CD80 Antibody

What is CD80 and what is its role in the immune system?

CD80 (also known as B7-1, B7, BB1) is a 60 kDa single chain type I glycoprotein belonging to the immunoglobulin superfamily. It plays a critical role in immune regulation through its interactions with receptor proteins on T cells. CD80 is expressed primarily by activated B cells, macrophages, dendritic cells, and can also be expressed by activated T cells .

CD80 has high affinity for binding to two T cell surface antigens: CD28 and CD152 (CTLA-4). The interaction with CD28 provides a costimulatory signal for T cell activation, while its interaction with CTLA-4 provides an inhibitory signal . This dual binding capability makes CD80 crucial in maintaining immune homeostasis through balancing activation and suppression signals.

Recent research has revealed that CD80 also interacts with PD-L1, forming cis-heterodimers on the same cell surface. This interaction can prevent PD-L1 from binding to PD-1 on T cells, thereby promoting T cell activation and enhancing anti-tumor immune responses . This complex network of interactions positions CD80 as a central regulator of adaptive immunity.

What types of CD80 antibodies are available for research purposes?

Research-grade CD80 antibodies are available in several formats to accommodate different experimental applications:

• Monoclonal antibodies: Clones such as 2D10.4 are widely used for their high specificity to human CD80 . These recognize specific epitopes and are useful for applications requiring consistent detection of particular CD80 domains.

• Polyclonal antibodies: These recognize multiple epitopes on CD80 (e.g., rabbit polyclonal antibodies) and can provide enhanced sensitivity, particularly in applications like Western blotting .

• Conjugated antibodies: These include:

  • Fluorochrome-conjugated antibodies (FITC, PE, APC, Alexa Fluor, Brilliant Violet) for flow cytometry

  • Biotin-conjugated antibodies for detection with streptavidin systems

  • Enzyme-linked antibodies for colorimetric applications

• Functional grade antibodies: These are specially purified to remove endotoxins and other contaminants (containing <0.001 ng/μg endotoxin) for use in functional assays where cell viability and physiological responses are essential .

The choice of antibody depends on the specific application, with considerations for species reactivity, application compatibility, and detection strategy.

How does CD80 differ from CD86 in function and expression patterns?

CD80 and CD86 (B7-2) are both ligands for CD28 and CTLA-4, but they exhibit important differences in expression, binding properties, and function:

CD80 is rapidly induced on activated B cells, Epstein-Barr Virus (EBV) transformed B cell lines, Burkitt's lymphoma cell lines, freshly isolated follicular B lymphoma cells, T cells, and monocytes . Both CD80 and CD86 are essential for T cell activation and can substitute for each other in this process, but their different expression patterns and binding affinities suggest specialized roles in orchestrating immune responses .

How should CD80 antibodies be validated for specificity and functionality?

Thorough validation of CD80 antibodies is essential for reliable research outcomes. A comprehensive validation approach should include:

• Positive and negative controls:

  • Use cell lines with known CD80 expression (activated B cells, dendritic cells as positive controls; resting T cells as negative controls)

  • Include CD80 knockout or knockdown cells when available

  • Test CD80-transfected cells versus non-transfected parent cells

• Cross-reactivity testing:

  • Test against related proteins (particularly CD86, which shares structural similarities)

  • Validate across relevant species if cross-reactivity is claimed

• Functional validation:

  • Blocking assays: Confirm that the antibody blocks CD80-CD28 or CD80-CTLA-4 interactions in mixed lymphocyte reactions

  • T cell activation assays: Verify that the antibody affects T cell proliferation or cytokine production in co-culture experiments

• Application-specific validation:

  • For flow cytometry: Compare with isotype controls and determine the optimal titration (typically ≤1 μg per test)

  • For Western blot: Confirm band size (approximately 60 kDa for glycosylated form, 33 kDa for non-glycosylated)

  • For immunohistochemistry: Compare with known expression patterns in lymphoid tissues

Whenever possible, use multiple antibody clones targeting different epitopes to confirm results, particularly for novel findings or contradictory observations.

What are the optimal conditions for using CD80 antibodies in flow cytometry?

For optimal flow cytometry results when studying CD80 expression:

• Sample preparation:

  • Use freshly isolated cells when possible, particularly for primary immune cells

  • For human peripheral blood mononuclear cells, activate B cells (with CD40L, IL-4, or LPS) to upregulate CD80

  • Use proper cell concentration (typically 1×10^5 to 1×10^8 cells per test in 100 μL final volume)

• Staining protocol:

  • Block Fc receptors to reduce non-specific binding

  • Titrate antibody carefully (≤1 μg per test is typically recommended)

  • Include compensation controls when using multiple fluorochromes

  • Add viability dye to exclude dead cells, which can increase background staining

• Controls:

  • Include isotype control antibodies matched for species, isotype, and fluorochrome

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

  • Include positive controls (activated B cells or dendritic cells)

• Analysis considerations:

  • Gate on live, single cells before analyzing CD80 expression

  • Consider CD80 expression level (median fluorescence intensity) rather than just percent positive

  • Co-stain with lineage markers (CD19 for B cells, CD11c for dendritic cells) to identify specific CD80+ cell populations

For specific clones such as 2D10.4, the antibody has been tested by flow cytometric analysis of normal human peripheral blood cells and shows reliable detection of CD80+ populations .

How can researchers troubleshoot inconsistent CD80 antibody staining in flow cytometry?

When facing inconsistent CD80 antibody staining in flow cytometry experiments, consider these systematic troubleshooting approaches:

• Sample-related issues:

  • CD80 expression varies with cell activation status—ensure consistent activation protocols

  • Cell viability affects staining—include viability dye and gate on live cells only

  • Sample age and storage conditions can impact results—use freshly prepared samples when possible

• Antibody-related factors:

  • Antibody concentration: Perform titration to determine optimal concentration for your specific cells

  • Clone selection: Different clones recognize different epitopes—some may be affected by glycosylation or conformational changes

  • Conjugate deterioration: Check fluorochrome integrity with rainbow beads and store antibodies according to manufacturer recommendations

• Protocol adjustments:

  • Fixation sensitivity: Some epitopes are fixation-sensitive—test different fixation methods or use unfixed cells

  • Blocking conditions: Insufficient blocking leads to high background—optimize blocking reagent and duration

  • Washing stringency: Inadequate washing causes non-specific binding—increase wash volume and number of washes

• Instrument considerations:

  • PMT voltage optimization: Set voltages to place negative populations in the first decade

  • Detector sensitivity: Use more sensitive detectors for dim signals

  • Daily quality control: Run calibration beads to ensure consistent instrument performance

When studying CD80 on primary cells, stimulation time is critical as peak expression occurs at different timepoints depending on cell type—typically 24-48 hours for B cells and 48-72 hours for dendritic cells . Document all protocol variables meticulously to ensure reproducibility across experiments.

How does CD80 contribute to cancer immune evasion mechanisms?

CD80 plays multifaceted roles in tumor immunity that vary by cancer type and microenvironment:

These findings highlight the context-dependent role of CD80 in cancer and suggest that CD80-targeting strategies must be tailored to specific tumor types and microenvironments.

How can CD80 antibodies be utilized in cancer immunotherapy research?

CD80 antibodies offer versatile applications in cancer immunotherapy research:

• Targeting approaches:

  • Blocking antibodies: Can be used to modulate CD80 interactions with CD28, CTLA-4, or PD-L1

  • Anti-CD80 immunotoxins: Constructs containing anti-CD80 monoclonal antibodies and toxic components (e.g., saponin) have shown strong cytotoxicity against CD80+ B cell lines like Raji cells, Reed-Sternberg cells, and CD80-transfected epithelial cell lines

• Combination therapy research:

  • CD80 antibodies can be combined with PD-1/PD-L1 inhibitors to potentially enhance T cell responses

  • Integration with CAR-T therapy: CD80 antibodies may enhance CAR-T efficacy by modulating the immunosuppressive microenvironment

• Biomarker applications:

  • Monitoring CD80 expression in tumors before and after treatment

  • Using CD80 expression patterns to stratify patients for immunotherapy trials

  • Tracking CD80+ immune cell populations in peripheral blood during immunotherapy

• Mechanistic studies:

  • Investigating the impact of modulating specific CD80 interactions on anti-tumor immunity

  • Studying how CD80 expression in different tumor compartments affects immune cell infiltration and function

Research in glioblastoma has shown that low CD80 expression in tumor stem cells may inhibit activation of the CD28 molecule on T cells, suggesting a mechanism for immune evasion . Similarly, in pancreatic cancer, TGF-β treatment upregulates CD80 expression, which is required for migration and invasion of tumor cells . These findings highlight the potential of CD80-targeted approaches in addressing tumor-specific immune evasion mechanisms.

What is the role of CD80 in autoimmune diseases and how can CD80 antibodies aid in their study?

CD80 plays significant roles in various autoimmune conditions, offering opportunities for therapeutic intervention and research:

• Multiple Sclerosis (MS):

  • CD80+ lymphocytes increase significantly during MS exacerbation

  • Following IFN-β treatment, CD80+ lymphocyte numbers decrease substantially, suggesting CD80+ cells may serve as indicators of treatment efficacy

  • CD80+ B cells have been identified as potential therapeutic targets for both HTLV-1-related myelopathy/tropical spastic paraparesis and multiple sclerosis

• Glomerular Diseases:

  • CD80 is expressed on antigen-presenting cells in patients with Minimal Change Nephropathy

  • Increased urinary CD80 correlates with frequent recurrence of Minimal Change Nephropathy

  • CD80 inhibitors (abatacept) show promising results in treatment, supporting CD80 as a therapeutic target

• Systemic Lupus Erythematosus (SLE):

  • Elevated CD80 expression contributes to T cell hyperactivation and autoantibody production

  • CD80 upregulation is associated with disease activity and tissue damage

CD80 antibodies facilitate autoimmune disease research through:

• Mechanistic studies:

  • Flow cytometric analysis of CD80+ cell populations in patient samples compared to healthy controls

  • Assessment of CD80 blockade effects on T cell activation in ex vivo assays

  • Investigation of CD80-dependent pathways in animal models of autoimmunity

• Biomarker development:

  • Monitoring CD80+ cells in peripheral blood as disease activity markers

  • Assessing urinary CD80 levels in kidney diseases

  • Correlating CD80 expression patterns with clinical outcomes

• Therapeutic development:

  • Preclinical testing of CD80-targeting approaches in animal models

  • Investigation of combination therapies targeting multiple costimulatory pathways

  • Development of CD80 modulation strategies with reduced systemic immunosuppression

The therapeutic efficacy of CD80 pathway inhibition in autoimmune conditions underscores its central role in immune dysregulation and its potential as a target for precision medicine approaches in autoimmunity.

How do post-translational modifications affect CD80 function and antibody recognition?

CD80 undergoes several post-translational modifications that significantly impact its function and detection:

• Glycosylation:

  • CD80 contains multiple N-linked glycosylation sites that increase its apparent molecular weight from the predicted 33 kDa to the observed 60 kDa

  • Glycosylation patterns affect protein stability, half-life, and binding properties

  • Different glycoforms may present different epitopes, affecting antibody recognition

  • Tissue-specific glycosylation can result in variable antibody binding efficiency across sample types

• Phosphorylation:

  • The cytoplasmic domain of CD80 contains potential phosphorylation sites

  • Phosphorylation status can affect subcellular localization and signaling

  • Activated immune cells may exhibit different phosphorylation patterns than resting cells

• Implications for antibody-based studies:

  • Western blot analysis may reveal multiple bands representing different glycoforms

  • Treatment with glycosidases may be necessary to confirm CD80 identity in complex samples

  • Fixation methods for flow cytometry or immunohistochemistry may differentially affect epitope exposure

• Experimental considerations:

  • Expression systems matter: bacterial systems lack glycosylation machinery, while mammalian systems provide more physiologically relevant modifications

  • Cell activation state influences post-translational modification patterns

  • Sample preparation methods may alter modifications and affect antibody binding

When selecting CD80 antibodies for specific applications, researchers should consider which epitopes are targeted and whether these epitopes might be affected by post-translational modifications in their experimental system. Testing multiple antibody clones that recognize different domains can help overcome detection issues related to post-translational modifications.

What methodological approaches can resolve contradictory findings in CD80 research?

Contradictory findings in CD80 research often stem from methodological differences and biological complexity. Systematic approaches to resolve these contradictions include:

• Standardization of experimental systems:

  • Cell types: Results from different cell types (primary cells vs. cell lines vs. transfected cells) may not be directly comparable

  • Species considerations: Human and mouse CD80 have structural and functional differences

  • Activation states: CD80 expression and function differ dramatically between resting and activated cells

• Context-specific analysis:

  • Tumor microenvironment: CD80 function in tumors may differ from its role in normal tissues

  • Disease context: CD80 may have opposing roles in different diseases

  • Cell-specific effects: CD80 on dendritic cells vs. B cells vs. tumor cells may have distinct functions

• Contradictory tumor prognosis associations:

  • In lung adenocarcinoma, elevated CD80 improves patient prognosis

  • In breast cancer and squamous cell carcinoma, CD80 overexpression correlates with poor prognosis

  • Resolution approach: Stratify analyses by cancer subtype, stage, and molecular features

• Methodological standardization:

  • Use multiple antibody clones and detection methods to confirm findings

  • Implement genetic approaches (CRISPR knockout/knockin) to validate antibody specificity

  • Apply dose-response studies to identify threshold effects

• Enhanced reporting standards:

  • Document antibody clones, concentrations, and incubation conditions in detail

  • Clearly describe cell activation status and culture conditions

  • Include comprehensive controls and statistical analyses

For example, the contradictory findings regarding CD80's role in different cancer types suggest context-dependent functions rather than truly contradictory mechanisms. In some cancers, CD80's interaction with CD28 predominates, enhancing anti-tumor immunity, while in others, its interaction with CTLA-4 or effects on tumor cell properties may be more significant .

How can researchers optimize CD80 antibody-based assays for detection of low expression?

Detecting low levels of CD80 expression requires specialized approaches to enhance sensitivity without sacrificing specificity:

• Flow cytometry optimization:

  • Signal enhancement:

    • Use high-brightness fluorochromes (PE, APC, Brilliant Violet dyes)

    • Implement indirect staining with primary and secondary antibodies for signal amplification

    • Choose antibody clones with optimal affinity (e.g., 2D10.4)

  • Instrument settings:

    • Optimize PMT voltages for maximum resolution of dim populations

    • Use instruments with high sensitivity detectors

  • Analysis approaches:

    • Employ fluorescence-minus-one controls for accurate gating

    • Consider median fluorescence intensity rather than percent positive

    • Use biexponential scaling for better visualization of dim signals

• Sample preparation enhancements:

  • Enrichment techniques:

    • Magnetic separation of target cell populations to increase frequency

    • Density gradient centrifugation to remove irrelevant cells

  • Ex vivo stimulation:

    • Short-term culture with CD40L, IL-4, or LPS to upregulate CD80 on B cells

    • Treatment with GM-CSF and IL-4 for dendritic cells

• Western blot sensitivity improvements:

  • Protein concentration methods:

    • Immunoprecipitation before Western blotting

    • TCA precipitation to concentrate proteins

  • Detection enhancements:

    • Highly sensitive ECL substrates

    • Longer exposure times with digital imaging systems

• Immunohistochemistry enhancements:

  • Signal amplification:

    • Polymer-based detection systems

    • Tyramide signal amplification

    • Extended primary antibody incubation (overnight at 4°C)

  • Background reduction:

    • Extensive blocking with appropriate sera

    • Optimization of antibody concentration through careful titration

    • Including detergents in wash buffers to reduce non-specific binding

When studying rare cell populations or samples with low CD80 expression, combining multiple optimization approaches provides the best strategy for reliable detection while maintaining specificity.

What are the considerations for developing CD80-targeted therapeutics based on antibody research?

Translating CD80 antibody research into therapeutic development requires careful consideration of several factors:

• Target specificity and biology:

  • CD80 interactions with multiple binding partners (CD28, CTLA-4, PD-L1) create complex signaling networks

  • Therapeutic antibodies must be designed with specific blocking or agonistic properties

  • Disease-specific expression patterns must inform targeting strategies (e.g., CD80 is upregulated in B cell lymphomas and certain solid tumors)

• Epitope selection:

  • Antibodies targeting different CD80 domains can produce distinct functional outcomes

  • Epitopes at CD28/CTLA-4 binding interfaces may block both stimulatory and inhibitory signals

  • Epitopes distant from binding interfaces can be used for diagnostic or cell-depleting strategies

• Format considerations:

  • Full IgG antibodies provide extended half-life but limited tissue penetration

  • Fab and scFv fragments offer better tissue penetration but shorter half-life

  • Bispecific formats can engage CD80 and another target simultaneously

  • Antibody-drug conjugates can deliver cytotoxic payloads to CD80+ cells

• Preclinical testing:

  • In vitro functional assays must assess impact on:

    • T cell activation and cytokine production

    • Cancer cell killing by immune effector cells

    • Regulatory T cell function

  • Animal models should recapitulate human CD80 biology

  • Toxicity assessment must consider expression on normal immune cells

• Biomarker development:

  • CD80 expression levels may predict response to CD80-targeted therapy

  • Monitoring CD80+ cell populations during treatment can provide pharmacodynamic information

  • Soluble CD80 levels in serum or other fluids may serve as accessible biomarkers

Anti-CD80 immunotoxins containing monoclonal antibodies and toxic components (e.g., saponin) have demonstrated strong cytotoxicity against CD80+ cell lines including B-cell lymphoma Raji cells and CD80-transfected epithelial cell lines . The efficacy of CD80 inhibitors (abatacept) in treating certain autoimmune conditions further supports the therapeutic potential of CD80-targeted approaches .