TNFRSF4 Mouse

What is the molecular structure of mouse TNFRSF4/OX40?

Mouse OX40/TNFRSF4 is a 256 amino acid type I transmembrane precursor protein with a distinctive structure consisting of:

• A 19 amino acid signal peptide

• A 192 amino acid extracellular domain containing 4 TNFR-cysteine rich repeats

• A 25 amino acid transmembrane domain

• A 36 amino acid cytoplasmic region

The protein undergoes significant post-translational modifications, particularly glycosylation, which increases its apparent molecular weight from the predicted 24.2 kDa to approximately 48-55 kDa when analyzed by Bis-Tris PAGE . The extracellular domain (Val20-Pro211) represents the functional portion commonly used in recombinant protein studies .

How does mouse TNFRSF4 compare with its human and rat counterparts?

Mouse OX40/TNFRSF4 shares approximately 63% amino acid sequence identity with its human counterpart and 90% with its rat counterpart . This relatively high conservation between species, particularly with rat, makes mouse models valuable for studying OX40 biology, though researchers should be aware of the differences when translating findings to human applications. The mouse protein contains the same functional domains as the human version, including the cysteine-rich repeats in the extracellular domain and the cytoplasmic signaling region, but species-specific variations can affect ligand binding kinetics and downstream signaling efficiency .

What are the expression patterns of TNFRSF4 in mouse immune cells?

TNFRSF4 is primarily expressed on activated CD4+ T cells, appearing 24-72 hours after activation, rather than on resting T cells . It is also expressed on activated CD8+ T cells, though typically at lower levels than on CD4+ T cells . Importantly, OX40 is constitutively expressed on regulatory T cells (Tregs), where it enhances sensitivity to IL-2 and promotes Treg proliferation, though it has been shown to decrease their immunosuppressive activity on effector T cells . The absence of expression on resting T cells makes OX40 an attractive target for specifically modulating activated immune responses without broadly affecting the entire T cell compartment .

What are the most reliable methods for detecting mouse TNFRSF4 expression?

Several validated methods are available for detecting mouse TNFRSF4 expression, each with specific applications:

Flow Cytometry: The gold standard for cellular expression analysis, using PE-conjugated antibodies such as Goat Anti-Mouse OX40/TNFRSF4. Researchers typically use activated mouse splenocytes stained with Rat Anti-Mouse CD3 APC-conjugated Monoclonal Antibody along with anti-OX40 antibodies to identify OX40-expressing T cell populations . This method allows for multi-parameter analysis to correlate OX40 expression with other markers.

ELISA: Sandwich ELISA kits provide quantitative measurement of mouse OX40 in various sample types including cell lysates, culture supernatants, plasma, serum, and tissue homogenates. These assays demonstrate high precision with coefficient of variation (CV) values typically between 3.4% and 6.9% .

Western Blot: Useful for protein size confirmation and semi-quantitative analysis, particularly with recombinant proteins. Western blotting can confirm the presence of both monomeric and potential multimeric forms of OX40 .

How can I evaluate OX40-OX40L interaction in mouse models?

Evaluating OX40-OX40L interactions in mouse models can be approached through several complementary methods:

Binding Assays: Immobilized Mouse OX40 Ligand with hFc Tag (5μg/ml) can be used in plate-based assays to generate dose-response curves with biotinylated Mouse OX40 (His-tagged). The EC50 of this interaction has been measured at approximately 1.36 μg/ml by ELISA . Similarly, immobilized OX40 His at 2 μg/mL can bind Biotin-OX40L His with a linear range of 1.22-19.53 ng/mL .

Functional Assays: Researchers can use anti-OX40 antibodies with agonist activity to stimulate OX40 signaling. The ED50 for this effect typically ranges from 0.4-1.2 μg/mL . These assays can measure downstream effects such as T cell proliferation, cytokine production, or survival.

In vivo Models: OX40-OX40L interactions can be studied through knockout models or through blocking/stimulating antibodies administered to mice. These approaches allow for assessment of physiological relevance in immune responses, inflammation, or disease models .

What are the considerations when using mouse TNFRSF4 knockout models?

When working with OX40/TNFRSF4 knockout (KO) mouse models, researchers should consider several important factors:

Phenotypic Characterization: TNFRSF4 KO mice show specific defects in CD4+ T cell responses, which should be thoroughly characterized before using these models for specific disease studies . This includes assessing basal T cell populations, activation status, and responses to various stimuli.

Compensatory Mechanisms: As with many immune receptor knockouts, compensatory upregulation of related costimulatory molecules may occur. Researchers should evaluate the expression of other TNFR family members and costimulatory molecules to account for potential compensatory effects that might confound experimental interpretation .

Strain Background: The genetic background of the knockout mice can significantly influence the phenotype. Researchers should document and consider the strain background when interpreting results and comparing data across studies .

Conditional vs. Global Knockouts: Consider whether a global or conditional knockout is more appropriate for your research question. Cell-specific deletion using Cre-loxP systems can help distinguish direct from indirect effects of OX40 deletion .

How does TNFRSF4 function in mouse models of autoimmunity and inflammation?

TNFRSF4 plays complex roles in mouse models of autoimmunity and inflammation:

Allergic Airway Inflammation: OX40-OX40L signaling has been implicated in the development and persistence of allergic airway inflammation in mouse models of asthma . Blocking this pathway can reduce inflammatory cell infiltration and cytokine production in the airways.

Autoimmune Diseases: Studies show OX40-OX40L interactions contribute to pathogenesis in multiple autoimmune disease models including experimental autoimmune encephalomyelitis (EAE, a multiple sclerosis model), collagen-induced arthritis, and inflammatory bowel disease models. In these contexts, OX40 signaling typically promotes the expansion and survival of autoreactive T cells .

Graft-versus-Host Disease (GVHD): The OX40-OX40L pathway contributes to GVHD pathogenesis in mouse models of allogeneic transplantation. Interrupting this pathway can reduce disease severity by limiting donor T cell expansion and inflammatory cytokine production .

Regulatory T Cell Function: Though OX40 is constitutively expressed on Tregs and enhances their sensitivity to IL-2 (promoting proliferation), it has also been shown to decrease their immunosuppressive activity on effector T cells . This dual effect creates a complex role in autoimmune regulation that depends on the specific disease context.

What is the current understanding of TNFRSF4 in mouse cancer immunotherapy models?

Research in mouse cancer models has revealed TNFRSF4 as a promising immunotherapeutic target:

Anti-tumor Immunity: OX40 agonists have been shown to augment anti-tumor immunity in several mouse cancer models by enhancing T cell expansion, survival, and effector functions . This approach helps overcome T cell exhaustion and promotes durable anti-tumor responses.

Combination Therapies: Studies indicate that combining OX40 agonists with other immune checkpoint modulators (such as PD-1/PD-L1 blockers) or with conventional therapies (radiation, chemotherapy) often produces synergistic effects in mouse tumor models . These combinations can convert immunologically "cold" tumors to "hot" tumors more susceptible to immune attack.

Memory T Cell Generation: OX40 stimulation enhances the generation of memory T cells in mouse models, which is critical for long-term tumor control and prevention of recurrence . This aspect is particularly important for developing durable cancer immunotherapies.

Tumor Microenvironment Modulation: Beyond direct T cell effects, OX40 engagement can reshape the tumor microenvironment by altering cytokine profiles and reducing immunosuppressive cell populations in mouse models .

How do OX40 agonists and antagonists affect immune responses in mouse models?

OX40 agonists and antagonists demonstrate distinct immunomodulatory effects in mouse models:

Agonists:

• Enhance T cell survival and expansion, particularly of antigen-specific CD4+ and CD8+ T cells

• Increase cytokine production, especially Th1 and Th2 cytokines depending on the context

• Promote memory T cell generation and long-term immunity

• Typical ED50 for agonist antibodies ranges from 0.4-1.2 μg/mL in functional assays

• Can diminish Treg suppressive function while potentially expanding Treg populations

Antagonists:

• Reduce inflammatory responses in autoimmune and allergic disease models

• Decrease T cell-mediated tissue damage in graft rejection models

• Limit pathogenic T cell expansion and cytokine production

• Particularly effective at preventing costimulatory signals during initial T cell activation

• Help maintain tolerance in models of autoimmunity

The choice between agonist and antagonist approaches depends on the desired outcome: enhancing immunity (cancer, infections) or suppressing immunity (autoimmunity, transplantation) .

What signaling pathways are activated downstream of mouse TNFRSF4?

Mouse TNFRSF4 engagement activates several key signaling pathways:

TRAF-Mediated Signaling: Upon OX40L binding, OX40 recruits TNFR-associated factors (TRAFs), particularly TRAF2, TRAF3, and TRAF5, to form a TCR-independent signaling complex . This recruitment is mediated by the cytoplasmic QEE motif present in the mouse OX40 protein.

NF-κB Pathway: A primary downstream effect of OX40 engagement is activation of both canonical and non-canonical NF-κB pathways. One component of the signaling complex, PKCθ, is particularly important for NF-κB activation . This leads to expression of survival factors and inflammatory mediators.

PI3K/Akt Pathway: OX40 signaling enhances the PI3K/Akt pathway, which can directly augment TCR signaling. This pathway promotes T cell survival by upregulating anti-apoptotic molecules like Bcl-2 and Bcl-xL .

MAPK Pathways: OX40 activates multiple MAPK pathways including p38, ERK, and JNK, contributing to T cell proliferation, differentiation, and cytokine production.

Understanding these pathways is crucial for developing targeted immunomodulatory strategies and predicting potential off-target effects of OX40-directed therapies .

How do mouse models inform the therapeutic targeting of TNFRSF4 in human disease?

Mouse models have provided critical insights for therapeutic targeting of TNFRSF4 in human disease:

Translational Considerations: Despite the 63% sequence homology between mouse and human OX40 , researchers must carefully interpret mouse model results when translating to human applications. Differences in expression patterns, binding affinities, and downstream signaling can affect therapeutic outcomes.

Predictive Biomarkers: Studies in mouse models have helped identify potential biomarkers that predict response to OX40-targeted therapies, including baseline OX40 expression levels on tumor-infiltrating lymphocytes and the composition of the tumor microenvironment .

Toxicity Profiles: Mouse models have revealed potential on-target toxicities of OX40 modulation, particularly autoimmune-like phenomena. These observations have informed safety monitoring in human clinical trials of OX40-targeted agents .

Combination Strategies: Mouse studies demonstrating synergy between OX40 modulation and other immunotherapies (checkpoint inhibitors, cytokine therapies, conventional treatments) have directly informed the design of human clinical trials testing similar combinations .

Dosing and Scheduling: Mouse pharmacodynamic studies have provided insights into optimal dosing schedules for OX40-targeted therapies, revealing the importance of timing relative to antigen exposure and other treatments .

What are the methodological challenges in studying TNFRSF4 in complex mouse disease models?

Researchers face several methodological challenges when studying TNFRSF4 in complex mouse disease models:

Temporal Expression Dynamics: Since OX40 is only expressed 24-72 hours after T cell activation , experimental timing is critical. Researchers must carefully design sampling timepoints to capture the relevant biology, particularly in acute disease models.

Distinguishing Cell-Type Specific Effects: OX40 is expressed on multiple T cell subsets including conventional CD4+, CD8+, and regulatory T cells . Cell-specific conditional knockout models or adoptive transfer approaches may be necessary to dissect the relative contribution of OX40 signaling in each population.

Ligand Availability: The regulated expression of OX40L on antigen-presenting cells creates another layer of complexity . Researchers must consider both receptor and ligand expression patterns when interpreting results from disease models.

Technical Detection Limitations: Flow cytometric detection of OX40 requires careful optimization of staining protocols and appropriate controls, as demonstrated in the protocols for staining membrane-associated proteins . Similarly, ELISA measurements should account for sample type-specific matrix effects, as indicated by the validation data showing different detection ranges depending on sample type .

What are the optimal storage and handling conditions for mouse TNFRSF4 reagents?

Proper storage and handling of mouse TNFRSF4 reagents is critical for maintaining their functionality:

Recombinant Proteins: Lyophilized recombinant mouse OX40 proteins should be stored at -20°C to -80°C for up to 12 months from the date of receipt. After reconstitution, aliquoting is strongly recommended to minimize freeze-thaw cycles, with reconstituted protein stable at -80°C for approximately 3 months . Reconstitution to concentrations exceeding 100 μg/ml is typically recommended for optimal stability .

Antibodies: For anti-mouse OX40/TNFRSF4 antibodies, storage at -20°C to -70°C in a manual defrost freezer is recommended, with stability for up to 12 months from the date of receipt. Once reconstituted, antibodies maintain stability for approximately 1 month at 2-8°C under sterile conditions, or 6 months at -20°C to -70°C .

ELISA Kits: Components must be stored according to manufacturer instructions, typically with detection antibodies and standards requiring -20°C storage, while plate components and buffers can often be stored at 2-8°C .

Avoid Repeated Freeze-Thaw Cycles: This is particularly important for all protein reagents, as repeated freezing and thawing can lead to protein denaturation, aggregation, and loss of biological activity .

How should researchers validate mouse TNFRSF4 antibodies for specific applications?

Thorough validation of mouse TNFRSF4 antibodies is essential for generating reliable research data:

Positive and Negative Controls: Always include appropriate positive controls (activated mouse T cells or recombinant OX40 protein) and negative controls (naive T cells or isotype-matched control antibodies). For flow cytometry, comparing staining patterns between activated splenocytes and appropriate isotype controls is essential, as demonstrated in the protocols using Goat Anti-Mouse OX40/TNFRSF4 PE-conjugated Antibody alongside Normal Goat IgG Phycoerythrin Control .

Cross-Reactivity Testing: Evaluate potential cross-reactivity with related TNFR family members. For example, some anti-mouse OX40 antibodies show less than 2% cross-reactivity with related proteins like mouse EDAR and mouse 4-1BB in direct ELISAs and Western blots .

Application-Specific Validation: Validate antibodies specifically for the intended application. An antibody that works well for Western blotting may not be suitable for flow cytometry or functional assays. For example, antibodies used for agonist activity should be validated specifically for this purpose, with typical ED50 values in the range of 0.4-1.2 μg/mL .

Titration Experiments: Perform antibody titrations to determine optimal concentrations for each specific application and cell type. This is particularly important for flow cytometry and functional assays where antibody concentration can significantly impact results .

What technical considerations are important when measuring soluble mouse TNFRSF4 in biological samples?

Measuring soluble mouse TNFRSF4 in biological samples requires attention to several technical considerations:

Sample Type Variability: Different biological sample types (serum, plasma, cell culture supernatants, tissue homogenates) can affect assay performance. Validation data for mouse OX40 ELISA shows variation in detection parameters across sample types, with precision (CV) values ranging from 3.4% to 6.9% depending on the sample matrix .

Sample Collection and Processing: Standardize collection procedures, including anticoagulant use for plasma (EDTA or heparin), processing times, and storage conditions. For example, ELISA kits have been validated for both EDTA and heparin plasma samples .

Assay Sensitivity and Range: Choose methods with appropriate sensitivity for the expected concentration range in your samples. Mouse OX40 ELISA kits typically have detection ranges that can measure from approximately 224 pg/mL to 1463 pg/mL .

Interference Testing: Consider potential interference from heterophilic antibodies, rheumatoid factor, or other components in complex biological samples that might affect measurement accuracy .

Standard Curve Optimization: Prepare standards in the same matrix as samples when possible, or use appropriate diluents that mimic the sample matrix to minimize matrix effects .

Validation Across Sample Types: When transitioning to a new sample type, perform spike-and-recovery experiments to validate the assay in the specific matrix of interest .