TNFRSF4 Antibody

What is TNFRSF4 and why is it significant in immunological research?

TNFRSF4 (also known as OX40 or CD134) is a type I transmembrane glycoprotein of approximately 50 kDa that is expressed primarily on activated T cells . It functions as a co-stimulatory molecule within the tumor necrosis factor receptor superfamily.

The significance of TNFRSF4 stems from its critical role in:

• T cell activation and proliferation

• Generation of memory T cells

• Regulation of T cell-mediated immune responses

• Potential applications as an immunotherapeutic target

Recent research has highlighted TNFRSF4 as a promising biomarker in various cancers, particularly in relation to tumor microenvironment (TME) assessment and prognosis prediction .

What are the primary applications of TNFRSF4 antibodies in research settings?

TNFRSF4 antibodies serve multiple research applications, each requiring specific considerations:

The selection should be guided by the specific research question, sample type, and anticipated downstream analyses.

How should researchers validate TNFRSF4 antibody specificity for their experimental systems?

Validation of TNFRSF4 antibody specificity is crucial for experimental reliability. A comprehensive validation approach should include:

• Positive and negative controls:

  • Positive: PHA-stimulated human T cells or regulatory T cells (Tregs), which show upregulated TNFRSF4 expression

  • Negative: Unstimulated PBMCs or irrelevant cell types

• Transfected cell line testing:

  • Compare HEK293 cells transfected with human TNFRSF4 against irrelevant transfectants

  • Expected result: Strong signal in TNFRSF4-transfected cells, minimal background in control transfectants

• Antibody isotype controls:

  • Use appropriate isotype controls (e.g., IgG1 kappa for Ber-ACT35 clone)

• Cross-reactivity assessment:

  • Test against intended species (Human, Non-Human Primate) and verify specificity

  • Consider epitope conservation when transitioning between model systems

• Multiple detection methods:

  • Confirm target detection using at least two independent techniques (e.g., flow cytometry and Western blot)

Proper validation ensures confidence in observed results and minimizes experimental artifacts.

How can TNFRSF4 antibodies be effectively used for studying tumor microenvironment dynamics?

TNFRSF4 antibodies have emerged as valuable tools for characterizing tumor microenvironment (TME) components, particularly immune infiltrates. Methodological approaches include:

• Multiplex immunohistochemistry (m-IHC):

  • Co-stain TNFRSF4 with other immune markers such as CD4, CD8, and FOXP3

  • This enables simultaneous visualization of TNFRSF4 expression in relation to specific T cell subsets within the TME

  • Protocol optimization typically requires antibody titration and sequential staining approaches

• Flow cytometric analysis of tumor-infiltrating lymphocytes (TILs):

  • Utilize PE, FITC, or APC-conjugated TNFRSF4 antibodies in multi-parameter panels

  • Include markers for T cell subsets (CD3, CD4, CD8), activation status, and exhaustion

  • This approach allows quantification of TNFRSF4+ immune cells within the TME

• Correlation with clinical outcomes:

  • Statistical methods such as Kaplan-Meier analysis can assess TNFRSF4 expression in relation to disease-specific survival (DSS)

  • Research suggests that elevated TNFRSF4 expression may signify favorable clinical outcomes in certain cancers like endometrial cancer

• Deconvolution algorithms:

  • Computational approaches like CIBERSORT can be applied to estimate the abundance of tumor-infiltrating immune cells (TICs) and their relationship with TNFRSF4 expression

These methods collectively provide insights into how TNFRSF4 contributes to immune surveillance and anti-tumor responses.

What methodological considerations are important when using TNFRSF4 antibodies for flow cytometry?

Flow cytometric analysis using TNFRSF4 antibodies requires careful attention to several key methodological aspects:

• Sample preparation optimization:

  • For primary human T cells: Stimulation with PHA (1 μg/mL for 3-5 days) significantly increases TNFRSF4 expression, improving detection sensitivity

  • For PBMCs: Pre-gating on CD3+ cells helps identify T cell populations of interest

• Antibody selection and panel design:

  • Choose fluorochrome conjugates based on instrument configuration and panel complexity

  • PE conjugates (like ABIN2749174) typically offer high signal-to-noise ratio for TNFRSF4 detection

  • Consider brightness hierarchy when designing multicolor panels

• Protocol optimization:

  • Titrate antibodies to determine optimal concentration

  • Include appropriate compensation controls

  • Use viability dyes to exclude dead cells, which can bind antibodies non-specifically

• Gating strategy recommendations:

  • Set quadrant markers based on isotype control staining

  • For transfected cell lines, include GFP co-expression for identifying positively transfected populations

  • When analyzing tumor samples, include lineage markers to distinguish T cell subsets

• Data interpretation considerations:

  • Compare expression levels between activated and non-activated T cells

  • Assess co-expression with other activation markers

  • Consider both percentage of positive cells and mean fluorescence intensity

Following these guidelines ensures robust and reproducible flow cytometry results when studying TNFRSF4 expression patterns.

How do different TNFRSF4 antibody clones compare in terms of epitope recognition and functional applications?

Different TNFRSF4 antibody clones recognize distinct epitopes and demonstrate varying functional characteristics that affect their research applications:

Key considerations for clone selection:

• Epitope accessibility:

  • Certain epitopes may be masked in fixed tissues or denatured samples

  • Conformation-dependent epitopes may be lost in Western blotting

• Functional requirements:

  • For neutralization studies, specifically validated clones like 977974 are preferred

  • The neutralization potency (ND50) varies between clones and should be considered for experimental design

• Cross-reactivity needs:

  • When working with non-human primates, Ber-ACT35 provides consistent results

  • For studies requiring broader species coverage, OX40-3108 offers advantages

Understanding these differences enables researchers to select the most appropriate clone for specific experimental requirements.

What is the significance of TNFRSF4 as a prognostic biomarker, and how can researchers effectively evaluate its expression in clinical specimens?

TNFRSF4 has emerged as a potential prognostic biomarker in several cancers, particularly endometrial cancer. To effectively evaluate its expression in clinical specimens:

• Tissue microarray (TMA) analysis approaches:

  • Use standardized IHC protocols with validated antibodies

  • Implement quantitative scoring systems based on staining intensity and percentage of positive cells

  • Consider automated image analysis for objective quantification

• Correlation with clinicopathological features:

  • Analyze TNFRSF4 expression in relation to tumor stage, grade, histological subtype, and molecular classification

  • Recent research indicates that elevated TNFRSF4 expression tends to signify favorable clinical outcomes in endometrial cancer

• Prognostic assessment methodology:

  • Apply disease-specific survival (DSS) analysis, defined as time between surgery and cancer-related death

  • Utilize Kaplan-Meier analysis with log-rank tests to evaluate prognostic impact

  • Consider multivariate Cox regression to adjust for confounding factors

• Integration with immune marker profiles:

  • Evaluate TNFRSF4 in conjunction with CD4, CD8, and FOXP3

  • Calculate correlation coefficients (Spearman or Pearson) between TNFRSF4 and immune markers

  • This approach provides insights into the immunological context of TNFRSF4 expression

• Meta-analysis of expression data:

  • Integrate findings across multiple datasets using forest plot visualization

  • Apply inclusion criteria: (1) confirmed diagnosis, (2) tumor and normal control samples, (3) available TNFRSF4 expression data

These methodological approaches enable robust evaluation of TNFRSF4 as a prognostic biomarker in clinical research contexts.

How can researchers design effective neutralization assays using anti-TNFRSF4 antibodies?

Neutralization assays are crucial for evaluating the functional activity of TNFRSF4-targeting therapeutic approaches. A methodological framework includes:

• Cell line selection and preparation:

  • Use HT1080 human fibrosarcoma cells transfected with human TNFRSF4

  • Maintain stable expression through appropriate selection markers

  • Include wild-type HT1080 cells as negative controls

• Stimulation protocol:

  • Recombinant Human OX40L/TNFSF4 (10-20 ng/mL) can be used to stimulate CXCL8/IL-8 secretion in a dose-dependent manner

  • Optimize stimulation time (typically 24-48 hours) for maximum cytokine production

• Neutralization assay setup:

  • Pre-incubate cells with increasing concentrations of anti-TNFRSF4 antibody

  • Add recombinant OX40L/TNFSF4 at the optimized concentration

  • Include appropriate controls:

    • Isotype control antibody

    • Unstimulated cells

    • Stimulated cells without neutralizing antibody

• Readout measurements:

  • Quantify CXCL8/IL-8 using ELISA (e.g., Human CXCL8/IL-8 Quantikine ELISA Kit)

  • Calculate neutralization dose (ND50) - the antibody concentration that inhibits 50% of cytokine production

  • For reference, clone 977974 has an ND50 of 0.6-3.0 μg/mL , while polyclonal antibodies may show ND50 of 0.025-0.16 μg/mL

• Data analysis:

  • Generate dose-response curves using non-linear regression

  • Compare neutralization efficacy between different antibody clones

  • Assess correlation between epitope binding and neutralization capacity

This framework allows systematic evaluation of antibody-mediated TNFRSF4 neutralization, which is particularly relevant for therapeutic development.

What are the most common technical challenges when working with TNFRSF4 antibodies and how can they be addressed?

Researchers frequently encounter specific technical challenges when working with TNFRSF4 antibodies. Here are methodological solutions:

• Low signal intensity in flow cytometry:

  • Cause: Insufficient expression of TNFRSF4 on resting T cells

  • Solution: Activate T cells with PHA (1 μg/mL for 3-5 days) or CD3/CD28 stimulation to upregulate TNFRSF4 expression

  • Alternative: Use brighter fluorochromes (PE or APC) for detection of low-level expression

• High background in immunohistochemistry:

  • Cause: Non-specific binding or endogenous peroxidase activity

  • Solution: Optimize blocking conditions (5-10% serum from the same species as secondary antibody)

  • Alternative: Use more specific detection systems like VisUCyte™ HRP Polymer detection reagents

• Multiple bands in Western blot:

  • Cause: Post-translational modifications, proteolytic cleavage, or non-specific binding

  • Solution: Use reducing conditions and Immunoblot Buffer Group 1 for consistent detection of the ~45-50 kDa band

  • Validation: Compare band patterns in activated T cells or Tregs versus control cells

• Cross-reactivity in multi-species studies:

  • Cause: Epitope differences between species

  • Solution: Select clones verified for cross-reactivity (e.g., OX40-3108 for human and chimpanzee)

  • Validation: Test antibody specificity in each species before comparative studies

• Inconsistent neutralization results:

  • Cause: Variability in recombinant protein activity or cell responsiveness

  • Solution: Establish dose-response curves for each new lot of recombinant OX40L/TNFSF4

  • Control: Include a standard neutralizing antibody with known ND50 as reference

These troubleshooting approaches enhance experimental reliability when working with TNFRSF4 antibodies across different applications.

How can researchers optimize multiplex immunohistochemistry protocols that include TNFRSF4 antibodies?

Multiplex immunohistochemistry (m-IHC) with TNFRSF4 antibodies requires careful optimization for successful simultaneous detection of multiple markers. Key methodological considerations include:

• Panel design strategy:

  • Combine TNFRSF4 with functionally relevant markers (CD4, CD8, FOXP3) for comprehensive T cell phenotyping

  • Consider antibody species origin to minimize cross-reactivity (e.g., use antibodies raised in different species)

  • Select fluorophores with minimal spectral overlap for clear discrimination

• Sample preparation optimization:

  • For FFPE tissues: Antigen retrieval conditions critically affect TNFRSF4 detection

  • Test different retrieval methods (citrate pH 6.0 vs. EDTA pH 9.0)

  • Optimize fixation protocols to preserve epitope integrity

• Sequential staining approach:

  • Order antibodies from lowest to highest abundance target

  • Consider placing TNFRSF4 early in the sequence if expression levels are low

  • Include thorough washing steps between antibodies to prevent cross-reactivity

• Signal amplification methods:

  • For low-abundance TNFRSF4: Implement tyramide signal amplification (TSA)

  • Carefully titrate primary antibodies to determine optimal concentration

  • Include single-color controls to assess bleed-through and optimize exposure settings

• Validation and controls:

  • Use serial sections with single-marker IHC as reference

  • Include biological controls (tonsil tissue shows reliable TNFRSF4 expression)

  • Implement computational analysis for objective quantification of co-localization

• Recommended antibody combinations:

  • TNFRSF4 (clone Ber-ACT35) + CD4 + CD8 + FOXP3 for T cell subset analysis

  • TNFRSF4 + proliferation markers (Ki67) + activation markers for functional assessment

This optimization framework enables robust m-IHC protocols that can reveal the spatial and functional relationships between TNFRSF4+ cells and other immune components within tissues.