Recombinant Human Tumor necrosis factor receptor superfamily member 9 (TNFRSF9), partial (Active)

What is TNFRSF9 and what are its alternative nomenclatures in scientific literature?

TNFRSF9, also known as 4-1BB, CD137, CDw137, and ILA (Induced By Lymphocyte Activation), is an inducible co-stimulatory receptor primarily expressed on activated T cells. It functions as a critical mediator in T cell activation, proliferation, differentiation, and apoptosis processes. TNFRSF9 also plays significant roles in the pathogenesis of certain autoimmune diseases and mediates anti-tumor immune responses of T cells and NK cells .

Researchers should be aware that literature searches may require using multiple nomenclatures to ensure comprehensive review of existing research. When reporting experimental findings, it is recommended to include all relevant nomenclatures in keywords and abstracts to enhance discoverability.

What is the molecular structure of recombinant human TNFRSF9 protein used in research applications?

Commercially available recombinant human TNFRSF9 typically consists of amino acids Leu24-Gln186 of the native protein. The recombinant form often contains an N-terminal linker and a C-terminal tag (commonly a 10xHis-tag) when expressed in mammalian cells. The protein has an apparent molecular weight of approximately 28 kDa on gel electrophoresis and demonstrates high purity (>95%) with low endotoxin content (<1EU/μg) in research-grade preparations .

For functional validation, recombinant TNFRSF9 can bind to its ligand TNFSF9 with EC50 values ranging from 1.011-3.702 ng/mL in ELISA assays, confirming biological activity suitable for immune and antibody screening experiments .

How should researchers assess functional activity of recombinant TNFRSF9 preparations?

Methodological approach for functional assessment:

• Perform binding assays with natural ligand TNFSF9 using ELISA to determine EC50 values

• Verify protein integrity via Western blot analysis under reducing and non-reducing conditions

• Conduct cellular activation assays using TNFRSF9-responsive T cell lines

• Analyze downstream signaling events (NF-κB activation, cytokine production)

• Compare activity with established reference standards

Functional recombinant TNFRSF9 should demonstrate consistent binding kinetics with its ligand and induce expected biological responses in relevant cell systems .

How does TNFRSF9 deficiency impact immune responses, particularly against viral pathogens?

TNFRSF9 deficiency presents a critical research model for understanding this receptor's immunological significance. Case studies of biallelic TNFRSF9 mutations reveal that CD137 deficiency severely compromises host defense against viral infections, particularly Epstein-Barr virus (EBV).

Immunological consequences include:

• Markedly reduced or abrogated expression of CD137 on activated T, B, and NK cells

• Impaired CD8+ T cell activation and function

• Reduced expression and release of critical cytokines (IFN-γ, TNF-α)

• Diminished expression of cytolytic molecules (perforin, granzyme B)

• Compromised cytotoxic activity against infected cells

These observations highlight TNFRSF9's essential role in anti-viral immunity, particularly for controlling persistent viral infections. Researchers investigating primary immunodeficiencies should consider TNFRSF9 deficiency in patients presenting with severe EBV-associated lymphoproliferative disorders (LPD) .

What experimental approaches can elucidate TNFRSF9 signaling mechanisms in immune cells?

To comprehensively investigate TNFRSF9 signaling pathways, researchers should employ a multi-modal approach:

• Genetic manipulation techniques:

  • CRISPR/Cas9-mediated gene editing of TNFRSF9 or downstream effectors

  • Overexpression systems using wildtype or mutant TNFRSF9 constructs

  • siRNA/shRNA knockdown for transient signaling studies

• Biochemical signaling analysis:

  • Phosphoproteomic analysis of downstream effectors (p38MAPK pathway)

  • Co-immunoprecipitation to identify novel interaction partners

  • Western blot analysis of activated signaling components

• Functional readouts:

  • Cytokine production profiling (IFN-γ, TNF-α, IL-2)

  • Cytotoxicity assays against relevant targets

  • Proliferation and survival assays following TNFRSF9 engagement

• Advanced imaging:

  • Live-cell imaging of TNFRSF9 clustering and immunological synapse formation

  • Super-resolution microscopy to visualize receptor distribution patterns

Research has established that TNFRSF9 signaling impacts p38MAPK phosphorylation, which subsequently regulates PAX6 expression. This signaling axis represents a targetable pathway in diseases where TNFRSF9 function is dysregulated .

How does TNFRSF9 expression influence tumor progression and what are the underlying mechanisms?

TNFRSF9 demonstrates significant tumor-suppressive functions in several cancer models, particularly breast cancer. The mechanisms through which TNFRSF9 regulates tumor progression include:

• Regulation of the p38MAPK/PAX6 signaling axis:

  • TNFRSF9 downregulation increases phosphorylated p38 (p-p38) levels

  • Elevated p-p38 subsequently upregulates PAX6 expression

  • PAX6 promotes tumor cell proliferation and invasion

  • P38 phosphorylation inhibitors can reverse these effects, suppressing proliferation and invasion while promoting apoptosis

• Expression patterns in tumor microenvironments:

  • TNFRSF9 is frequently downregulated in breast cancer tissues and cell lines

  • Knockdown of TNFRSF9 promotes breast cancer cell development

  • In melanoma brain metastases, TNFRSF9 is expressed on tumor cells, endothelial cells, and tumor-infiltrating lymphocytes

• Spatial expression patterns:

  • In some tumors, TNFRSF9 expression increases with distance from blood vessels

  • This pattern may be linked to hypoxia-driven regulation of TNFRSF9

  • The relationship between hypoxia and TNFRSF9 expression warrants further investigation

These findings position TNFRSF9 as a potential biomarker and therapeutic target in cancer research.

What methodological approaches should researchers use to study TNFRSF9 expression in tumor samples?

When investigating TNFRSF9 expression in tumor tissues, researchers should implement a comprehensive analysis strategy:

• Tissue preparation and analysis:

  • Formalin-fixed paraffin-embedded (FFPE) and fresh-frozen tissue processing

  • Laser capture microdissection for isolation of specific cellular compartments

  • Multiplex immunohistochemistry to simultaneously assess TNFRSF9 expression with other markers

  • Spatial transcriptomics to map expression patterns relative to vascular structures and hypoxic regions

• Expression quantification:

  • Digital pathology with automated quantification algorithms

  • Cell-type specific scoring systems (tumor cells vs. endothelial cells vs. immune infiltrates)

  • Gradient analysis to assess expression patterns relative to blood vessels or hypoxic regions

• Correlation with clinical parameters:

  • Tumor size, grade, and stage

  • Mutation status of relevant cancer genes (e.g., BRAF V600E in melanoma)

  • Patient survival and treatment response data

  • Immune infiltration patterns and markers

• Epigenetic analysis:

  • DNA methylation profiling of the TNFRSF9 promoter

  • Chromatin accessibility studies (ATAC-seq)

  • Histone modification analysis at the TNFRSF9 locus

This systematic approach enables comprehensive characterization of TNFRSF9 expression patterns in tumors and facilitates identification of clinically relevant associations .

How is TNFRSF9 being utilized in CAR-T cell therapy design and what are the key considerations?

TNFRSF9 (CD137/4-1BB) has emerged as a crucial component in second-generation chimeric antigen receptor (CAR) T cell therapy designs. Researchers should consider the following aspects when incorporating TNFRSF9 into CAR constructs:

• Structural integration:

  • Position of the TNFRSF9 co-stimulatory domain relative to CD3ζ signaling domain

  • Optimization of linker sequences between domains

  • Potential for domain mutations to enhance or modulate signaling strength

• Functional characteristics of TNFRSF9-containing CARs:

  • Enhanced T cell persistence compared to CD28-based CARs

  • Balanced effector-memory phenotype development

  • Reduced T cell exhaustion during repeated antigen exposure

  • Distinct cytokine production profiles compared to other co-stimulatory domains

• Experimental validation approaches:

  • In vitro cytotoxicity against target cells expressing varying antigen densities

  • Long-term persistence assays in serial re-stimulation models

  • Detailed phenotypic characterization using multiparameter flow cytometry

  • In vivo efficacy and persistence in relevant preclinical models

• Clinical translation considerations:

  • Optimized manufacturing protocols specific for TNFRSF9-containing CARs

  • Monitoring for unique toxicity profiles

  • Biomarkers to predict and assess response to TNFRSF9-based CAR-T cells

TNFRSF9 incorporation in CAR-T cells has demonstrated significant advantages for treating certain malignancies, particularly in terms of long-term persistence and memory formation, making it a valuable component in current CAR designs .

What are the emerging strategies for therapeutic targeting of TNFRSF9 in cancer immunotherapy?

Several innovative approaches are being explored for therapeutic targeting of TNFRSF9 in cancer immunotherapy:

• Agonistic antibodies:

  • Development of antibodies with optimal binding kinetics and epitope specificity

  • Engineering of antibody Fc regions to enhance or minimize FcγR engagement

  • Bispecific antibody formats linking TNFRSF9 engagement to tumor targeting

• Targeted delivery strategies:

  • Antibody-cytokine fusion proteins (immunocytokines)

  • Tumor-targeting scFv:TNF fusion proteins that enhance TNFRSF9 signaling

  • Protease-activated prodrug approaches using tumor-selective proteases

  • Hypoxia-responsive TNFRSF9 targeting agents based on observed hypoxia-driven expression

• Combination therapeutic approaches:

  • Synergistic pairing with checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Combination with conventional therapies (radiation, chemotherapy)

  • Sequential timing strategies to optimize immune activation

  • Coupling with NF-κB inhibitors or proteasome inhibitors to modulate signaling outcomes

• Biomarker development:

  • TNFRSF9 DNA methylation as a predictive biomarker for immunotherapy response

  • Expression patterns on tumor and immune cells as response indicators

  • Soluble TNFRSF9 as a potential blood-based biomarker

These approaches highlight the complexity and potential of TNFRSF9-targeted immunotherapeutic strategies, with particular emphasis on overcoming previous clinical challenges related to toxicity and efficacy .

How do genetic variations in TNFRSF9 influence protein function and disease susceptibility?

Genetic variations in TNFRSF9 have significant implications for protein function and disease susceptibility, as evidenced by recent discoveries:

• Pathogenic mutations:

  • Biallelic heterozygous mutations (e.g., NM_001561.5: c.208+1→AT and c.452C>A [p.T151K]) can cause CD137 deficiency

  • These hypomorphic mutations result in markedly reduced or abrogated CD137 expression on activated immune cells

  • Functional consequences include impaired T cell activation, reduced cytokine production, and diminished cytotoxic activity

  • Clinical manifestations include increased susceptibility to severe EBV-associated lymphoproliferative disease

• Experimental approaches to study genetic variations:

  • Site-directed mutagenesis to generate TNFRSF9 variants

  • Stable cell line creation expressing wild-type vs. mutant TNFRSF9

  • CRISPR/Cas9 knock-in models recapitulating human mutations

  • Patient-derived primary cell analyses

• Structure-function relationship analyses:

  • Mapping mutations to functional domains

  • Computational modeling of protein structural changes

  • Ligand binding assays for variant proteins

  • Signaling pathway activation assessments

• Clinical correlation strategies:

  • Genotype-phenotype correlation studies

  • Response to immunomodulatory therapies based on variant status

  • Development of personalized therapeutic approaches for patients with TNFRSF9 mutations

Understanding these genetic variations provides critical insights for researchers investigating primary immunodeficiencies and developing targeted therapeutics for patients with TNFRSF9-related disorders .

What is the significance of TNFRSF9 DNA methylation patterns in cancer biology and immunotherapy response?

TNFRSF9 DNA methylation represents an emerging epigenetic biomarker with significant implications for cancer biology and immunotherapy response prediction:

• Methylation analysis methodologies:

  • Bisulfite sequencing of TNFRSF9 promoter and regulatory regions

  • Methylation-specific PCR for targeted analysis

  • Genome-wide methylation arrays incorporating TNFRSF9 CpG sites

  • Integration with transcriptomic data to correlate methylation with expression

• Cancer-specific methylation patterns:

  • Differential methylation between tumor and normal adjacent tissues

  • Correlation with TNFRSF9 expression levels in various tumor types

  • Association with tumor progression and clinical outcomes

  • Relationship to tumor immune microenvironment characteristics

• Predictive value for immunotherapy:

  • Correlation between TNFRSF9 methylation status and response to immune checkpoint inhibitors

  • Integration into multiparameter predictive models

  • Potential as a companion diagnostic for TNFRSF9-targeted therapies

  • Longitudinal assessment during treatment to monitor response

• Mechanistic insights:

  • Relationship between hypoxia and TNFRSF9 methylation

  • Impact of epigenetic modifying drugs on TNFRSF9 expression

  • Interaction with transcription factors and chromatin remodeling complexes

These findings provide rationale for further investigating TNFRSF9 DNA methylation as a predictive biomarker for immunotherapy response, potentially enabling more precise patient selection for various immunotherapeutic approaches .

What are the optimal storage and handling conditions for recombinant TNFRSF9 protein?

Proper storage and handling of recombinant TNFRSF9 protein is critical for maintaining biological activity and experimental reproducibility:

• Storage recommendations:

  • Store lyophilized recombinant TNFRSF9 at -20°C

  • Avoid repeated freeze-thaw cycles of reconstituted protein

  • For reconstituted protein, prepare single-use aliquots

  • Typical formulation includes PBS (pH 7.4) with stabilizers such as 0.01% SKL and 5% trehalose

• Reconstitution protocols:

  • Use sterile, filtered buffer to reconstitute lyophilized protein

  • Allow protein to equilibrate to room temperature before reconstitution

  • Gently mix by swirling rather than vortexing to prevent protein denaturation

  • Briefly centrifuge to collect all material at the bottom of the vial

• Quality control measures:

  • Verify protein concentration after reconstitution

  • Perform activity testing using standardized binding assays

  • Check for aggregation using dynamic light scattering

  • Monitor endotoxin levels for experiments sensitive to bacterial contaminants

• Shipping and transfer conditions:

  • Transport on blue ice to maintain temperature

  • Validate activity after shipping or transfer between laboratories

  • Document freeze-thaw cycles and storage durations

Adherence to these storage and handling guidelines will ensure optimal experimental outcomes when working with recombinant TNFRSF9 protein .

How can researchers effectively design experiments to investigate the TNFRSF9/p38MAPK/PAX6 signaling axis?

Investigation of the TNFRSF9/p38MAPK/PAX6 signaling axis requires a systematic experimental approach:

• Pathway validation experiments:

  • siRNA/shRNA knockdown of TNFRSF9 followed by assessment of p-p38 and PAX6 levels

  • Overexpression of TNFRSF9 with concurrent monitoring of downstream components

  • P38 inhibitor studies (e.g., SB203580) to verify the dependence of PAX6 upregulation on p38 phosphorylation

  • Rescue experiments using constitutively active or dominant-negative constructs

• Signaling kinetics analysis:

  • Time-course experiments following TNFRSF9 activation

  • Quantitative assessment of phosphorylation status at multiple timepoints

  • Single-cell analysis to account for heterogeneity in signaling responses

  • Correlation between signaling dynamics and functional outcomes

• Cell models and conditions:

  • Comparison across multiple cell lines with varying baseline TNFRSF9 expression

  • Primary cells versus established cell lines

  • Consideration of microenvironmental factors (hypoxia, inflammatory cytokines)

  • 3D culture systems to better recapitulate in vivo signaling

• Functional readouts:

  • Cell proliferation, invasion, and apoptosis assays

  • Gene expression profiling to identify additional pathway components

  • Interaction with other signaling pathways (crosstalk mapping)

  • Systems biology approaches to model pathway dynamics

This comprehensive approach enables detailed characterization of this important signaling axis and provides opportunities to identify novel therapeutic targets for diseases where TNFRSF9 function is dysregulated .

What are the emerging technologies for studying TNFRSF9 function in complex tissue microenvironments?

Cutting-edge technologies are revolutionizing the study of TNFRSF9 function within complex tissue microenvironments:

• Spatial multi-omics approaches:

  • Spatial transcriptomics to map TNFRSF9 expression patterns relative to vascular structures and cell types

  • Multiplexed ion beam imaging (MIBI) for simultaneous detection of multiple proteins

  • Imaging mass cytometry for high-dimensional spatial phenotyping

  • Digital spatial profiling to quantify protein and RNA abundance with spatial context

• Advanced organoid and tissue models:

  • Patient-derived tumor organoids with preserved immune components

  • Microfluidic organ-on-chip systems modeling tumor-immune interactions

  • 3D bioprinting of tissue models with controlled TNFRSF9 expression

  • Ex vivo tissue slice cultures maintaining native architecture

• Single-cell analysis platforms:

  • Single-cell RNA sequencing with TNFRSF9 pathway gene signatures

  • CyTOF mass cytometry for high-parameter protein profiling

  • Cellular indexing of transcriptomes and epitopes (CITE-seq)

  • Spatial single-cell sequencing technologies

• In vivo imaging technologies:

  • Intravital microscopy with fluorescently labeled TNFRSF9 antibodies

  • PET imaging with radiolabeled TNFRSF9-targeting agents

  • Photoacoustic imaging for deep tissue visualization

  • Bioluminescence resonance energy transfer (BRET) systems for pathway activation monitoring

These technologies provide unprecedented insights into TNFRSF9 biology within native tissue contexts, enabling more translational research approaches .

How might TNFRSF9-based therapies be optimized to overcome current limitations in clinical applications?

Optimization of TNFRSF9-based therapies requires innovative approaches to address current clinical limitations:

• Reducing systemic toxicity:

  • Development of tumor-selective activation strategies

  • Incorporation of protease-sensitive linkers activated in tumor microenvironments

  • Engineering of prodrug-like antibody formats with inhibitory domains shed at tumor sites

  • Spatially restricted activation through tumor-targeting bispecific constructs

• Enhancing efficacy:

  • Rational combination with checkpoint inhibitors or conventional therapies

  • Optimization of dosing schedules and administration routes

  • Development of TNFRSF9 agonists with optimized receptor crosslinking properties

  • Engineering of novel scaffold formats beyond traditional antibodies

• Precision medicine approaches:

  • Implementation of TNFRSF9 methylation as a biomarker for patient selection

  • Development of companion diagnostics to identify optimal responders

  • Monitoring of tumor immune microenvironment changes during treatment

  • Adaptive trial designs with biomarker-guided treatment modifications

• Manufacturing and stability improvements:

  • Enhanced production systems for complex biologics

  • Formulation optimization for improved stability and delivery

  • Development of extended-release or depot formulations

  • Novel delivery systems for improved tumor penetration