to overcome resistance mechanisms

• Biomarker development:

  • CCR8 expression levels are being evaluated as potential predictive biomarkers for immunotherapy response

  • Anti-CCR8 antibodies enable accurate assessment of CCR8 expression in clinical samples

  • This facilitates patient stratification for personalized therapy approaches

• Novel antibody engineering approaches:

  • Development of bispecific antibodies targeting CCR8 and other immune targets

  • Antibody-drug conjugates delivering cytotoxic agents specifically to CCR8+ cells

  • Engineering of antibodies with enhanced effector functions for more effective depletion

The development of highly specific monoclonal antibodies against CCR8, such as C8Mab-2 and C8Mab-3, provides valuable tools for advancing these therapeutic strategies .

What methodological considerations are important when using CCR8 antibodies for multiplex assays?

When incorporating CCR8 antibodies into multiplex assays, researchers should address the following methodological considerations:

• Antibody compatibility assessment:

  • Test for interference between antibodies in the multiplex panel

  • Evaluate potential cross-reactivity with other targets

  • Verify that detection systems do not interfere with each other

• Signal optimization strategies:

  • Adjust antibody concentrations individually within the multiplex context

  • Consider signal amplification methods for low-abundance targets

  • Implement appropriate compensation controls for spectral overlap

• Sample processing adaptations:

  • Optimize fixation and permeabilization protocols to preserve all target epitopes

  • Determine optimal blocking conditions to minimize background across all targets

  • Establish appropriate washing protocols to reduce non-specific binding

• Panel design principles:

  • Assign brightest fluorophores to lowest-expressed targets

  • Consider antigen density when selecting fluorophores

  • Plan panel based on biological questions and expression patterns

• Analysis complexity management:

  • Implement appropriate gating strategies for complex populations

  • Use dimensionality reduction techniques (tSNE, UMAP) for high-parameter data

  • Apply consistent analysis frameworks across experiments

Successful multiplex applications have been demonstrated with CCR8 antibodies in flow cytometry, where human peripheral blood cells were simultaneously stained for CCR8 and CD14 to identify specific cellular populations .

How should researchers interpret CCR8 expression heterogeneity in primary samples?

Interpreting CCR8 expression heterogeneity in primary samples requires a nuanced analytical approach:

• Biological significance assessment:

  • Correlate CCR8 expression patterns with functional characteristics of cell subpopulations

  • Determine whether expression differences represent distinct cellular states or continuum of activation

  • Compare expression profiles across different tissue compartments (blood vs. tissue-resident cells)

• Technical variation control:

  • Standardize sample collection, processing, and staining protocols

  • Include internal controls to normalize across experiments

  • Validate findings using complementary techniques (flow cytometry, immunohistochemistry, mRNA analysis)

• Contextual interpretation frameworks:

  • Consider CCR8 expression in relation to disease state, treatment status, or experimental conditions

  • Evaluate expression changes longitudinally when possible

  • Compare with established reference ranges for specific cell populations

• Single-cell resolution analysis:

  • Implement single-cell techniques to resolve population heterogeneity

  • Correlate CCR8 expression with other markers of cell state or function

  • Use computational approaches to identify distinct cellular clusters based on expression profiles

When analyzing CCR8 expression in primary cells, researchers should consider that studies have shown differential expression patterns across cell types, with particularly high expression in certain regulatory T cell populations in cancer contexts .

What approaches can differentiate between functional and non-functional CCR8 expression?

Distinguishing functional from non-functional CCR8 expression requires functional assessment beyond mere protein detection:

• Receptor signaling evaluation:

  • Measure calcium flux in response to CCR8 ligands (CCL1/I-309)

  • Assess downstream phosphorylation events in signaling cascades

  • Quantify G-protein activation following receptor stimulation

• Migratory capacity assessment:

  • Conduct chemotaxis assays using transwell systems with CCL1 as chemoattractant

  • Compare migration indices between different cell populations

  • Perform antibody neutralization studies to confirm specificity

• Receptor internalization dynamics:

  • Track CCR8 surface expression following ligand exposure

  • Measure receptor recycling kinetics after internalization

  • Assess the impact of receptor mutations on trafficking patterns

• Molecular interaction studies:

  • Investigate protein-protein interactions with signaling complexes

  • Evaluate receptor oligomerization status

  • Assess post-translational modifications affecting receptor function

A standardized chemotaxis assay protocol has been developed that measures the migration of CCR8-expressing cells toward CCL1/I-309 in a dose-dependent manner. This assay can be combined with neutralizing antibodies to confirm receptor functionality, as demonstrated with rat anti-human CCR8 monoclonal antibody (MAB1429) .