TNFRSF25 Human

How is TNFRSF25 expression regulated in different cell types?

TNFRSF25 is expressed constitutively in the form of randomly spliced transcripts, primarily in lymphoid cells, but also in other cell types including certain tumor cells . Within the immune compartment, TNFRSF25 expression is particularly important on T cells where it serves as a co-stimulatory receptor . Expression analysis typically employs flow cytometry, with human PBMCs first blocked with human serum before incubation with biotinylated anti-hTNFRSF25 antibodies . The dynamic regulation of TNFRSF25 during immune responses and in pathological conditions remains an active area of investigation for researchers.

What is the identified ligand for TNFRSF25 and how does it activate signaling?

The ligand for TNFRSF25 has been identified as TL1A (TNFSF15) . TL1A has co-stimulatory activity for TNFRSF25-expressing T cells through the activation of NF-κB and suppression of apoptosis by up-regulation of c-IAP2 . This interaction plays a critical role in T-cell activation and immune regulation. TL1A expression can be detected on human umbilical vein endothelial cells and other tissues . Multimeric forms of TL1A have been developed as TNFRSF25 agonists that can co-stimulate CD8+ T cells and reduce tumor growth, even in the absence of Fc-FcγR interactions .

What are the effective approaches for detecting TNFRSF25 expression in experimental settings?

Researchers employ several complementary methodologies to detect TNFRSF25 expression:

• Flow cytometry: EL4 cells stably transduced with TNFRSF25 (EL4-TNFRSF25) can be incubated with anti-TNFRSF25 antibodies at 4°C for 30 minutes, followed by staining with PE-labeled secondary antibodies . For human samples, PBMCs should undergo blocking with 10% human serum before antibody incubation.

• Western blotting: Using verified antibodies against TNFRSF25, such as those available from commercial sources that have been validated for specificity .

• RT-PCR: Analysis of TNFRSF25 mRNA expression using specific primers targeting conserved regions of the transcript.

• Immunohistochemistry: For tissue samples, allowing visualization of TNFRSF25 distribution in situ.

Each method offers distinct advantages, and researchers should select based on their specific experimental questions and available samples.

How can recombinant TNFRSF25 proteins and agonists be generated for functional studies?

The generation of recombinant TNFRSF25 proteins and agonists involves several sophisticated approaches:

• Extracellular domain fusion proteins: The extracellular domain (ECD) of human or mouse TNFRSF25 can be fused to a C-terminal His(×6)-Avi-Tag for purification and detection .

• Fc-fusion proteins: hFc-hTL1A fusion proteins can be generated by adding an N-terminal human IgG1 Fc tag to human TL1A ECD (L72-L251) .

• Multimerization strategies: hFc-TNC-TL1A fusion proteins incorporate a chicken tenascin C (TNC) tag between the hFc tag and TL1A ECD to enhance multimerization and potency .

• Antibody engineering: Agonistic antibodies can be optimized by modifying the CH1-hinge region, as demonstrated with 1A6-m1, where replacing the CH1-hinge with that of human IgG2 enhanced antitumor effects .

• Variable region cloning: The variable heavy chain (VH) and variable light chain (VL) gene sequences of IgG antibodies can be subcloned into appropriate expression vectors for antibody production .

These approaches enable researchers to create tools for studying TNFRSF25 biology and developing potential therapeutic agents.

What experimental models are most suitable for studying TNFRSF25 function in vivo?

Based on published research, several complementary models have proven valuable for TNFRSF25 functional studies:

• Syngeneic mouse tumor models: These provide a physiologically relevant system to evaluate the antitumor effects of TNFRSF25 agonists, including both antibodies and TL1A proteins .

• T-cell expansion models: In vivo models that assess how TNFRSF25 signaling affects T-cell proliferation, activation, and effector function .

• FcγR-deficient mice: Essential for understanding the dependency of TNFRSF25 agonistic antibodies on Fc gamma receptor interactions .

• Allergic lung inflammation models: Used to study the role of TNFRSF25 in Th2-mediated inflammatory responses, particularly relevant for understanding its role in asthma .

• Adoptive transfer models: NKT-deficient mice can become susceptible to lung inflammation upon adoptive transfer of wild-type NKT cells, but remain resistant with transfer of dominant-negative TNFR25 transgenic NKT cells .

Each model system offers distinct advantages, and researchers should carefully select based on their specific research questions.

How does TNFRSF25 activation balance pro-survival versus pro-apoptotic signaling pathways?

TNFRSF25 signaling represents a sophisticated molecular switch between cell death and survival:

• Pro-apoptotic signaling: TNFRSF25 contains a death domain that can recruit TRADD and FADD adaptor proteins, leading to caspase activation and apoptotic cell death . This pathway is activated under specific cellular contexts and can contribute to immune homeostasis.

• Pro-survival signaling: Alternatively, TNFRSF25 can signal through TRADD, TRAF2, and subsequent NF-κB activation to promote cell survival and activation . This pathway is particularly important for T-cell co-stimulation and effector function.

• Regulatory mechanisms: The balance between these opposing outcomes appears context-dependent, influenced by factors including cell type, activation state, and the presence of additional co-stimulatory or co-inhibitory signals.

• Therapeutic implications: Understanding this signaling duality is crucial for therapeutic targeting, as different disease contexts may require either enhancement or inhibition of TNFRSF25 signaling.

Research continues to elucidate the molecular determinants that govern this signaling bifurcation.

What is the role of TNFRSF25 in T helper cell differentiation and cytokine production?

TNFRSF25 plays critical roles in T helper cell biology with significant immunological implications:

• Th2 response regulation: The TNFRSF25/TNFSF15 pair functions as a critical trigger for allergic lung inflammation, a cardinal feature of asthma . TNFR25 signals are required for Th2-polarized CD4 cells to exert their effector function.

• Cytokine production: TNFRSF25 co-stimulates IL-13 production by glycosphingolipid-activated NKT cells and promotes Th2 cytokine production more broadly .

• T-cell activation: Agonistic antibodies like 1A6-m1 activate CD8+ T cells and antigen-specific T cells, leading to enhanced effector function and tumor regression .

• Regulatory T cells: Activating TNFRSF25 by 1A6-m1 expanded splenic regulatory T (Treg) cells, although interestingly it did not influence intratumoral Treg cells . This differential effect has important implications for therapeutic applications.

These findings position TNFRSF25 as a key regulator of T-cell differentiation and function with potential therapeutic applications in allergic conditions and cancer.

How do Fc-FcγR interactions influence the activity of TNFRSF25-targeting antibodies?

Research has revealed complex interactions between antibody Fc regions and Fcγ receptors that fundamentally affect TNFRSF25 targeting:

• Dual receptor requirements: The antitumor effects of the anti-mTNFRSF25 agonistic antibody 1A6-m1 require engagement of both inhibitory FcγRIIB and activating FcγRIII . This dual dependency distinguishes TNFRSF25 from some other TNFR family members.

• Fc engineering impacts: Replacing 1A6-m1's CH1-hinge region with that of human IgG2 (h2) conferred enhanced antitumor effects . This demonstrates how structural modifications to the Fc region can optimize therapeutic efficacy.

• Alternative approaches: Multimeric TL1A fusion proteins can function as TNFRSF25 agonists and reduce tumor growth even in the absence of Fc-FcγR interactions . This provides an alternative strategy when Fc-mediated effects are undesirable.

• Experimental validation: These dependencies have been systematically evaluated using Fc variants and FcγR-deficient mice in both T-cell expansion models and tumor models .

These findings highlight the importance of considering Fc engineering in developing TNFRSF25-targeting therapeutics and suggest strategies for optimizing clinical outcomes.

What mechanisms underlie the antitumor effects of TNFRSF25 agonists?

TNFRSF25 agonists demonstrate antitumor activity through multiple immune-enhancing mechanisms:

• CD8+ T-cell activation: TNFRSF25 agonists directly activate CD8+ T cells, enhancing their cytotoxic function against tumor cells . This represents a primary mechanism of antitumor activity.

• Antigen-specific T-cell responses: Agonistic antibodies like 1A6-m1 enhance antigen-specific T-cell responses, leading to tumor regression . This specificity helps direct the immune response toward malignant cells.

• Long-term immune memory: TNFRSF25 activation induces long-term antitumor immune memory, suggesting potential for durable clinical responses and protection against recurrence .

• Differential effects on Treg populations: Interestingly, while TNFRSF25 agonists expand splenic regulatory T cells, they don't influence intratumoral Treg cells . This selective effect may help preserve antitumor immunity within the tumor microenvironment.

• Minimal observed side effects: TNFRSF25 agonists exhibited antitumor effects in syngeneic mouse tumor models without causing observed side effects , suggesting a potentially favorable therapeutic window.

These multifaceted mechanisms position TNFRSF25 as a promising target for cancer immunotherapy.

How does TNFRSF25 contribute to allergic inflammatory conditions?

TNFRSF25 plays a central role in allergic inflammation through several key mechanisms:

• Th2 response amplification: TNFRSF25/TNFSF15 pair functions as a critical trigger for allergic lung inflammation, a cardinal feature of asthma . TNFR25 signals are required for Th2-polarized CD4 cells to exert their effector function.

• IL-13 production: TNFRSF25 co-stimulates IL-13 production by glycosphingolipid-activated NKT cells . IL-13 is a key mediator of allergic inflammatory responses, particularly in asthma.

• Early signaling events: TNFR25 appears to provide an early signal for Th2 cytokine production in the lung . This temporal positioning makes it a potential upstream target for therapeutic intervention.

• Therapeutic potential: Antibody blockade of TNFSF15 (TL1A) inhibits lung inflammation and production of Th2 cytokines even when administered days after airway antigen exposure . Similarly, blockade of TNFR25 by a dominant-negative transgene confers resistance to lung inflammation in mice .

These findings suggest that, contrary to the agonistic approach used in cancer immunotherapy, antagonizing TNFRSF25 signaling might be beneficial in treating allergic conditions.

What approaches can optimize TNFRSF25-targeting therapeutics for clinical development?

Optimizing TNFRSF25-targeting therapeutics involves several sophisticated strategies:

• Epitope selection: Research identified the anti-mTNFRSF25 agonistic antibody 1A6-m1, which exhibited greater antitumor activity than a higher-affinity antibody targeting an overlapping epitope . This suggests that specific epitope targeting may be more important than mere binding affinity.

• Fc engineering: Replacing 1A6-m1's CH1-hinge region with that of human IgG2 conferred enhanced antitumor effects . This structural modification approach represents a promising optimization strategy.

• Multimerization approaches: For ligand-based therapeutics, enhancing multimerization through protein engineering (such as incorporating TNC domains) improves agonistic activity . These multimeric TL1A fusion proteins can effectively reduce tumor growth.

• FcγR engagement optimization: The antitumor effects of TNFRSF25 agonistic antibodies require engagement of both inhibitory FcγRIIB and activating FcγRIII . Designing antibodies with optimal FcγR binding properties is therefore crucial.

• Combination strategies: Though not explicitly covered in the search results, combining TNFRSF25 agonists with other immunotherapeutic approaches (checkpoint inhibitors, adoptive cell therapies) may enhance efficacy through complementary mechanisms.

These optimization strategies provide a roadmap for developing next-generation TNFRSF25-targeting therapeutics with improved efficacy and safety profiles.

What unresolved questions remain regarding TNFRSF25 biology and function?

Despite significant progress, several fundamental questions about TNFRSF25 remain unanswered:

Addressing these questions will be essential for fully leveraging TNFRSF25 as a therapeutic target.

What methodological advances would accelerate TNFRSF25 research?

Several technological and methodological developments could significantly advance TNFRSF25 research:

• Single-cell analysis techniques: Implementing single-cell RNA sequencing and CyTOF would enable better characterization of TNFRSF25 expression and function across diverse immune cell subsets.

• Humanized mouse models: Developing improved humanized mouse models would enhance the translational relevance of preclinical studies on TNFRSF25-targeting therapeutics.

• Structural biology approaches: Crystal structures of TNFRSF25 in complex with different agonists would facilitate rational design of more effective therapeutic agents.

• CRISPR-based screening: Employing genome-wide CRISPR screens could identify novel regulators and modifiers of TNFRSF25 signaling.

• Ex vivo human systems: Developing robust ex vivo systems using human samples would bridge the gap between animal models and clinical applications.

These methodological advances would provide deeper insights into TNFRSF25 biology and accelerate the development of effective therapeutics.

How might TNFRSF25 targeting be incorporated into combination immunotherapy strategies?

The potential for TNFRSF25 in combination immunotherapy presents exciting opportunities:

• Checkpoint inhibitor combinations: Combining TNFRSF25 agonists with PD-1/PD-L1 or CTLA-4 inhibitors could enhance T-cell activation while simultaneously removing inhibitory signals.

• CAR-T cell therapy enhancement: TNFRSF25 agonists might improve the persistence and effector function of adoptively transferred CAR-T cells through co-stimulatory signaling.

• Radiation therapy combinations: TNFRSF25 agonists could potentially enhance the abscopal effect of radiation therapy by amplifying T-cell responses against tumor antigens released during radiation-induced cell death.

• Cancer vaccine adjuvants: TNFRSF25 agonists might serve as effective adjuvants for cancer vaccines, enhancing antigen-specific T-cell responses.

• Dual-targeting approaches: Bispecific antibodies targeting both TNFRSF25 and tumor-associated antigens could increase specificity and reduce off-target effects.

These combination strategies represent promising directions for enhancing the efficacy of cancer immunotherapy while managing potential adverse effects.