TNFRSF14 Human, His

What is TNFRSF14 and what are its major functional domains?

TNFRSF14, also known as Herpesvirus Entry Mediator (HVEM), is a human cell surface receptor belonging to the TNF-receptor superfamily. It serves as a receptor for two TNF superfamily ligands: lymphotoxin α (TNF-β) and LIGHT (TNFSF14), which initiate positive signaling pathways . The protein consists of an extracellular domain (amino acids Leu39-Val202 in the mature protein) that mediates ligand binding, a transmembrane domain, and a cytoplasmic region that facilitates downstream signaling . When expressed as a recombinant protein with a histidine tag, the theoretical molecular weight is approximately 18.5 kDa, though the observed weight in human tissues is around 26.5 kDa due to post-translational modifications .

How does TNFRSF14 function within the immune system?

TNFRSF14 plays a complex role in immune regulation by delivering negative signals to T cells through interaction with the B and T Lymphocyte Attenuator (BTLA) molecule . This interaction is critical for maintaining immune homeostasis and preventing excessive T-cell activation. Simultaneously, TNFRSF14 can mediate positive signaling through interactions with LIGHT and lymphotoxin α. The receptor is expressed at high levels on activated monocytes, macrophages, and THP-1 cells (a human macrophage-like cell line), suggesting a significant role in myeloid cell function . Research has shown that TNFRSF14 activation in monocytes/macrophages can induce the expression of proatherogenic cytokines and matrix metalloproteinases, highlighting its importance in inflammatory processes .

What methodologies effectively quantify TNFRSF14 expression in tissue samples?

For quantitative analysis of TNFRSF14 expression in tissue samples, researchers should employ:

• Flow cytometry: Using fluorescently-labeled antibodies against TNFRSF14, expression can be quantified at the single-cell level. This approach has successfully demonstrated that approximately 50% of follicular lymphoma B cells express HVEM at levels above isotype control .

• Immunohistochemistry: This method is particularly useful for analyzing TNFRSF14 expression patterns within tissue architecture. Studies have utilized this technique to reveal high expression levels in the shoulder regions of atherosclerotic plaques, particularly in areas rich in foam cells and HLA-DR–positive cells .

• Western blot analysis: This approach has been effective for comparing TNFRSF14 expression between different regions of atherosclerotic plaques, revealing higher expression in atheromatous regions compared to fibrous regions .

Each method provides distinct advantages, and selection should be based on experimental requirements for spatial resolution versus quantitative precision.

How do TNFRSF14 genetic aberrations affect its function in immune responses?

TNFRSF14 genetic aberrations, found in approximately 40% of follicular lymphoma (FL) patients, significantly impact immune function in several ways:

• Expression levels: FL B cells with dual TNFRSF14 aberrations show virtually undetectable HVEM expression compared to wild-type cells where ~50% express HVEM at levels above isotype control. Single aberrations result in intermediate expression levels .

• Allogeneic T-cell stimulation: FL B cells with dual TNFRSF14 aberrations demonstrate enhanced capacity to stimulate allogeneic T-cell responses. Specifically:

  • Higher frequencies of alloreactive CD4+ T cells (17% vs. 8% in primary allostimulation, 28% vs. 13% in secondary allostimulation)

  • Modest increase in activated alloreactive CD8+ T cells

• Clinical implications: These aberrations associate with higher incidence of acute graft-versus-host disease (GVHD) in patients undergoing allogeneic hematopoietic stem cell transplantation, suggesting practical applications for stratified transplantation approaches .

This represents the first demonstration of how an acquired genetic lesion impacts tumor cells' ability to stimulate allogeneic T-cell immune responses, with significant implications for adoptive immunotherapy strategies.

What experimental design considerations are crucial when evaluating TNFRSF14 as an immune checkpoint target?

Researchers evaluating TNFRSF14 as an immune checkpoint target should incorporate these methodological considerations:

• Genetic modification models: Use CRISPR/Cas9 genetic editing to establish TNFRSF14-deficient tumor cell lines for comparison with TNFRSF14-expressing lines in appropriate animal models (e.g., PBMC-humanized NSG mice) .

• Antibody characterization: When developing blocking antibodies, thoroughly assess:

  • Direct effects on tumor cells versus immune-mediated effects

  • Agonist versus antagonist activity on human T cells

  • Interactions with accessory cells, including macrophages that may participate in tumor control

• Controls: Include tumor lines deficient for TNFRSF14 when testing therapeutic antibodies to confirm that effects are dependent on target expression .

• Microenvironment factors: Consider that the tumor microenvironment may influence TNFRSF14 signaling outcomes, as studies have shown different effects in humanized versus non-humanized mouse models .

• In vitro validation: Complement in vivo studies with in vitro analyses of antibody effects on tumor survival, with and without relevant immune cell populations .

How can researchers optimize quality control for recombinant TNFRSF14-His protein?

To ensure high-quality recombinant TNFRSF14-His protein for research applications, implement this comprehensive quality control pipeline:

These quality control measures, as implemented for commercial TNFRSF14-His preparations, ensure that experimental results accurately reflect the biological properties of the protein rather than artifacts from the production process .

What mechanisms explain TNFRSF14's role in atherosclerosis?

TNFRSF14 contributes to atherosclerosis through several coordinated mechanisms:

• Expression pattern: Immunohistochemical analysis reveals high TNFRSF14 expression in atherosclerotic plaque regions rich in macrophages/foam cells, particularly in shoulder regions. Expression is specifically observed in CD68-positive foam cells and is absent in normal human aorta .

• Cytokine induction: Activation of TNFRSF14 in THP-1 cells (macrophage-like cell line), particularly in combination with IFN-γ, induces synergistic production of proatherogenic cytokines including TNF-α and interleukin-8 .

• Matrix remodeling: TNFRSF14 activation induces expression of matrix metalloproteinases (MMP-1, MMP-9, MMP-13) and tissue inhibitors of metalloproteinase-1 and -2, potentially decreasing plaque stability by promoting extracellular matrix degradation .

• Ligand interaction: TNFSF14 (LIGHT), the ligand for TNFRSF14, is expressed at higher levels in inflammatory regions of atherosclerotic plaques. This interaction likely stimulates TNFRSF14-mediated inflammatory reactions in foam cells .

• Cellular activation: Upregulation of TNFRSF14 occurs concomitantly with CD14 (a monocyte activation marker) in activated peripheral blood monocytes, suggesting its regulation is linked to monocyte activation states .

These findings collectively indicate that TNFRSF14 mediates atherosclerosis by orchestrating proatherogenic cytokine production and matrix degradation in activated monocytes/macrophages within plaque microenvironments.

How does TNFRSF14 function as a targetable immune checkpoint in cancer?

TNFRSF14 functions as a targetable immune checkpoint through these mechanisms:

• Negative T-cell signaling: TNFRSF14 delivers negative signals to T cells through interaction with BTLA, creating an immunosuppressive environment that benefits tumor growth .

• Prognostic association: TNFRSF14 expression has been associated with worse prognosis in various malignancies, supporting its role in tumor immune evasion .

• Experimental validation: Genetic deletion of TNFRSF14 or blocking TNFRSF14/BTLA interaction with monoclonal antibodies profoundly impacts tumor growth in mice reconstituted with human T cells, demonstrating its potential as a therapeutic target .

• Mechanism of action: Blocking TNFRSF14 increases proliferation and number of tumor-infiltrating lymphocytes, enhancing anti-tumor immune responses .

• Expression requirement: Therapeutic effects of anti-TNFRSF14 antibodies are lost with TNFRSF14-deficient tumors, confirming that expression by tumor cells is necessary for checkpoint blockade efficacy .

• Macrophage involvement: Surprisingly, anti-TNFRSF14 antibodies also show effects in non-humanized mouse models, suggesting additional mechanisms potentially involving myeloid cells .

These findings establish TNFRSF14 as a novel immune checkpoint with therapeutic potential through both T cell-dependent and potentially myeloid cell-dependent mechanisms.

What are the optimal conditions for producing and purifying functional TNFRSF14-His?

For optimal production of functional TNFRSF14-His protein:

• Expression system: Use human HEK293 cells for expression, as this mammalian system provides appropriate post-translational modifications essential for proper folding and function of human TNFRSF14 .

• Construct design:

  • Target the extracellular domain (Leu39-Val202) of human TNFRSF14

  • Position the His-tag at the C-terminus to minimize interference with ligand binding regions

• Purification strategy: Implement a multi-step purification protocol that achieves >95% purity as determined by both Tris-Bis PAGE and HPLC analysis .

• Quality assessment: Validate functionality through binding assays with known ligands (e.g., CD160) to ensure that the purified protein maintains its biological activity .

• Storage formulation: Lyophilize the purified protein in appropriate buffer conditions that maintain stability and activity upon reconstitution .

This approach has successfully produced recombinant TNFRSF14-His with a theoretical molecular weight of 18.5 kDa and high biological activity as measured by ligand binding assays .

What methodological approaches best detect TNFRSF14 aberrations in patient samples?

To effectively identify and characterize TNFRSF14 aberrations in patient samples:

• Next-generation sequencing: Sequence the TNFRSF14 gene to identify mutations, particularly those resulting in altered protein expression or function. This approach has successfully identified aberrations in 40% of follicular lymphoma patients .

• Flow cytometry: Quantitatively assess HVEM protein expression on cell surfaces using fluorescently-labeled antibodies. This approach effectively distinguishes between wild-type, single aberration, and dual aberration expression patterns .

• Functional validation: Implement in vitro alloreactivity assays to assess the functional impact of identified aberrations. Compare the capacity of wild-type and aberrant TNFRSF14-expressing cells to stimulate allogeneic T-cell responses .

• Phenotypic characterization: Analyze additional cell surface molecules associated with antigen-presenting capacity (MHC class I, MHC class II, CD80, CD86, CD58) to determine if aberrations specifically affect TNFRSF14 or have broader impacts on cell phenotype .

• Clinical correlation: Associate identified aberrations with clinical outcomes, such as incidence of acute GVHD in transplantation settings, to establish clinical relevance .

This multi-modal approach provides comprehensive characterization of both the genetic basis and functional consequences of TNFRSF14 aberrations.

What experimental system best models TNFRSF14-mediated T-cell regulation in vitro?

To effectively model TNFRSF14-mediated T-cell regulation in vitro:

• Allostimulation assays: Use a mixed lymphocyte reaction system where TNFRSF14-expressing or TNFRSF14-deficient cells stimulate allogeneic T cells. This approach has successfully demonstrated that:

  • Wild-type versus TNFRSF14-aberrant cells induce different frequencies of activated T cells

  • CD4+ and CD8+ T cells respond differently to TNFRSF14-mediated regulation

  • Primary and secondary stimulation reveal progressive differences in T-cell activation

• T-cell activation monitoring:

  • Assess CD25 upregulation as a marker of T-cell activation

  • Evaluate regulatory T-cell populations using CD25+CD127lo surface expression pattern and FOXP3 expression

  • Distinguish between regulatory and effector T-cell populations to accurately interpret results

• Influence of inflammatory environment: Model the proinflammatory post-transplant environment through in vitro activation conditions and assess how this affects TNFRSF14 expression and function .

• Antibody blocking studies: Incorporate blocking antibodies against TNFRSF14 or its ligands to dissect specific interaction effects on T-cell activation .

This system effectively recapitulates the impact of TNFRSF14 on T-cell regulation and provides a platform for testing therapeutic interventions targeting this pathway.

How might TNFRSF14-targeted therapies improve transplantation outcomes?

TNFRSF14-targeted therapies could substantially impact transplantation outcomes through several mechanisms:

• Stratified immunosuppression: Patients with TNFRSF14 aberrations experience higher incidence of acute GVHD, suggesting they could benefit from more aggressive immunosuppression regimens to reduce harmful GVHD after transplantation .

• Enhanced donor-recipient matching: Incorporating TNFRSF14 status into matching algorithms could improve transplant compatibility beyond traditional HLA matching, potentially reducing GVHD risk .

• Targeted blockade approach: Developing TNFRSF14 blocking agents could specifically inhibit excessive alloreactive T-cell responses while preserving beneficial immune functions. This approach is supported by in vitro data showing that FL B cells with TNFRSF14 aberrations stimulate greater frequencies of alloreactive effector T cells .

• Preservation of anti-tumor effects: Careful modulation of TNFRSF14 signaling could potentially maintain beneficial graft-versus-tumor effects while reducing harmful GVHD, as TNFRSF14 influences both processes .

• Personalized therapy: TNFRSF14 genetic testing could allow for personalized immunosuppression strategies based on predicted GVHD risk, optimizing the balance between GVHD prevention and maintenance of beneficial immune functions .

These approaches represent promising avenues for improving transplantation outcomes through targeted manipulation of TNFRSF14 signaling pathways.

What are the most promising research directions for TNFRSF14 in cardiovascular disease?

Emerging research on TNFRSF14 in cardiovascular disease suggests these promising research directions:

• Plaque vulnerability biomarkers: TNFRSF14 expression patterns overlap with MMP-1, -9, and -13 in atherosclerotic plaques, suggesting potential as a biomarker for plaque instability and rupture risk .

• Targeted anti-inflammatory therapies: Development of agents that specifically inhibit TNFRSF14-induced production of proatherogenic cytokines (TNF-α, IL-8) in macrophages could provide more selective anti-inflammatory approaches than current therapies .

• Matrix remodeling pathway intervention: TNFRSF14 activation induces both matrix metalloproteinases and their inhibitors, suggesting complex regulation of matrix remodeling. Understanding and modulating this balance could stabilize vulnerable plaques .

• TNFSF14-TNFRSF14 axis targeting: The observed higher expression of TNFSF14 (LIGHT) in inflammatory regions of plaques suggests that disrupting this ligand-receptor interaction could reduce inflammatory processes driving atherosclerosis .

• Monocyte activation modulation: The concomitant upregulation of TNFRSF14 and CD14 during monocyte activation indicates that targeted intervention in this pathway might influence early stages of foam cell formation .

These research directions hold promise for developing novel diagnostic and therapeutic approaches for atherosclerosis by targeting the TNFRSF14 signaling network.

What challenges exist in translating TNFRSF14 research to clinical applications?

Despite promising results, several challenges must be addressed for successful clinical translation of TNFRSF14 research:

• Dual signaling complexity: TNFRSF14 can mediate both positive and negative signaling depending on which ligand it engages. Therapeutic interventions must carefully consider this dual nature to avoid unintended consequences .

• Context-dependent effects: Research indicates that TNFRSF14 blockade has different effects in different models (humanized versus non-humanized), suggesting complex interactions with the microenvironment that must be understood for successful translation .

• Expression heterogeneity: Variations in TNFRSF14 expression between patients and even within different regions of the same tissue (as seen in atherosclerotic plaques) complicate standardization of therapeutic approaches .

• Target specificity: Anti-TNFRSF14 antibodies must be highly specific to avoid off-target effects on related TNF-receptor family members, requiring extensive validation before clinical application .

• Biomarker development: Establishing reliable biomarkers to identify patients most likely to benefit from TNFRSF14-targeted therapies remains challenging but essential for personalized medicine approaches .

Addressing these challenges requires rigorous preclinical validation using diverse experimental models and careful correlation with human pathophysiology.