TNFRSF25 Antibody, HRP conjugated

What is TNFRSF25 and why is it an important research target?

TNFRSF25 (also known as DR3, LARD, WSL-1, APO3, TRAMP, and TR3) belongs to the tumor necrosis factor receptor superfamily. It is a glycoprotein with a 417 amino acid residue transmembrane precursor structure containing four cysteine-rich repeats in its extracellular domain and a death domain in its cytoplasmic region . TNFRSF25 is primarily expressed in tissues enriched with lymphocytes, particularly activated T cells, and functions as a receptor for TL1A (TNFSF15) . Its significance as a research target stems from its dual role in both costimulatory signaling for T cells and apoptotic pathway activation in certain cellular contexts, making it relevant for immunotherapy research, particularly in cancer treatment approaches .

What are the main applications for TNFRSF25 antibodies in research?

TNFRSF25 antibodies are valuable tools for multiple research applications including: (1) Western blot analysis for protein expression quantification; (2) Flow cytometry for identifying TNFRSF25-expressing cells, particularly in lymphocyte populations; (3) Immunohistochemistry for tissue localization studies; (4) Functional studies investigating T-cell activation and apoptosis pathways; and (5) Therapeutic development research exploring agonistic antibodies for cancer immunotherapy . HRP-conjugated variants specifically facilitate detection in techniques requiring enzymatic signal amplification such as Western blotting, ELISA, and immunohistochemistry, where the horseradish peroxidase enzyme catalyzes colorimetric, chemiluminescent, or fluorescent reactions for visualization.

How do TNFRSF25 expression patterns change during T-cell activation?

Naïve B and T cells primarily express multiple truncated TNFRSF25 isoforms but not the transmembrane isoform 1. Upon T-cell activation, expression shifts predominantly to the transmembrane TNFRSF25 isoform 1 . This differential expression pattern can be detected using appropriate antibodies and provides insight into T-cell activation status. When studying activation-dependent expression, researchers should consider using flow cytometry with anti-TNFRSF25 antibodies (including HRP-conjugated variants for certain applications) alongside activation markers to track this transition. This knowledge is particularly important when designing experiments to study TNFRSF25-mediated signaling in different T-cell activation states.

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

For optimal Western blot results with HRP-conjugated TNFRSF25 antibodies, researchers should: (1) Transfer proteins to PVDF or nitrocellulose membranes using standard protocols; (2) Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature; (3) Dilute the HRP-conjugated TNFRSF25 antibody according to manufacturer specifications (typically between 1:1000-1:5000) in blocking buffer; (4) Incubate membranes with diluted antibody for 1-2 hours at room temperature or overnight at 4°C; (5) Wash extensively with TBST (at least 3×10 minutes); and (6) Develop using appropriate chemiluminescent substrate. When analyzing TNFRSF25, be aware that the canonical protein has a reported mass of 45.4 kDa, but post-translational modifications such as glycosylation may affect migration patterns . For detecting specific isoforms (of which up to 12 have been reported), researchers should pay careful attention to band sizes and consider using positive controls with known expression patterns.

How can flow cytometry protocols be optimized for TNFRSF25 detection in primary lymphocytes?

For optimal flow cytometry detection of TNFRSF25 in primary lymphocytes, researchers should: (1) Isolate peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation; (2) Block cells with appropriate serum (10% human serum for human samples) at 4°C for 15 minutes to minimize non-specific binding; (3) Incubate with primary anti-TNFRSF25 antibodies at manufacturer-recommended concentrations; (4) For indirect staining with HRP-conjugated secondary antibodies, wash cells thoroughly and incubate with the secondary antibody at appropriate dilution; (5) Consider treatment with PMA and Calcium Ionomycin for 24 hours to upregulate TNFRSF25 expression on T cells before staining, as demonstrated in detection protocols . Multi-parameter analysis including markers like CD3 can help identify TNFRSF25 expression specifically on T-cell populations. Setting quadrant markers based on appropriate isotype control antibodies is essential for accurate assessment of positive populations .

What validation steps should be performed when using a new lot of TNFRSF25 antibody?

When validating a new lot of TNFRSF25 antibody, researchers should perform the following essential steps: (1) Positive control testing using cell lines with known TNFRSF25 expression (e.g., EL4 cells stably transduced with TNFRSF25 as referenced in the literature ); (2) Negative control testing using TNFRSF25-negative cells or knockout models; (3) Titration experiments to determine optimal working concentration by testing a range of antibody dilutions; (4) Comparison with previous lots or alternative validated antibodies targeting the same epitope; (5) Specificity testing by pre-blocking with recombinant TNFRSF25 protein; and (6) Application-specific validation in the experimental system of interest. For HRP-conjugated antibodies specifically, additional validation of the enzymatic activity should be performed using appropriate substrates to ensure signal generation is within expected ranges.

How can TNFRSF25 antibodies be used to investigate the differential effects on regulatory T cells versus effector T cells?

Studies have shown that TNFRSF25 activation can expand regulatory T (Treg) cells in certain contexts while also promoting effector T-cell activation in others . To investigate these differential effects, researchers can: (1) Isolate CD4+ T-cell populations and sort into CD25+FoxP3+ Tregs and conventional CD4+ T cells; (2) Treat both populations with TNFRSF25 agonistic antibodies or multimeric TL1A proteins; (3) Measure proliferation using flow cytometry-based assays with vital dyes; (4) Assess functional changes through cytokine production profiling and suppression assays; and (5) Analyze signaling pathway activation through phospho-flow or Western blot techniques using pathway-specific antibodies. Research has demonstrated that activating TNFRSF25 by agonistic antibodies like 1A6-m1 expanded splenic Treg cells while simultaneously activating CD8+ T cells and antigen-specific T cells against tumors . These differential effects appear to be context-dependent and influenced by the local microenvironment, with evidence suggesting that despite Treg expansion systemically, intratumoral Treg populations may remain unaffected .

What are the key considerations for developing TNFRSF25 agonistic antibodies for cancer immunotherapy?

Development of TNFRSF25 agonistic antibodies for cancer immunotherapy requires several critical considerations: (1) Epitope selection – research indicates that specific epitopes may confer differential agonistic activity even among antibodies with similar binding affinities ; (2) Antibody structure – the CH1-hinge region flexibility appears to influence agonistic activity, with evidence showing that replacing the CH1-hinge region with that of human IgG2 enhanced antitumor effects ; (3) Fc receptor engagement – studies have demonstrated that the antitumor effects of TNFRSF25 agonistic antibodies require engagement of both inhibitory FcγRIIB and activating FcγRIII ; (4) Alternative approaches – multimeric TL1A fusion proteins can serve as TNFRSF25 agonists that reduce tumor growth even without Fc-FcγR interactions ; and (5) Safety considerations – careful monitoring for potential systemic immune activation. Recent research has shown promising results with TNFRSF25 agonists exhibiting antitumor effects in syngeneic mouse tumor models without observed side effects , suggesting potential for clinical translation.

How do binding kinetics influence the functional activity of TNFRSF25 antibodies?

Binding kinetics significantly impact TNFRSF25 antibody functionality. Surface plasmon resonance (SPR) analysis has been used to characterize the kinetic parameters of antibodies binding to TNFRSF25 . Interestingly, research has identified that higher binding affinity does not necessarily correlate with enhanced agonistic activity or antitumor effects. For example, the anti-mTNFRSF25 agonistic antibody 1A6-m1 exhibited greater antitumor activity than a higher affinity anti-TNFRSF25 antibody targeting an overlapping epitope . This suggests that epitope specificity and the mode of receptor engagement may be more important than absolute binding affinity. When evaluating TNFRSF25 antibodies, researchers should assess both on-rate (kon) and off-rate (koff) constants, as these parameters may differentially affect receptor clustering, internalization rates, and subsequent signaling cascade activation. Additionally, multimerization of the ligand appears to enhance agonistic activity, as demonstrated by multimeric TL1A fusion proteins that showed potent effects on T-cell costimulation .

What are common issues when using HRP-conjugated antibodies for TNFRSF25 detection, and how can they be resolved?

Common issues with HRP-conjugated TNFRSF25 antibodies include: (1) High background signal – resolve by increasing blocking stringency (5-10% BSA or milk), adding 0.1-0.3% Tween-20 to wash buffers, and using fresh blocking reagents; (2) Weak or absent signal – troubleshoot by confirming TNFRSF25 expression in your sample (consider PMA/ionomycin stimulation to upregulate expression ), optimizing antibody concentration, extending incubation time, or using enhanced chemiluminescent substrates; (3) Non-specific bands in Western blotting – improve specificity by optimizing antibody dilution, including detergents in wash buffers, and using gradient gels for better protein separation; (4) Batch-to-batch variability – validate each new lot against previously verified materials and maintain consistent experimental conditions; and (5) Loss of HRP activity – store antibodies according to manufacturer recommendations (typically at -20°C to -70°C), avoid repeated freeze-thaw cycles, and use within the recommended shelf-life (typically 12 months from receipt at -20°C to -70°C, 1 month at 2-8°C after reconstitution, or 6 months at -20°C to -70°C after reconstitution) .

How can researchers distinguish between different TNFRSF25 isoforms using antibody-based detection methods?

To distinguish between the multiple TNFRSF25 isoforms (up to 12 have been reported ), researchers should: (1) Select antibodies with known epitope specificity, preferably those that can differentiate between full-length transmembrane isoform 1 and truncated variants; (2) Use high-resolution gel electrophoresis systems (e.g., gradient gels) to separate closely sized isoforms; (3) Combine with isoform-specific RT-PCR to correlate protein detection with transcript expression; (4) Consider using 2D gel electrophoresis for complex samples to separate isoforms that may have similar molecular weights but different isoelectric points; (5) Perform immunoprecipitation followed by mass spectrometry for definitive isoform identification; and (6) Use cells with known isoform expression profiles as positive controls (e.g., resting T cells primarily express truncated isoforms while activated T cells predominantly express transmembrane isoform 1 ). When analyzing data, carefully document band patterns and molecular weights, as post-translational modifications like glycosylation can further complicate isoform identification .

What strategies can overcome challenges in detecting low TNFRSF25 expression levels in certain cell types?

For detecting low-level TNFRSF25 expression, researchers can employ several strategies: (1) Signal amplification systems – utilize tyramide signal amplification (TSA) with HRP-conjugated antibodies to significantly enhance detection sensitivity; (2) Cell activation – stimulate cells with PMA and calcium ionomycin for 24 hours to upregulate TNFRSF25 expression before analysis, as demonstrated in both human PBMCs and mouse splenocytes ; (3) Enrichment techniques – perform immunomagnetic separation or FACS to enrich for lymphocyte populations where TNFRSF25 is more highly expressed; (4) Enhanced imaging – use high-sensitivity detection systems such as enhanced chemiluminescence for Western blots or high-sensitivity flow cytometers with PMT detectors; (5) Increase sample loading – for Western blot applications, optimize protein loading to maximize detection while maintaining good resolution; and (6) RNA-based validation – complement protein detection with qRT-PCR to confirm expression at the transcript level. When interpreting results with these enhanced methods, always include appropriate negative controls to confirm specificity of the detected signal.

How can TNFRSF25 antibodies be used to study the role of this receptor in autoimmune disease models?

TNFRSF25 plays dual roles in immune regulation that are relevant to autoimmune disease research. To study these roles, researchers can: (1) Use flow cytometry with anti-TNFRSF25 antibodies to monitor expression changes on different T-cell subsets during disease progression in models like experimental autoimmune encephalomyelitis (EAE) or collagen-induced arthritis; (2) Apply agonistic anti-TNFRSF25 antibodies in vivo to evaluate effects on Treg expansion and function, as TNFRSF25 activation has been shown to expand regulatory T cells in certain contexts ; (3) Combine with neutralizing antibodies against TL1A (the TNFRSF25 ligand) to assess the contribution of this signaling axis to disease pathogenesis; (4) Perform adoptive transfer studies with TNFRSF25-deficient T cells versus wild-type cells to determine cell-intrinsic effects; and (5) Use immunohistochemistry with HRP-conjugated antibodies to visualize TNFRSF25-expressing cells within inflamed tissues. Understanding the balance between TNFRSF25's role in expanding protective Treg populations versus its potential to enhance effector T-cell responses is crucial for interpreting results in autoimmune contexts.

What insights have TNFRSF25 antibodies provided into cancer immunosurveillance and immunotherapy?

TNFRSF25 antibodies have revealed several important insights into cancer immunotherapy: (1) Agonistic anti-TNFRSF25 antibodies exhibit significant antitumor effects in syngeneic mouse tumor models without observed toxicities ; (2) TNFRSF25 activation primarily works through CD8+ T-cell costimulation rather than direct tumor cell killing, as demonstrated by cell depletion studies showing CD8+ T-cell dependence of antitumor effects ; (3) Epitope specificity influences therapeutic efficacy, with certain antibodies showing superior activity despite lower binding affinity ; (4) Both antibody structure and Fc receptor engagement are critical for optimal activity, with dependency on both inhibitory FcγRIIB and activating FcγRIII ; (5) TNFRSF25 agonists can induce long-term antitumor immune memory, suggesting potential for durable therapeutic responses ; and (6) Alternative approaches using multimeric TL1A proteins can activate TNFRSF25 and reduce tumor growth even without Fc-FcγR interactions . These findings collectively position TNFRSF25 as a promising immunotherapeutic target that functions through T-cell costimulation mechanisms distinct from currently approved checkpoint inhibitor approaches.

What is the significance of TNFRSF25 in transplantation research, and how are antibodies used in this field?

TNFRSF25 has significant implications in transplantation research due to its role in regulatory T-cell biology. Previous studies have confirmed that TNFRSF25 agonists can expand regulatory T cells and inhibit allograft rejection in mouse models of organ transplantation and hematopoietic cell transplantation (HCT) . In this field, TNFRSF25 antibodies are utilized to: (1) Monitor expression patterns on graft-infiltrating lymphocytes using flow cytometry or immunohistochemistry; (2) Expand regulatory T cells ex vivo or in vivo through agonistic activity; (3) Study the balance between effector and regulatory responses in graft rejection; (4) Develop novel immunomodulatory strategies that leverage TNFRSF25's ability to expand Tregs while potentially preserving anti-pathogen immunity; and (5) Investigate differences between systemic and local immune regulation, as research has shown differential effects of TNFRSF25 activation on splenic versus tissue-resident Tregs . The potential to selectively expand regulatory T cells through TNFRSF25 activation represents a promising approach for inducing transplantation tolerance without broad immunosuppression.

How might single-cell analysis technologies enhance our understanding of TNFRSF25 biology when used with specific antibodies?

Single-cell technologies combined with TNFRSF25 antibodies offer transformative insights: (1) Single-cell RNA-seq paired with TNFRSF25 protein detection (CITE-seq) can reveal correlations between receptor expression and transcriptional states, identifying novel cell populations with unique TNFRSF25 expression patterns; (2) Mass cytometry (CyTOF) using metal-conjugated anti-TNFRSF25 antibodies enables high-dimensional analysis of expression patterns across diverse immune subsets alongside dozens of other markers; (3) Imaging mass cytometry or multiplexed immunofluorescence provides spatial context to TNFRSF25 expression within tissues, revealing potential intercellular communication networks; (4) Single-cell Western blotting can detect different TNFRSF25 isoforms at the individual cell level; and (5) Proximity ligation assays can investigate protein-protein interactions with TNFRSF25 in situ. These approaches are particularly valuable for understanding the heterogeneity of TNFRSF25 expression and signaling across different T-cell subsets and tissue microenvironments, potentially revealing new therapeutic opportunities beyond those observed in bulk population studies.

What are the latest developments in combining TNFRSF25 targeting with other immunomodulatory approaches?

Recent research suggests several promising combinatorial approaches: (1) TNFRSF25 agonists with checkpoint inhibitors – preliminary evidence suggests potential synergy between T-cell costimulation via TNFRSF25 and removal of inhibitory signals through checkpoint blockade; (2) TNFRSF25 targeting with cancer vaccines – enhancing antigen-specific T-cell responses through TNFRSF25 costimulation may boost vaccine efficacy; (3) Combination with adoptive cell therapies – pre-treating or engineering T cells to express or respond to TNFRSF25 signaling could enhance persistence and activity after transfer; (4) Bispecific antibodies targeting both TNFRSF25 and tumor antigens to direct T-cell responses specifically to malignant cells; and (5) Combination with conventional therapies like radiation that may upregulate TNFRSF25 expression on tumor-infiltrating lymphocytes. Research has demonstrated that TNFRSF25 agonistic antibodies can induce long-term antitumor immune memory , suggesting potential for durable responses with these combinatorial approaches. When designing such studies, careful attention to dosing and scheduling is essential to balance immune activation against potential toxicities.

How can structural biology approaches inform the design of next-generation TNFRSF25-targeting antibodies?

Structural biology provides critical insights for next-generation TNFRSF25 antibody design: (1) Epitope mapping through X-ray crystallography or cryo-EM of antibody-TNFRSF25 complexes can identify binding sites that promote optimal receptor clustering and signaling; (2) Structure-function analyses comparing antibodies with different agonistic potencies can reveal key structural determinants of activity (research has already identified the importance of CH1-hinge region flexibility in determining agonistic potential ); (3) Molecular dynamics simulations can predict how antibody binding affects receptor conformation and oligomerization; (4) Hydrogen-deuterium exchange mass spectrometry can map conformational changes induced by different antibodies; and (5) Structure-guided engineering approaches can optimize features like valency, flexibility, and FcγR binding. These approaches are particularly relevant given the observation that antibody affinity does not directly correlate with agonistic activity, as demonstrated by the superior antitumor effects of 1A6-m1 compared to higher-affinity antibodies targeting overlapping epitopes . Understanding the structural basis of these phenomena will enable rational design of antibodies with enhanced therapeutic properties.