TNFRSF4 Human

What is the molecular structure of human TNFRSF4/OX40?

Human OX40 (TNFRSF4) is a 48 kDa type I transmembrane glycoprotein with a complex structure comprising multiple domains. The protein consists of a 28 amino acid signal sequence, followed by a 185 amino acid extracellular domain (ECD) that contains four TNFR-Cys repeats and an O-glycosylated hinge region. This is complemented by a 20 amino acid transmembrane segment and a 41 amino acid cytoplasmic domain . The protein's functional region spans from Leu29 to Ala216, which is critical for its biological activity and is often targeted in antibody development . This structural arrangement facilitates TNFRSF4's role in immune signaling and T cell co-stimulation.

How does TNFRSF4 function within the T cell co-stimulatory network?

TNFRSF4 operates as a critical T cell co-stimulatory molecule within the tumor necrosis factor receptor superfamily. It coordinates with multiple other membrane-bound co-stimulators, including CD28, CD40, CD30, CD27, and 4-1BB (CD137) . This coordination creates a sophisticated signaling network that regulates T cell activation, proliferation, and survival. TNFRSF4 is not constitutively expressed on naïve CD4+ T cells but appears after T cell receptor (TCR) engagement by antigen-presenting cells (APCs), primarily dendritic cells and B cells . This expression is further dependent on co-stimulation via the CD40/CD40 ligand and CD28/B7 pathways, highlighting the integrated nature of T cell activation cascades. The temporal dynamics of TNFRSF4 expression are notable, reaching maximal levels at 2-5 days post-activation in naïve T cells, but appearing much more rapidly (approximately 4 hours) after reactivation of memory T cells .

What evolutionary conservation patterns exist for TNFRSF4 across species?

The extracellular domain of human TNFRSF4 shows significant but varying degrees of amino acid identity with other mammals: 71% with feline, 68% with canine, 67% with rabbit, and 64% with both mouse and rat orthologs . This moderate to high conservation across species suggests that TNFRSF4 plays an evolutionarily important role in adaptive immunity. The differential conservation rates across species domains may indicate selective pressures on specific functional regions of the protein. When designing cross-species experimental approaches, researchers should consider these conservation patterns, particularly when translating findings from animal models to human applications.

What are the optimal methods for detecting TNFRSF4 in human samples?

Multiple validated methods exist for TNFRSF4 detection, each with specific applications depending on research requirements:

• Flow Cytometry: Human peripheral blood mononuclear cells (PBMCs) can be effectively analyzed using anti-TNFRSF4 monoclonal antibodies (such as Clone #443318) . For optimal results, co-staining with T cell markers (e.g., CD3) is recommended to identify TNFRSF4-expressing T cell subpopulations. Secondary antibody approaches using phycoerythrin-conjugated detection systems provide sensitive visualization .

• Western Blot: This technique effectively detects TNFRSF4 in cell lysates, particularly from activated T cells. Under reducing conditions, TNFRSF4 appears as a specific band at approximately 45 kDa when using appropriate antibodies . This method is especially valuable for comparing expression levels between different cellular conditions or treatments.

• Immunohistochemistry: TNFRSF4 can be visualized in tissue sections, such as human tonsil, using specific antibodies followed by appropriate detection systems like DAB staining . This approach is particularly useful for examining TNFRSF4 expression in the context of tissue architecture.

• ELISA: Sandwich ELISA provides quantitative detection of soluble TNFRSF4 with high sensitivity (detection limit typically <10 pg/mL) . This method works well with multiple sample types including cell culture supernatant, serum, and plasma (both heparin and EDTA) .

How can researchers optimize ELISA protocols for TNFRSF4 detection?

When employing ELISA for TNFRSF4 quantification, researchers should consider the following optimization strategies:

• Sample Selection: Human TNFRSF4 ELISA kits are validated for cell culture supernatant, serum, and plasma (both heparin and EDTA anticoagulants) . The choice of sample type should align with specific research questions.

• Detection Range Considerations: Commercial kits typically offer detection ranges of 31.2-2000 pg/mL with minimum detection limits of approximately 31.2 pg/mL . Samples should be appropriately diluted to fall within this range.

• Specificity Verification: High-quality ELISA kits show no detectable cross-reactivity with other relevant proteins . When studying novel contexts, additional validation steps may be warranted.

• Equipment Requirements: Standardize instrumentation with microplate readers in standard size, automated plate washers, and appropriate pipetting systems. For large sample numbers, multichannel pipettes are recommended to maintain consistency and processing efficiency .

What are the key considerations for flow cytometric analysis of TNFRSF4?

Flow cytometry represents a powerful approach for studying TNFRSF4 expression on specific cell populations, but requires careful methodological consideration:

• Timing of Analysis: Given TNFRSF4's dynamic expression pattern (maximal at 2-5 days post-activation in naïve T cells and 4 hours post-reactivation in memory T cells), careful timing of analysis is critical .

• Activation Protocol Selection: For in vitro studies, standardized activation protocols using agents like PHA (1 μg/mL for 3-5 days) have been validated for inducing detectable TNFRSF4 expression on T cells .

• Co-staining Strategy: Co-staining with lineage markers (e.g., CD3 for T cells) and additional activation markers provides contextual information about the activation state of TNFRSF4-expressing cells .

• Control Selection: Appropriate isotype controls are essential for distinguishing specific TNFRSF4 staining from background .

How does TNFRSF4 expression relate to cisplatin resistance in cancer models?

In cisplatin-resistant neuroblastoma models, TNFRSF4 emerges as a significantly dysregulated gene that functions independently of MYCN amplification status . Studies using matched cisplatin-sensitive (KellyLuc) and resistant (KellyCis83Luc) cell lines have demonstrated differential TNFRSF4 expression patterns that correlate with treatment response. In metastatic models, cisplatin-resistant neuroblastoma cells exhibit greater metastatic potential, particularly to lymph nodes, compared to their sensitive counterparts . This suggests that TNFRSF4 dysfunction may contribute to both chemoresistance and enhanced metastatic capacity, highlighting its potential as both a biomarker and therapeutic target in treatment-resistant disease.

What mechanisms might explain TNFRSF4's role in cancer progression?

While the complete mechanistic understanding of TNFRSF4's role in cancer remains an active area of investigation, several potential pathways warrant consideration:

• Immune Evasion: Given TNFRSF4's established role in T cell co-stimulation, altered expression may disrupt normal anti-tumor immune responses, potentially enabling immune evasion by cancer cells.

• Cell-Intrinsic Signaling: The correlation between TNFRSF4 expression in tumor cells (rather than infiltrating lymphocytes) and clinical outcomes suggests cell-autonomous functions beyond its immune regulatory role .

• Metastatic Capacity: The increased lymph node metastasis observed in TNFRSF4-dysregulated models suggests potential involvement in invasion and migration pathways .

• Treatment Resistance: The association with cisplatin resistance indicates possible interactions with cellular pathways mediating therapeutic response, including potentially DNA damage repair mechanisms or apoptotic pathways.

How should researchers design studies to investigate TNFRSF4's role in tumor progression?

When investigating TNFRSF4's contributions to cancer progression, researchers should consider multi-faceted experimental approaches:

• Model Selection: Utilize both in vitro cellular models and in vivo systems that recapitulate key aspects of human disease. For example, matched cisplatin-sensitive and resistant cell lines provide valuable tools for studying treatment resistance mechanisms .

• Metastatic Assessment: Implement techniques to quantify metastatic burden, with particular attention to lymph node involvement. This may include bioluminescence imaging of labeled cells and confirmatory histopathological assessment .

• Correlation with Clinical Parameters: Connect experimental findings with clinical variables including age at diagnosis, disease stage, and risk stratification to establish clinical relevance .

• Mechanistic Dissection: Employ techniques to distinguish tumor cell-intrinsic versus immune-mediated effects of TNFRSF4, such as targeted expression modulation in tumor cells versus immune compartments.

What controls are essential for TNFRSF4 functional studies?

Proper control selection is crucial for robust TNFRSF4 research:

• Antibody Controls: For flow cytometry, appropriate isotype controls (e.g., Rat IgG for anti-TNFRSF4 antibodies) are essential to establish specific staining thresholds .

• Activation Controls: When studying TNFRSF4 in T cells, include both resting and activated populations to account for activation-dependent expression patterns. Standard activation protocols using PHA (1 μg/mL) for defined periods (3-5 days) provide appropriate comparisons .

• Cell Type Controls: Include both TNFRSF4-expressing and non-expressing cell populations, such as regulatory T cells versus other PBMC populations .

• Technical Controls: For quantitative techniques like ELISA, include standard curves with defined TNFRSF4 concentrations to ensure accurate quantification .

How can researchers effectively measure TNFRSF4-mediated functional responses?

Beyond expression analysis, functional assessment of TNFRSF4 activity provides critical insights:

• Cytokine Production: Measure downstream cytokine production (e.g., CXCL8/IL-8) following TNFRSF4 engagement to assess signaling pathway activation .

• Proliferation Assays: Quantify T cell proliferation in response to TNFRSF4 stimulation or blockade to evaluate its co-stimulatory function.

• Survival Analysis: Assess cellular survival outcomes in the context of TNFRSF4 manipulation to determine its contribution to cell fate decisions.

• Signaling Pathway Analysis: Employ phospho-flow cytometry or western blotting for key signaling mediators to dissect the molecular mechanisms downstream of TNFRSF4 activation.

How might TNFRSF4-targeted approaches be developed for cancer therapy?

Given TNFRSF4's emerging role in cancer progression and treatment resistance, several therapeutic strategies warrant exploration:

• Agonistic Approaches: For cancers where enhanced T cell responses would be beneficial, TNFRSF4 agonists might stimulate anti-tumor immunity. This approach would need to carefully balance enhanced T cell activation against potential autoimmune side effects.

• TNFRSF4 Modulation in Tumor Cells: Since TNFRSF4 expression in tumor cells (rather than immune cells) correlates with outcomes in neuroblastoma, approaches to modulate its expression or function specifically in cancer cells may have therapeutic potential .

• Combination with Chemotherapy: The association between TNFRSF4 dysregulation and cisplatin resistance suggests potential for combination therapies that target TNFRSF4 pathways alongside conventional chemotherapy .

• Biomarker-Guided Therapy: TNFRSF4 expression patterns may serve as biomarkers for patient stratification, identifying individuals most likely to benefit from specific therapeutic approaches.

What are the most promising directions for understanding TNFRSF4 biology?

Future research into TNFRSF4 biology should address several key knowledge gaps:

• Signaling Network Integration: Further elucidation of how TNFRSF4 signaling integrates with other co-stimulatory and co-inhibitory pathways would enhance our understanding of T cell regulation.

• Non-Immune Functions: Investigation of TNFRSF4's potential cell-autonomous functions in non-immune cells, particularly tumor cells, could reveal novel biological roles .

• Therapeutic Window Assessment: Determining the therapeutic window for TNFRSF4 modulation that enhances anti-tumor immunity without triggering autoimmune complications represents a critical research priority.

• Resistance Mechanism Dissection: Further characterization of how TNFRSF4 contributes to treatment resistance mechanisms may identify additional therapeutic vulnerabilities in resistant disease .

What technical advances would accelerate TNFRSF4 research?

Several methodological developments could significantly advance TNFRSF4 research:

• Single-Cell Analysis: Application of single-cell technologies to examine TNFRSF4 expression and function at single-cell resolution would reveal heterogeneity in expression patterns and responses.

• In Vivo Imaging: Development of non-invasive methods to visualize TNFRSF4 expression and activation in live animals would enable dynamic studies of its regulation.

• Structural Biology Approaches: Detailed structural studies of TNFRSF4 in complex with its ligand and downstream signaling mediators would inform rational drug design efforts.

• High-Throughput Screening Platforms: Development of screening approaches to identify modulators of TNFRSF4 expression or function could accelerate therapeutic discovery.