Recombinant Mouse Tumor necrosis factor receptor superfamily member 14 (Tnfrsf14), partial (Active)

What is TNFRSF14 and what are its primary functions in mouse models?

TNFRSF14, also known as HVEM (Herpes Virus Entry Mediator), is a 29-kD type II transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. In mice, TNFRSF14 plays crucial roles in immune and inflammatory responses by interacting with ligands including TNFSF14 (also called LIGHT). TNFRSF14 is expressed on various cell types including activated T lymphocytes and mast cells .

The protein functions as a key regulator of immune responses by limiting T-cell activation via ligation of B- and T-lymphocyte attenuator. This regulatory capacity has significant implications for research in transplantation biology, inflammatory diseases, and cancer models. In mice, TNFRSF14 has been demonstrated to contribute to various pathophysiological processes, including airway inflammation in asthma models and renal fibrosis development .

How does TNFRSF14 signaling differ between mice and humans?

An important methodological consideration for researchers is understanding the cross-species differences in TNFRSF14 signaling. While the core functions are conserved, there are notable receptor interaction differences:

• In humans, TNFSF14 can interact with three receptors: TNFRSF14 (HVEM), TNFRSF3 (lymphotoxin-beta receptor/LTβR), and TNFRSF6B (soluble decoy receptor 3/Dcr3)

• In mice, TNFSF14 interacts with only two receptors: TNFRSF14 and TNFRSF3

This divergence in receptor availability may lead to different signaling outcomes between species, which researchers should account for when designing experiments and interpreting results, particularly when considering translational applications.

What experimental models are effective for studying mouse TNFRSF14 function?

Several well-validated experimental models exist for investigating TNFRSF14 function:

• Unilateral Urethral Obstruction (UUO): This model has been effectively used to study TNFRSF14's role in renal fibrosis. Tnfsf14-deficient mice subjected to UUO surgery display markedly reduced renal fibrosis lesions and inflammatory cytokine expression compared to wild-type controls .

• Allergic Airway Inflammation Models: Both ovalbumin (OVA)-induced chronic airway inflammation and house dust mite (HDM)-induced asthma models have been used successfully to evaluate TNFRSF14's contribution to asthma pathology .

• Mast Cell-Deficient Mice with Selective Reconstitution: This sophisticated approach involves engrafting mast cells that either express or lack TNFRSF14 into genetically mast cell-deficient mice, allowing for precise determination of mast cell-specific TNFRSF14 contributions to disease phenotypes .

How can researchers effectively use recombinant mouse TNFRSF14 to study its role in T-cell responses?

When investigating TNFRSF14's impact on T-cell responses, researchers should consider the following methodological approaches:

• In vitro allogeneic response models: Human lymphoma B cells with TNFRSF14 aberrations show increased capacity to stimulate allogeneic T-cell responses, suggesting a similar approach could be effective with mouse cells. This model allows for controlled measurement of T-cell activation parameters in response to TNFRSF14 manipulation .

• Measurement endpoints: Key readouts should include T-cell proliferation, cytokine production profiles (particularly TH2-associated cytokines), and markers of T-cell activation.

• Blocking antibody studies: Administration of TNFRSF14-neutralizing antibodies can help distinguish between developmental effects and acute signaling requirements, as demonstrated in asthma models where TNFRSF14 blockade with a neutralizing antibody after antigen sensitization diminishes pathological features .

What methodological considerations are important when studying TNFRSF14 in fibrosis models?

When investigating TNFRSF14's role in fibrosis, researchers should implement the following approaches:

• Expression analysis: Assess TNFRSF14 levels in both affected tissues and circulation using techniques such as immunohistochemistry, qRT-PCR, and western blot analysis. In UUO-induced renal fibrosis, TNFSF14 and its receptors (HVEM and LTβR) were rapidly upregulated .

• Genetic manipulation: Compare wild-type and Tnfsf14-deficient mice in fibrosis models to establish causality. Tnfsf14 knockout mice subjected to UUO surgery displayed significantly reduced renal fibrosis lesions .

• Downstream signaling assessment: Examine the expression of critical pro-fibrotic molecules like sphingosine kinase 1 (Sphk1), which has been identified as a downstream mediator of TNFSF14-induced renal fibrosis .

• In vitro validation: Conduct primary cell culture experiments using recombinant TNFSF14 to confirm direct effects on target cells. For instance, recombinant TNFSF14 administration markedly upregulated Sphk1 expression in primary mouse renal tubular epithelial cells .

How does TNFRSF14 modulate mast cell function in inflammatory conditions?

TNFRSF14 expression on mast cells has significant implications for allergic and inflammatory research. Key methodological approaches include:

• Mast cell isolation and culture: Both mouse and human mast cells express TNFRSF14, making comparative studies feasible.

• IgE-dependent activation assays: TNFSF14:TNFRSF14 interactions can enhance IgE-mediated mast cell signaling and increase secretion of both pre-stored and de novo synthesized mediators .

• Selective reconstitution strategies: Engrafting mast cells that either express or lack TNFRSF14 into mast cell-deficient mice allows for precise determination of the contribution of mast cell-specific TNFRSF14 expression to disease phenotypes .

• Measurement endpoints: Key readouts should include degranulation markers (e.g., β-hexosaminidase release), cytokine/chemokine production, and lipid mediator synthesis.

How should researchers interpret conflicting roles of TNFRSF14 across different disease models?

TNFRSF14 demonstrates context-dependent functions that may appear contradictory across disease models:

• In follicular lymphoma: TNFRSF14 aberrations (mutations or deletions) are associated with increased T-cell stimulation and poor clinical outcomes, suggesting tumor-suppressive functions in this context .

• In renal fibrosis: TNFSF14 signaling through its receptors promotes fibrotic pathology, indicating a pro-pathological role .

• In bladder cancer: Increased TNFRSF14 expression correlates with good prognosis, suggesting anti-tumor properties in this malignancy .

When encountering seemingly contradictory data, researchers should:

• Consider tissue-specific expression patterns and signaling partners

• Evaluate the specific cellular context (e.g., immune cells versus epithelial cells)

• Analyze the disease stage being modeled (acute versus chronic)

• Determine whether genetic alterations affect protein expression versus function

• Examine potential differences in downstream signaling pathway activation

What are key considerations when comparing genetic knockout versus receptor blocking approaches?

When interpreting studies using different methods to manipulate TNFRSF14 signaling, researchers should consider:

• Developmental versus acute effects: Genetic knockouts eliminate TNFRSF14 throughout development, potentially triggering compensatory mechanisms, while blocking antibodies reveal acute signaling requirements. For example, studies have shown both approaches affect asthma pathology but may reveal different aspects of TNFRSF14 biology .

• Specificity considerations: Antibody-based blocking may have off-target effects or incomplete blockade, while genetic knockout provides complete elimination of the target protein.

• Timing differences: The temporal aspects of when TNFRSF14 signaling is disrupted (developmental versus post-maturation) may yield different phenotypes.

• Dosage effects: Genetic approaches create binary (present/absent) conditions, while antibody-based approaches may achieve partial inhibition depending on dosing strategies.

How can researchers distinguish between direct TNFRSF14 effects and secondary inflammatory responses?

Separating primary signaling effects from secondary inflammatory consequences requires systematic experimental approaches:

• Time-course experiments: Evaluate early versus late events following TNFRSF14 activation or inhibition.

• Cell-specific deletion models: Use cell type-specific Cre-loxP systems to delete TNFRSF14 in distinct populations, as demonstrated with mast cell-specific studies .

• Ex vivo and in vitro validation: Isolate specific cell populations to confirm direct responses to recombinant TNFRSF14 stimulation, as shown with primary mouse renal tubular epithelial cells .

• Pathway inhibition studies: Block specific downstream signaling components to determine which pathways are essential for observed phenotypes.

What validation approaches should be employed when extrapolating mouse TNFRSF14 findings to human diseases?

Translating mouse TNFRSF14 research to human applications requires several validation strategies:

• Parallel human sample analysis: Complement mouse studies with examination of human clinical specimens, as demonstrated in studies of renal fibrosis where researchers analyzed biopsy kidney tissues from patients with CKD (including membranous nephritis, focal segmental glomerulosclerosis, and thrombotic microangiopathy) .

• Comparative expression profiling: Compare expression patterns and regulation of TNFRSF14 between mouse models and human disease tissues.

• Functional conservation testing: Determine whether human TNFRSF14 can rescue phenotypes in mouse knockout models.

• Receptor-ligand interaction mapping: Given the different receptor landscape between species (three receptors in humans versus two in mice), detailed mapping of interaction patterns is essential .

• Cross-validation in human cell systems: Test findings from mouse models in human primary cells or organoid cultures when possible.

How can TNFRSF14 research inform therapeutic strategies for human diseases?

TNFRSF14 research has revealed several potential therapeutic applications:

• Transplantation medicine: TNFRSF14 aberrations in follicular lymphoma increase the ability of lymphoma cells to stimulate allogeneic T-cell responses and are associated with increased acute graft-versus-host disease (GVHD). This suggests that patients with TNFRSF14 aberrations may benefit from more aggressive immunosuppression to reduce harmful GVHD after transplantation .

• Fibrotic disease interventions: Targeting the TNFSF14-TNFRSF14 axis may represent a useful immunotherapeutic strategy for kidney fibrosis, potentially by disrupting the upregulation of pro-fibrotic factors like Sphk1 .

• Asthma treatments: TNFRSF14 represents a potential therapeutic target in asthma, as blockade reduces airway hyperreactivity, inflammation, and remodeling in mouse models .

• Cancer prognostics: TNFRSF14 mutations in follicular lymphoma may serve as prognostic markers for identifying high-risk patients as candidates for risk-adapted therapies .

What are the optimal storage and handling conditions for recombinant mouse TNFRSF14?

To maintain optimal activity of recombinant mouse TNFRSF14:

• Storage temperature: Store lyophilized protein at -20°C to -80°C and reconstituted protein in working aliquots at -80°C.

• Reconstitution medium: Use sterile buffers appropriate for the experimental system, typically PBS with carrier protein (e.g., 0.1% BSA) to prevent adsorption to tubes.

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles by preparing single-use aliquots.

• Working conditions: Maintain protein on ice during experimental setup and use within recommended time frames after thawing.

What quality control parameters should be evaluated when using recombinant mouse TNFRSF14?

Key quality control measures include:

• Purity assessment: Verify protein purity via SDS-PAGE analysis, aiming for >95% purity.

• Endotoxin testing: Confirm low endotoxin levels (<1.0 EU/μg), especially for immunology experiments where endotoxin contamination could confound results.

• Functional validation: Test bioactivity using established assays such as:

  • Binding assays with known ligands

  • Cell-based functional assays measuring downstream signaling

  • Comparison with reference standards when available

• Batch consistency: Maintain records of lot-to-lot variation and establish internal reference standards when possible.