TNFSF18 Antibody

What is TNFSF18 and what are its key biological functions?

TNFSF18, also known as GITRL (Glucocorticoid-Induced TNF-Related Ligand), is a cytokine belonging to the tumor necrosis factor (TNF) ligand superfamily. It functions primarily as a regulator of T-cell responses by binding to its receptor TNFRSF18/AITR/GITR . At the molecular level, TNFSF18 serves as a costimulator that lowers the threshold for T-cell activation and proliferation, making it a critical mediator in immune response regulation .

The protein plays several important roles in cellular signaling, including:

• Mediating activation of NF-kappa-B

• Triggering increased phosphorylation of STAT1

• Up-regulating expression of adhesion molecules VCAM1 and ICAM1

• Promoting leukocyte adhesion to endothelial cells

• Regulating monocyte migration from splenic reservoirs to inflammation sites

These functions make TNFSF18 a promising target for research into cancer immunotherapy, autoimmune disorders, and inflammatory conditions .

What is the discrepancy between calculated and observed molecular weights of TNFSF18?

Research consistently shows a notable discrepancy between the calculated and observed molecular weights of TNFSF18. The calculated molecular weight based on the amino acid sequence is approximately 20 kDa (177 amino acids) , while the observed molecular weight in experimental settings is typically around 44 kDa . This difference can be attributed to post-translational modifications, particularly glycosylation, which significantly increases the apparent molecular weight of the protein on SDS-PAGE gels.

When troubleshooting this discrepancy in experimental work, researchers should note:

• The 44 kDa band is the correct target in Western blot applications

• Variations in glycosylation patterns may occur between different cell types

• Deglycosylation experiments can confirm antibody specificity if needed

What are the common applications for TNFSF18 antibodies in research?

TNFSF18 antibodies are versatile research tools validated for multiple laboratory applications:

The antibody selection should be guided by specific experimental requirements, with special attention to the host species (typically rabbit) and isotype (IgG) for compatibility with secondary detection systems .

How should researchers optimize TNFSF18 antibody use for flow cytometry applications?

For flow cytometric analysis of TNFSF18 expression, researchers should implement several key methodological approaches:

First, consider cellular activation status - TNFSF18 expression is significantly upregulated upon activation. As demonstrated with HUVEC cells, treatment with 1μg/ml lipopolysaccharide for 24 hours substantially increases TNFSF18 detection by flow cytometry . This principle applies to various cell types, with expression patterns differing between resting and activated states.

For optimal staining protocols:

• Use fresh or properly preserved single-cell suspensions

• Implement a 1:500 dilution for rabbit monoclonal antibodies such as clone EPR23451-119

• Include proper compensation controls when multiplexing

• Consider fixation effects on epitope recognition

• Include both stimulated and unstimulated controls to establish baseline expression

• Validate surface versus intracellular staining protocols based on research questions

Expression analysis should account for cell-specific differences, as TNFSF18 shows variable expression patterns across immune and non-immune cell types .

What are the optimal conditions for using TNFSF18 antibodies in Western blot applications?

Achieving optimal results with TNFSF18 antibodies in Western blot requires attention to several critical parameters:

The observed molecular weight of TNFSF18 (44 kDa) differs substantially from the calculated weight (20 kDa), requiring appropriate positive controls for band validation . When performing Western blots:

• Sample preparation: Complete lysis buffers containing phosphatase and protease inhibitors are essential for preserving protein integrity

• Gel percentage: 10-12% SDS-PAGE gels provide optimal resolution for the 44 kDa band

• Transfer conditions: Semi-dry or wet transfer methods are both suitable, with optimization for higher molecular weight proteins

• Blocking: 5% non-fat milk or BSA in TBST (depending on antibody specifications)

• Primary antibody dilution: Start with 1:1000 dilution and adjust based on signal intensity

• Incubation time: Overnight at 4°C generally yields better specificity than shorter incubations

• Positive controls: Rat skeletal muscle lysate or SGC-7901 cells provide reliable positive controls

For troubleshooting poor signals, consider that glycosylation patterns may vary between tissues and cell lines, potentially affecting antibody recognition. Extended exposure times may be necessary for samples with low expression levels.

How can researchers effectively use TNFSF18 antibodies to investigate cancer immunotherapy mechanisms?

TNFSF18 antibodies serve as valuable tools for investigating cancer immunotherapy mechanisms, particularly those involving T-cell activation and tumor microenvironment interactions. When designing experiments in this field, researchers should consider:

TNFSF18's role as a potential therapeutic target stems from its ability to promote T-cell activation and proliferation, making it relevant for cancer immunotherapy research . Studies comparing GITR ligand fusion proteins (GITRL-FP) with anti-GITR antibodies have demonstrated the potential of targeting this pathway for therapeutic development .

Methodological approaches should include:

• Functional assays measuring T-cell activation via cytokine production

• Flow cytometric analysis of TNFSF18 expression on tumor-infiltrating lymphocytes

• Analysis of GITR receptor-ligand interactions using recombinant proteins

• Assessment of dose-response relationships with TNFSF18-targeting biologics

• Investigation of combination therapies with checkpoint inhibitors targeting PD-1, PD-L1, or CTLA-4

When evaluating TNFSF18-targeting approaches, researchers should assess EC50 values in cell-based reporter assays to determine agonistic potency, as was demonstrated with mGITRL-FP compared to anti-GITR antibodies . Additionally, tumor models such as CT26 and B16F10-Luc2 provide valuable platforms for in vivo evaluation of anti-tumor activities.

What are the optimal antigen retrieval methods for TNFSF18 immunohistochemistry?

Successful immunohistochemical detection of TNFSF18 requires careful optimization of antigen retrieval techniques. Based on validated protocols, researchers should implement:

Primary recommendation: Tris-EDTA (TE) buffer at pH 9.0 for heat-induced epitope retrieval (HIER) . This alkaline buffer effectively exposes TNFSF18 epitopes that may be masked during fixation processes.

Alternative approach: Citrate buffer at pH 6.0 can serve as an alternative when TE buffer optimization proves challenging .

Methodology considerations include:

• Tissue fixation: Formalin-fixed, paraffin-embedded (FFPE) tissues require consistent fixation times (12-24 hours)

• Section thickness: 4-5 μm sections provide optimal results

• Heating method: Water bath, microwave, or pressure cooker (pressure cooker often yields most consistent results)

• Heating time: 20 minutes at full retrieval temperature

• Cooling period: Allow gradual cooling for 20-30 minutes before proceeding with immunostaining

• Antibody dilution: Begin with 1:50-1:100 for initial optimization, then adjust based on results

• Detection system: High-sensitivity polymer detection systems recommended for low-expressing samples

The antibody has been validated for detection of TNFSF18 in human thyroid cancer tissue and mouse testis tissue , which can serve as positive controls when establishing the protocol.

How do researchers distinguish between TNFSF18 and other TNF family members in experimental assays?

First, antibody selection is critical - use antibodies raised against recombinant fusion proteins containing specific sequences corresponding to amino acids 52-177 of human GITR Ligand/TNFSF18 (NP_005083.2) . This region contains unique sequences that differentiate TNFSF18 from other TNF superfamily members.

Experimental strategies should include:

• Sequential immunoprecipitation to assess cross-reactivity

• Competitive binding assays with recombinant proteins

• Knockout or knockdown validation in cell lines

• Inclusion of both positive and negative control tissues/cells

• Peptide blocking experiments to confirm specificity

When designing multiplex assays, researchers should consider:

• The amino acid sequence CSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPP is specific to TNFSF18

• Cross-reactivity testing is essential when detecting multiple TNF family members simultaneously

• Validation with recombinant protein standards at various concentrations

• Careful selection of non-overlapping fluorophores for fluorescence-based detection systems

For functional differentiation, TNFSF18's specific binding to TNFRSF18/AITR/GITR receptor provides a distinctive characteristic that can be leveraged in binding specificity assays .

What strategies can address inconsistent TNFSF18 antibody staining results?

Inconsistent staining results with TNFSF18 antibodies can arise from multiple sources. Implementing a systematic troubleshooting approach includes:

For Western blot applications:

• Ensure proper sample preparation with complete protease inhibitors

• Validate protein loading with housekeeping protein controls

• Confirm transfer efficiency with reversible staining methods

• Consider longer transfer times for the 44 kDa glycosylated form

• Test multiple antibody concentrations (1:200-1:5000 dilution range)

For immunohistochemistry:

• Compare antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

• Optimize antibody incubation time and temperature

• Validate with known positive controls (human thyroid cancer tissue or mouse testis tissue)

• Consider signal amplification systems for low-expressing samples

For flow cytometry:

• Confirm cell viability before staining

• Compare surface vs. intracellular staining protocols

• Test staining after cellular activation (e.g., LPS treatment for 24 hours)

• Optimize fixation conditions that preserve epitope recognition

Batch-to-batch variation in antibody production may also contribute to inconsistency. Researchers should document lot numbers and validate each new lot against previous results with established positive controls.

How can researchers validate the specificity of TNFSF18 antibodies in their experimental system?

Validating TNFSF18 antibody specificity requires multiple complementary approaches to ensure experimental rigor:

A comprehensive validation strategy should include:

• Positive and negative control samples:

  • Use rat skeletal muscle as a positive control for Western blot

  • SGC-7901 cells serve as validated positive controls for Western blot

  • Human thyroid cancer and mouse testis tissues provide reliable positive controls for IHC

• Peptide competition assays:

  • Pre-incubate antibody with recombinant TNFSF18 protein

  • Compare staining patterns with and without blocking peptide

  • Specific binding should be eliminated or significantly reduced

• Genetic validation approaches:

  • Test antibody on TNFSF18 knockout or knockdown samples

  • Overexpression systems can confirm specific band migration patterns

  • CRISPR-Cas9 modified cell lines provide stringent controls

• Cross-platform validation:

  • Confirm expression using multiple techniques (WB, IHC, IF, FC)

  • Correlate protein detection with mRNA expression data

  • Compare results from multiple antibodies targeting different epitopes

• Molecular weight confirmation:

  • Verify the 44 kDa observed molecular weight in Western blots

  • Understand that the calculated 20 kDa weight will not be observed due to glycosylation

How can TNFSF18 antibodies be utilized to study T-cell activation mechanisms?

TNFSF18 antibodies provide valuable tools for investigating T-cell activation mechanisms, offering insights into both basic immunology and therapeutic applications. Researchers should consider these methodological approaches:

For in vitro T-cell activation studies:

• Isolate primary T cells from peripheral blood or mouse splenocytes

• Compare TNFSF18 expression in naive versus activated T-cell populations

• Use flow cytometry with anti-TNFSF18 antibodies (1:500 dilution) to quantify surface expression during activation time course

• Correlate TNFSF18 expression with activation markers (CD25, CD69, etc.)

• Implement functional readouts like cytokine production (IL-2, IFN-γ) to assess T-cell function

For investigating the TNFSF18-GITR pathway:

• Compare the EC50 value of GITR ligand fusion proteins with anti-GITR antibodies in cell-based reporter assays

• Assess dose-response relationships in T-cell activation models

• Evaluate the impact of receptor-ligand interactions on downstream signaling events (NF-κB activation, STAT1 phosphorylation)

• Consider combination studies with other costimulatory or coinhibitory pathways

The ability of TNFSF18 to function as a costimulator that lowers the threshold for T-cell activation provides a foundation for investigating its role in enhancing immune responses, particularly in contexts where T-cell activation is therapeutically desirable, such as cancer immunotherapy .

What are the considerations when using TNFSF18 antibodies to investigate endothelial cell biology?

TNFSF18 plays important roles in endothelial cell biology, particularly in mediating interactions between activated T-lymphocytes and endothelial cells . When designing experiments in this area, researchers should consider:

Endothelial cell models:

• HUVEC (Human Umbilical Vein Endothelial Cells) provide a well-established model system

• Pre-treatment with 1μg/ml lipopolysaccharide for 24 hours significantly upregulates TNFSF18 expression for flow cytometric detection

• Consider comparison between different endothelial cell types (microvascular, arterial, etc.)

Functional assays:

• Adhesion assays measuring leukocyte attachment to endothelial monolayers

• Transendothelial migration studies to assess barrier function

• Expression analysis of adhesion molecules (VCAM1, ICAM1) induced by TNFSF18 signaling

Technical considerations:

• Flow cytometric analysis requires careful compensation when multiplexing

• Consider both surface and intracellular staining protocols

• Compare constitutive versus induced expression patterns

• Implement appropriate endothelial cell markers for co-staining

The role of TNFSF18 in promoting leukocyte adhesion to endothelial cells and its relationship with adhesion molecule expression (VCAM1, ICAM1) highlights its importance in inflammatory responses and vascular biology . These interactions represent potential targets for therapeutic intervention in inflammatory conditions.

How can TNFSF18 antibodies contribute to cancer immunotherapy research?

TNFSF18 antibodies enable critical investigations into cancer immunotherapy mechanisms, particularly through their ability to probe the GITR-GITRL pathway. When designing cancer immunotherapy experiments, researchers should consider:

Research approaches for TNFSF18 in cancer immunotherapy:

• Compare agonistic properties of various TNFSF18-targeting constructs using in vitro cell-based reporter assays

• Evaluate dose-dependent effects on tumor growth inhibition

• Assess the impact of Fc isotype on antitumor activity through comparative studies

• Investigate combination strategies with checkpoint inhibitors targeting PD-L1, PD-1, or CTLA-4

• Analyze tumor-infiltrating lymphocyte phenotypes following TNFSF18 pathway modulation

Experimental models:

• The CT26 tumor model provides a validated system for evaluating antitumor activity

• B16F10-Luc2 represents an alternative model for comparative studies

• Consider patient-derived xenograft models for translational relevance

The development of novel GITR ligand fusion proteins (GITRL-FP) designed to maximize valency demonstrates ongoing innovation in targeting this pathway for therapeutic benefit . Comparison studies between these fusion proteins and traditional antibody approaches offer insights into optimal targeting strategies.

What methodological approaches best leverage TNFSF18 antibodies for studying inflammatory conditions?

TNFSF18's role in inflammation makes TNFSF18 antibodies valuable tools for investigating inflammatory conditions. Researchers should implement these methodological approaches:

Tissue-specific analysis:

• Immunohistochemistry with optimized antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Compare expression patterns between healthy and inflamed tissues

• Consider multiplex staining to identify TNFSF18-expressing cell populations

• Correlation of protein expression with disease severity markers

Cellular models:

• Flow cytometric analysis of cells from inflammatory microenvironments

• In vitro systems modeling specific inflammatory contexts

• Assessment of TNFSF18 regulation in response to inflammatory mediators

Mechanistic investigations:

• Analysis of NF-κB activation downstream of TNFSF18 signaling

• Phosphorylation studies of STAT1 in response pathway activation

• Expression analysis of adhesion molecules (VCAM1, ICAM1) in relationship to TNFSF18 levels

The involvement of TNFSF18 in regulating monocyte migration from splenic reservoirs to inflammation sites offers potential therapeutic targets for inflammatory diseases . Understanding these mechanisms requires careful experimental design and appropriate utilization of TNFSF18 antibodies for detection and functional studies.