TNFRSF25 Antibody

What is TNFRSF25 and why is it significant in immunological research?

TNFRSF25, also known as Death Receptor 3 (DR3), is a member of the tumor necrosis factor receptor superfamily with significant immunological functions. The human canonical protein consists of 417 amino acid residues with a molecular mass of 45.4 kDa and exists in up to 12 different isoforms. It is predominantly expressed in thymocytes and lymphocytes, functioning as a receptor for TNFSF15 (TL1A) . TNFRSF25 has emerged as a critical player in allergic lung inflammation and asthma pathogenesis, making it a valuable research target . Its role in both promoting cell survival through NF-κB activation and inducing apoptosis through death domain signaling makes it particularly relevant for studying immune regulation mechanisms .

What are the common applications for TNFRSF25 antibodies in research?

TNFRSF25 antibodies are versatile tools employed across multiple immunological research applications. Most commonly, these antibodies are utilized in Western blot experiments to detect and quantify TNFRSF25 protein expression levels in various cell types . Additionally, they are frequently employed in flow cytometry for identifying TNFRSF25-expressing cells, particularly activated T cells where TNFRSF25 expression increases after activation . Immunohistochemistry applications allow for tissue localization studies, while ELISA techniques enable quantitative detection in complex samples. For functional studies, TNFRSF25 antibodies can be used to modulate receptor signaling, either blocking or activating the pathway to study downstream effects on cytokine production, particularly IL-17 and Th2 cytokines .

How can I validate the specificity of a TNFRSF25 antibody for my experiments?

Validating TNFRSF25 antibody specificity requires a multi-faceted approach. Begin with positive and negative control samples - TNFRSF25 is highly expressed in lymphoid tissues, particularly activated T cells, which serve as excellent positive controls, while non-lymphoid tissues typically show minimal expression . Perform a Western blot analysis looking for a specific band at approximately 45.4 kDa, corresponding to the canonical form, though be aware that alternative spliced variants may appear at different molecular weights .

For more rigorous validation, employ knockdown or knockout systems: compare antibody staining between wild-type samples and those with TNFRSF25 knockdown/knockout. Cross-reactivity testing is also crucial, especially when studying orthologs in different species, as TNFRSF25 has been reported in mouse, rat, bovine, frog, chimpanzee, and chicken species . Finally, epitope mapping can provide definitive evidence of specificity by identifying the exact binding site of the antibody on the TNFRSF25 protein.

What are the key considerations when selecting a TNFRSF25 antibody for flow cytometry?

When selecting a TNFRSF25 antibody for flow cytometry applications, researchers should consider several critical factors. First, epitope accessibility is paramount - choose antibodies targeting extracellular domains of TNFRSF25 for live cell applications, as these regions remain accessible without permeabilization . Confirm that the antibody has been specifically validated for flow cytometry, as not all antibodies that work in Western blot will perform adequately in flow applications .

The fluorophore conjugation should align with your experimental setup - consider brightness requirements and potential spectral overlap with other fluorophores in your panel. Cross-reactivity with the species under investigation must be verified; for mouse studies, ensure the antibody specifically recognizes mouse TNFRSF25 . Finally, consider the activation state of your cells - TNFRSF25 expression increases after T cell activation, so detection sensitivity may vary between resting and activated lymphocytes .

How can TNFRSF25 antibodies be utilized to investigate the relationship between TNFRSF25 signaling and cytokine production in T cells?

TNFRSF25 antibodies provide powerful tools for dissecting the complex relationship between TNFRSF25 signaling and cytokine production in T cells. For agonistic studies, researchers can use stimulatory TNFRSF25 antibodies (potentially cross-linked for enhanced signaling) to activate the receptor pathway in isolated CD4+ T cells. This approach has revealed that TNFRSF25 signals significantly enhance both Th2 and Th17 cytokine production . Flow cytometry with intracellular cytokine staining can then quantify IL-17, IL-13, and other cytokines at the single-cell level.

For antagonistic approaches, blocking TNFRSF25 antibodies can interrupt the TNFRSF25-TL1A interaction, mirroring the phenotype observed in dominant-negative TNFRSF25 transgenic models . Time-course experiments coupling TNFRSF25 antibody treatments with RNA-seq or cytokine profiling can elucidate the temporal dynamics of cytokine regulation. Importantly, these experiments should include appropriate controls, such as isotype antibodies and comparative analysis with TL1A knockout or TNFRSF25 transgenic models to validate antibody effects . This approach has revealed that TNFRSF25 not only regulates cytokine production but also enhances expression of its own ligand (TL1A) in CD4+ T cells, suggesting a positive feedback mechanism .

What are the most effective protocols for using TNFRSF25 antibodies in studying allergic inflammation models?

For studying allergic inflammation models using TNFRSF25 antibodies, researchers should implement multi-phase protocols that capture both preventive and therapeutic interventions. In preventive approaches, administer blocking TNFRSF25 antibodies (1-2 mg/kg) or anti-TL1A antibodies prior to allergen challenge in mouse models of asthma or allergic inflammation . For therapeutic assessment, introduce antibodies after established allergen sensitization, as research has shown efficacy even when administered days after airway antigen exposure .

Comprehensive readout methods should include: (1) Bronchoalveolar lavage fluid analysis for inflammatory cell infiltration, particularly eosinophils; (2) Lung histopathology with scoring for peribronchial/perivascular inflammation; (3) Cytokine profiling focused on Th2 cytokines (especially IL-13) and Th17 cytokines; (4) Flow cytometry of lung-infiltrating lymphocytes with TNFRSF25 antibodies to track receptor expression changes during inflammation .

For mechanistic studies, combine TNFRSF25 antibody treatment with adoptive transfer experiments using wild-type or dominant-negative TNFRSF25 transgenic T cells or NKT cells into recipient mice (particularly effective in NKT-deficient mice which are normally resistant to allergic lung inflammation) . This approach can definitively establish the cell-specific contributions of TNFRSF25 signaling to inflammatory processes.

How can TNFRSF25 antibodies be employed to investigate the differential roles of TNFRSF25 splice variants?

Investigating the differential roles of TNFRSF25 splice variants requires specialized antibody-based approaches. First, develop or source isoform-specific antibodies targeting unique epitopes present in particular splice variants, such as those absent in the Δ5,6 variant that lacks exons 5 and 6 . These can be validated using overexpression systems of individual isoforms.

Employ these isoform-specific antibodies in Western blot and immunoprecipitation experiments to quantify the relative expression patterns of different variants across tissues and activation states. The canonical form appears at 45.4 kDa, while other variants will show distinct molecular weights . For functional studies, use selective immunodepletion where isoform-specific antibodies remove particular variants from biological samples before functional assays.

Complementary approaches should include comparing cells from transgenic mice overexpressing specific variants (such as full-length TNFRSF25 versus the Δ5,6 variant) in cytokine production assays . Research has shown that while both full-length and Δ5,6 variants can increase IL-17 secretion compared to wild-type cells, the full-length form produces a more pronounced effect . This suggests functional differences between splice variants that can be further explored using selective antibody targeting combined with downstream signaling analysis (e.g., NF-κB activation, caspase recruitment).

What are the critical considerations for developing humanized anti-TNFRSF25 antibodies for potential therapeutic applications?

Developing humanized anti-TNFRSF25 antibodies for therapeutic purposes requires stringent consideration of multiple factors. Begin with epitope selection targeting functional domains that modulate TNFRSF25-TL1A interaction or downstream signaling. For humanization strategy, variable domain resurfacing approaches have proven successful with other TNF-family targeted antibodies .

During the humanization process, identify and preserve non-conserved surface residues in framework regions that might impact binding affinity. For example, when humanizing anti-TNF-α antibodies, researchers retained residues H63, H67, H82, H18, H27, H30, H72, H79 in the heavy chain and L2, V59 in the light chain to maintain antigen binding properties . Apply similar structural analysis to TNFRSF25-targeting antibodies.

Rigorous validation must include:

• Binding affinity comparisons between murine original and humanized versions using surface plasmon resonance

• Functional assays measuring the antibody's ability to block or stimulate TNFRSF25 signaling in human T cells

• Cross-reactivity assessment against other TNF receptor family members

• Stability testing under physiological conditions

Finally, evaluate therapeutic potential in humanized mouse models of allergic inflammation or autoimmunity, where TNFRSF25 signaling has been implicated as a key mediator .

What are the optimal conditions for using TNFRSF25 antibodies in Western blot applications?

For optimal Western blot detection of TNFRSF25, careful sample preparation and protocol optimization are essential. Begin with proper sample preparation: lyse cells or tissues in RIPA buffer supplemented with protease inhibitors, as TNFRSF25 can undergo proteolytic processing . For T cell samples, consider activation status, as TNFRSF25 expression increases after activation .

Gel electrophoresis parameters should accommodate the 45.4 kDa molecular weight of canonical TNFRSF25, though be prepared to identify multiple bands corresponding to the up to 12 different isoforms that have been reported . A 10-12% polyacrylamide gel typically provides optimal resolution. During transfer, PVDF membranes may offer better protein retention than nitrocellulose for this target.

For blocking and antibody incubation, a typical protocol includes:

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Incubate with primary anti-TNFRSF25 antibody (typically 1:500-1:2000 dilution) overnight at 4°C

• Wash 3-5 times with TBST

• Incubate with species-appropriate HRP-conjugated secondary antibody (typically 1:5000) for 1 hour at room temperature

• Develop using enhanced chemiluminescence

Include positive controls (activated lymphocytes or thymocytes) and consider using TNFRSF25 knockout/knockdown samples as negative controls for definitive band identification.

How can I optimize immunoprecipitation protocols using TNFRSF25 antibodies to study protein-protein interactions?

Optimizing immunoprecipitation (IP) protocols for TNFRSF25 requires careful consideration of cellular context and interaction dynamics. For studying TNFRSF25 protein interactions, begin with an appropriate lysis buffer that preserves protein-protein interactions - typically a non-denaturing buffer containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), and protease/phosphatase inhibitors.

Cross-linking may be necessary to capture transient interactions: treat cells with membrane-permeable crosslinkers like DSP (dithiobis[succinimidyl propionate]) before lysis to stabilize complexes involving TNFRSF25, particularly when studying interactions with adapter proteins like TRADD, FADD, or TRAF2 .

For the IP procedure:

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• Incubate cleared lysates with 2-5 μg of anti-TNFRSF25 antibody overnight at 4°C

• Add protein A/G beads and incubate for 2-4 hours

• Wash extensively (4-5 times) with lysis buffer containing reduced detergent concentration

• Elute bound proteins for downstream analysis

For detecting known interaction partners, probe Western blots with antibodies against TRADD, FADD, TRAF2, or caspases, which have been documented to interact with TNFRSF25 . IP followed by mass spectrometry can identify novel interaction partners, particularly those that may differ between splice variants of TNFRSF25.

What are the best approaches for using TNFRSF25 antibodies in multi-parameter flow cytometry to identify and characterize specific T cell subsets?

For multi-parameter flow cytometry using TNFRSF25 antibodies, strategic panel design and optimized protocols are essential. When designing your antibody panel, position TNFRSF25 in a channel with appropriate sensitivity, as expression levels can vary with T cell activation status . Pair TNFRSF25 with key T cell subset markers including:

• Basic T cell markers: CD3, CD4, CD8

• Activation markers: CD25, CD69, CD44, CD62L

• Subset-specific markers: FOXP3 (Tregs), GATA3 (Th2), RORγt (Th17)

• Cytokine detection: intracellular IL-17, IL-13 (after stimulation)

For optimal staining, consider this protocol:

• Isolate cells and rest briefly in protein-containing buffer

• Surface stain with antibody cocktail including anti-TNFRSF25 (typically PE or APC-conjugated) for 30 minutes at 4°C

• For intracellular targets, fix and permeabilize cells using commercially available kits

• Stain with intracellular antibodies for 45-60 minutes at 4°C

• Wash and analyze promptly

Include fluorescence-minus-one (FMO) controls for TNFRSF25 to accurately set gates, particularly important as expression can be dynamic. For functional correlation, consider combining with phospho-flow techniques to detect downstream signaling events like NF-κB activation . This integrated approach can reveal how TNFRSF25 expression correlates with functional states across T cell subsets, particularly in Th2 and Th17 populations where TNFRSF25 signaling enhances cytokine production .

How can TNFRSF25 antibodies be used in combination with genetic approaches to study receptor function?

Combining TNFRSF25 antibodies with genetic approaches creates powerful systems for dissecting receptor function. A comprehensive strategy should include parallel antibody and genetic manipulation experiments to cross-validate findings.

For knockdown/knockout validation studies:

• Use TNFRSF25 antibodies to confirm protein depletion in siRNA knockdown or CRISPR/Cas9 knockout models

• Compare phenotypes between antibody blockade and genetic deletion approaches

• Perform rescue experiments where TNFRSF25 variants are re-expressed in knockout cells, then use antibodies to confirm expression and functional restoration

For transgenic model applications:

• Use TNFRSF25 antibodies to quantify expression levels in transgenic mice overexpressing full-length TNFRSF25, splice variants like Δ5,6, or dominant-negative TNFRSF25

• Compare functional outcomes between antibody blockade in wild-type mice versus dominant-negative transgenic mice

• Perform adoptive transfer experiments where cells from transgenic mice are transferred to recipients, then tracked using TNFRSF25 antibodies

In particularly informative experiments, researchers have shown that NKT-deficient mice (normally resistant to allergic lung inflammation) become susceptible upon adoptive transfer of wild-type NKT cells, but remain resistant when receiving dominant-negative TNFRSF25 transgenic NKT cells . Using TNFRSF25 antibodies to track these transferred populations provides crucial information about their persistence and localization, complementing the genetic approach.

What are common challenges when using TNFRSF25 antibodies and how can they be addressed?

Researchers commonly encounter several challenges when working with TNFRSF25 antibodies. For detection sensitivity issues, particularly in Western blot applications, enhance signal by using high-sensitivity ECL substrates or signal amplification systems, as TNFRSF25 can be expressed at relatively low levels in resting lymphocytes . If multiple bands appear, this may represent detection of different splice variants among the 12 reported isoforms - validate using positive controls with known expression patterns or isoform-specific controls.

Background noise in immunohistochemistry or immunofluorescence can be minimized by extensive blocking (3-5% BSA with normal serum from the secondary antibody species) and extended washing steps. For flow cytometry applications where signal-to-noise ratio is insufficient, try signal amplification methods such as biotin-streptavidin systems or use of brighter fluorophores like PE or APC.

Cross-reactivity with other TNF receptor family members may occur due to structural similarities. Address this by comparing staining patterns with TNFRSF25 knockout samples or by pre-absorbing the antibody with recombinant proteins of potentially cross-reactive family members. Finally, for functional studies, conflicting results may arise from using different antibody clones - some may be agonistic while others antagonistic, so characterize the functional properties of your specific antibody clone before interpreting results.

How should researchers interpret conflicting results between different TNFRSF25 antibody clones?

When confronted with conflicting results between different TNFRSF25 antibody clones, a systematic analytical approach is essential. Begin by characterizing the epitope specificity of each antibody clone - differences may arise from recognition of distinct domains of TNFRSF25 (extracellular, transmembrane, or intracellular), leading to detection of different subsets of splice variants .

Perform side-by-side validation experiments:

• Compare detection patterns in Western blots under identical conditions

• Assess binding to recombinant TNFRSF25 protein by ELISA

• Evaluate staining patterns by flow cytometry on identical samples

• Test antibodies on TNFRSF25 knockout/knockdown samples as negative controls

Functional effects may vary dramatically between clones - some antibodies may act as receptor agonists (mimicking TL1A binding), while others may be antagonistic or functionally neutral. These differences can be leveraged experimentally; for instance, an antagonistic antibody might phenocopy the effects seen in dominant-negative TNFRSF25 transgenic models .

For critical experiments, confirm key findings with at least two independent antibody clones, or validate with orthogonal methods such as genetic approaches. When publishing, clearly report the clone, manufacturer, and lot number of antibodies used, as variations between lots can contribute to experimental inconsistencies.

What strategies can improve reproducibility when working with TNFRSF25 antibodies across different experimental systems?

Improving reproducibility with TNFRSF25 antibodies across experimental systems requires standardized protocols and rigorous validation approaches. Establish standard operating procedures (SOPs) for each application (Western blot, flow cytometry, immunoprecipitation) with detailed parameters including antibody concentrations, incubation times, temperatures, and buffer compositions.

Implement antibody validation hierarchies:

• Tier 1: Verify antibody performance against recombinant TNFRSF25 protein

• Tier 2: Confirm specificity using genetic controls (knockout/knockdown)

• Tier 3: Cross-validate with orthogonal methods (e.g., mRNA expression)

For cross-laboratory consistency, create and distribute reference samples - aliquots of characterized cell lysates or fixed cells with known TNFRSF25 expression levels. These serve as calibration standards across experiments and locations.

When extending studies across species, be aware that despite conservation, structural differences exist between human, mouse, and other species' TNFRSF25 orthologs . Validate antibody cross-reactivity for each species individually, and consider species-specific positive controls (e.g., activated mouse T cells for mouse studies) .

Finally, maintain detailed records of antibody performance across different lots, and consider creating a laboratory-specific validation report for each antibody that documents optimal conditions and expected results for your specific experimental systems.

How are TNFRSF25 antibodies being utilized in studies of autoimmune diseases beyond asthma and allergic inflammation?

TNFRSF25 antibodies are increasingly being applied to investigate diverse autoimmune conditions beyond their established role in asthma and allergic inflammation. In multiple sclerosis research, these antibodies help elucidate how TNFRSF25 signaling contributes to neuroinflammation through enhanced IL-17 production by Th17 cells . This approach has revealed potential therapeutic opportunities targeting the TNFRSF25/TL1A axis to modulate disease progression.

For inflammatory bowel disease studies, TNFRSF25 antibodies are employed to investigate how this signaling pathway affects intestinal immune homeostasis. The dual ability of TNFRSF25 to promote both Th2 and Th17 responses positions it as a potential regulator of immune balance in the gut microenvironment .

In rheumatoid arthritis research, these antibodies help track TNFRSF25 expression in synovial tissues and infiltrating lymphocytes, providing insights into how this receptor contributes to joint inflammation. The methodological approach typically combines tissue immunohistochemistry with flow cytometric analysis of synovial fluid lymphocytes, correlating TNFRSF25 expression with disease activity markers.

For each autoimmune condition, blocking antibodies against TNFRSF25 or its ligand TL1A provide valuable tools to assess therapeutic potential, complementing genetic approaches using dominant-negative TNFRSF25 transgenic models or TL1A knockout systems .

What role can TNFRSF25 antibodies play in investigating the intersection between TNFRSF25 signaling and different T cell polarization states?

TNFRSF25 antibodies offer sophisticated tools for dissecting the complex relationship between TNFRSF25 signaling and T cell polarization states. Using multi-parameter flow cytometry panels that combine TNFRSF25 detection with master transcription factor staining (GATA3 for Th2, RORγt for Th17, T-bet for Th1, FOXP3 for Tregs), researchers can correlate receptor expression with polarization status at the single-cell level .

Functional studies utilizing agonistic TNFRSF25 antibodies have revealed differential effects across T helper subsets: significantly enhanced IL-17 production in Th17 cells and increased Th2 cytokines in Th2-polarized cells, with minimal impact on Th1 responses . This suggests a selective role in promoting inflammatory (Th17) and allergic (Th2) immune responses.

For temporal dynamics investigation, researchers can employ TNFRSF25 antibodies in time-course experiments during T cell polarization, revealing when receptor expression changes and how this correlates with commitment to specific lineages. When combined with signaling inhibitors targeting NF-κB or other downstream pathways, these studies can elucidate the molecular mechanisms by which TNFRSF25 differentially influences polarization outcomes.

Particularly intriguing is the finding that TNFRSF25 signaling enhances expression of its own ligand (TL1A) in CD4+ T cells , suggesting a positive feedback mechanism that may amplify specific polarization programs - a phenomenon that can be further explored using antibody-based detection and blocking approaches.

How can TNFRSF25 antibodies contribute to understanding the receptor's role in tumor immunology and cancer therapy?

TNFRSF25 antibodies provide valuable tools for investigating this receptor's complex roles in tumor immunology. Initial characterization studies should employ immunohistochemistry and flow cytometry with anti-TNFRSF25 antibodies to profile expression patterns across tumor tissues, tumor-infiltrating lymphocytes (TILs), and peripheral blood lymphocytes from cancer patients . The finding that TNFRSF25 is expressed in certain tumor cells themselves adds complexity to its potential roles .

For functional investigations, researchers can use agonistic TNFRSF25 antibodies to activate this pathway in ex vivo TILs, assessing effects on proliferation, cytokine production, and cytotoxic function. Given TNFRSF25's ability to enhance both Th2 and Th17 responses , it may differentially affect anti-tumor immunity depending on the cancer microenvironment.

The dual signaling capacity of TNFRSF25 - promoting both apoptosis through its death domain and survival through NF-κB activation - presents intriguing therapeutic possibilities. Engineered antibodies that selectively bias signaling toward the apoptotic pathway could potentially target TNFRSF25-expressing tumors while sparing normal tissues.

For translational applications, bispecific antibodies linking TNFRSF25 targeting with immune checkpoint blockade could potentially enhance anti-tumor immunity by simultaneously activating T cells through TNFRSF25 while removing inhibitory signals. This approach would require extensive preclinical validation using humanized antibody formats similar to those developed for other TNF family targets .