Recombinant Human Tumor necrosis factor ligand superfamily member 9 (TNFSF9), partial (Active)

What is TNFSF9 and what are its key biological functions?

TNFSF9, also known as 4-1BB Ligand or CD137L, is a type II transmembrane protein belonging to the tumor necrosis factor (TNF) superfamily that plays a crucial role in immune response activation. The protein consists of an intracellular domain, a transmembrane segment, and an extracellular domain (ECD) that mediates its biological activity. TNFSF9 binds to its receptor TNFRSF9 (4-1BB/CD137) which is expressed on activated CD4+ and CD8+ T cells, thymocytes, NK cells, monocytes, neutrophils, dendritic cells, and eosinophils . Upon binding, TNFSF9 transduces a co-stimulatory signal that promotes proliferation, activation, and survival of CD4+ and CD8+ T cells .

The protein plays a distinct temporal role in T cell responses, with CD28 being important for initial T cell expansion, while TNFSF9-TNFRSF9 interactions act later in the response . Additionally, this signaling supports the survival and responsiveness of memory T cells during viral infection . TNFSF9 can also induce reverse signaling in monocytes to stimulate inflammatory cytokine production, and in macrophages, TNFSF9 associates with TLR4 to enhance inflammatory responses .

What structural forms of TNFSF9 exist and how do they affect experimental design?

TNFSF9 exists in both membrane-bound and soluble forms, each with distinct biological properties that researchers must consider when designing experiments. The membrane-bound form is approximately 50 kDa and consists of an 82 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 206 amino acid extracellular domain in rat models . A smaller, 26 kDa soluble form can be released from the surface of activated cells while retaining bioactivity .

For experimental applications, recombinant forms typically consist of the extracellular domain with various tags. For example, commercially available human TNFSF9 may include the region from amino acids 71-254 with an N-terminal tag . When designing experiments, researchers should carefully consider which form best represents their biological question of interest. Membrane-bound TNFSF9 studies may require cell-based systems, while soluble TNFSF9 effects can be studied using recombinant proteins in solution.

What is the optimal reconstitution protocol for recombinant TNFSF9?

The optimal reconstitution protocol for recombinant TNFSF9 depends on the specific formulation. For lyophilized preparations, the following methodology is recommended:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water

• For carrier-containing formulations, reconstitute at 250 μg/mL in PBS

• For carrier-free formulations, reconstitute in PBS with consideration for the final application

• Consider adding glycerol (5-50% final concentration) if preparing for long-term storage

For optimal results, avoid repeated freeze-thaw cycles by preparing single-use aliquots. When working with reconstituted protein, maintain at 4°C for up to one week . For experimental consistency, verify protein concentration after reconstitution using appropriate protein assay methods.

How does TNFSF9 expression correlate with tumor immune microenvironment characteristics?

TNFSF9 expression has significant associations with tumor immune microenvironment (TIME) characteristics, particularly in renal cell carcinoma (RCC). High TNFSF9 expression correlates with several important immune parameters:

Gene pathway analysis reveals that tumors with high TNFSF9 expression show upregulation of adaptive immune responses, response to bacteria, and extracellular matrix-associated proteins . This suggests that TNFSF9-high tumors feature enhanced antigen presentation and regulated immune responses. Interestingly, despite having increased CD8+ T cells and interferon-gamma levels, these tumors also contain more dysfunctional T cells and regulatory T cells (Tregs) . This seemingly contradictory finding suggests a complex immune regulatory environment where both immune activation and suppression mechanisms are enhanced.

What methodologies are most effective for studying TNFSF9's role in T-B cell interactions?

The study of TNFSF9's role in T-B cell interactions requires specialized methodologies that can detect subtle changes in cellular interactions and activation states. Based on research findings, the following approaches are recommended:

• Immune Cell Population Analysis: CIBERSORT algorithm analysis reveals that TNFSF9-high tumors have increased T follicular helper (TFH) cells and plasma B cells, suggesting enhanced T-B interactions . Researchers should employ multiparameter flow cytometry with markers for TFH cells (CXCR5, PD-1, ICOS) and B cell subsets.

• Functional Co-stimulation Assays: To measure TNFSF9's co-stimulatory effects, researchers can use recombinant TNFSF9 in combination with anti-CD3 stimulation to assess IL-2 secretion by T cells. The ED50 for this effect is typically 5-30 ng/mL . This approach isolates TNFSF9-specific effects from other co-stimulatory pathways.

• Binding Interaction Studies: Characterize the binding interactions between TNFSF9 and its receptor using functional ELISA methods. Typical protocols involve coating TNFRSF9 at 100 ng/mL and measuring binding of recombinant TNFSF9 with an ED50 of 1-6 ng/mL .

• Gene Expression Pathway Analysis: Tools like Metascape can identify upregulated adaptive immune response pathways in TNFSF9-high samples . This approach contextualizes TNFSF9's role within broader immune signaling networks.

How can TNFSF9 be utilized as a predictive biomarker for immunotherapy response?

TNFSF9 shows significant potential as a predictive biomarker for immunotherapy response, particularly for patients receiving combination nivolumab plus ipilimumab (NIVO+IPI) therapy for metastatic renal cell carcinoma (mRCC). The methodology for developing and utilizing TNFSF9 as a biomarker includes:

• Expression Analysis: In clinical studies, TNFSF9 expression discriminated between response and non-response groups with 88.89% sensitivity and 87.50% specificity (AUC = 0.9444) . This outperformed PD-L1 expression (AUC = 0.75) .

• Threshold Determination: Establishing expression thresholds that optimize predictive value requires ROC curve analysis with validation across multiple cohorts.

• Multiparameter Integration: Combining TNFSF9 with other potential biomarkers (TAP1, CD8A) may enhance predictive power .

• Implementation Protocol:

  • Collect tumor tissue samples prior to immunotherapy initiation

  • Measure TNFSF9 expression using validated RNA-seq or immunohistochemistry methods

  • Classify patients as high or low TNFSF9 expressors based on established thresholds

  • Integrate with clinical decision-making for therapy selection

This approach leverages the finding that TNFSF9-high tumors demonstrate enhanced adaptive immune responses, increased T and B cell infiltration, and particular sensitivity to CTLA-4 inhibition due to their elevated Treg populations .

How do carrier proteins affect the stability and activity of recombinant TNFSF9?

Carrier proteins significantly impact the stability and experimental utility of recombinant TNFSF9. Bovine Serum Albumin (BSA) is commonly added as a carrier protein to enhance stability, increase shelf-life, and allow storage at more dilute concentrations . The effects of carrier proteins include:

For cell or tissue culture applications and ELISA standards, the BSA-containing formulation is generally recommended . In contrast, carrier-free protein is preferable for applications where BSA might interfere with the experimental system . Researchers should carefully consider these factors when selecting recombinant TNFSF9 preparations for their specific experimental designs.

What methods are most effective for detecting the soluble form of TNFSF9 in experimental and clinical samples?

Detecting soluble TNFSF9 (sTNFSF9) in experimental and clinical samples requires specialized methodologies due to its relatively low abundance (26 kDa form) compared to the membrane-bound version . The following approaches have proven effective:

• ELISA-Based Detection:

  • Sandwich ELISA using capture and detection antibodies specific to TNFSF9

  • Sensitivity can be enhanced using biotin-streptavidin amplification systems

  • Typical detection ranges are in the pg/mL to ng/mL range

• Western Blot Analysis:

  • Sample concentration may be required prior to electrophoresis

  • Use reducing conditions to distinguish monomeric sTNFSF9

  • Molecular weight markers should focus on the 25-30 kDa range

• Functional Binding Assays:

  • Measure binding to recombinant TNFRSF9 proteins coated on plates

  • Assess biological activity through downstream signaling events

• Clinical Sample Processing:

  • For serum/plasma: collect in appropriate anticoagulant tubes and process within 30 minutes

  • Centrifuge at 1000-2000g for 10 minutes at 4°C

  • Store aliquots at -80°C to prevent degradation

  • Avoid repeated freeze-thaw cycles

When interpreting results, researchers should consider that a 26 kDa soluble form of TNFSF9 can be released from activated cells while retaining bioactivity . This soluble form may have distinct biological effects compared to the membrane-bound version.

How does TNFSF9 expression influence response to combination immunotherapy?

TNFSF9 expression significantly impacts patient response to combination immunotherapy, particularly the nivolumab plus ipilimumab (NIVO+IPI) regimen in metastatic renal cell carcinoma. Research findings demonstrate several key relationships:

• Predictive Value: TNFSF9 expression discriminates between responders and non-responders with 88.89% sensitivity and 87.50% specificity (AUC = 0.9444), outperforming PD-L1 as a biomarker (AUC = 0.75) .

• Immune Microenvironment Characteristics: High TNFSF9 expression correlates with:

  • Increased adaptive immune responses

  • Higher immune and ESTIMATE scores

  • Greater tumor infiltration by CD8+ T cells, B cells, and dendritic cells

• T Cell Function: TNFSF9-high tumors show complex T cell dynamics:

  • Increased CD8+ T cell infiltration and interferon-gamma levels

  • Higher levels of dysfunctional T cells

  • Increased regulatory T cell (Treg) populations

• Therapeutic Mechanism Insight: The increased Treg population in TNFSF9-high tumors suggests why dual checkpoint inhibition (NIVO+IPI) is particularly effective. While PD-1 inhibition (nivolumab) addresses T cell exhaustion, CTLA-4 inhibition (ipilimumab) likely suppresses Treg activation, creating a synergistic effect specifically beneficial in TNFSF9-high tumors .

These findings suggest that TNFSF9 expression analysis could be integrated into clinical decision-making to identify patients most likely to benefit from combination immunotherapy approaches.

What experimental systems best model TNFSF9-dependent immune activation in cancer immunotherapy research?

Modeling TNFSF9-dependent immune activation in cancer immunotherapy research requires specialized experimental systems that recapitulate the complex interactions between tumor cells, antigen-presenting cells, and effector T cells. Based on the current understanding of TNFSF9 biology, the following experimental systems are recommended:

• 3D Co-culture Systems:

  • Composition: Tumor cells, dendritic cells, T cells

  • Rationale: Models TNFSF9-mediated interactions between antigen-presenting cells and T cells in a tumor-like context

  • Readouts: T cell proliferation, cytokine production, tumor cell killing

• Humanized Mouse Models:

  • Implementation: Immune-deficient mice reconstituted with human immune cells

  • Advantages: Allows study of human TNFSF9-TNFRSF9 interactions in vivo

  • Assessment Methods: Flow cytometry of tumor-infiltrating lymphocytes, cytokine profiling, tumor growth kinetics

• Ex Vivo Tumor Slice Cultures:

  • Approach: Fresh tumor tissue maintained in culture with preserved architecture

  • Benefits: Maintains intact tumor microenvironment with endogenous TNFSF9 expression

  • Analyses: Immunohistochemistry, flow cytometry, cytokine profiling

• In Vitro Functional Assays:

  • Setup: Purified T cells stimulated with anti-CD3 plus recombinant TNFSF9

  • Applications: Precisely quantify TNFSF9's co-stimulatory effects

  • Typical Conditions: ED50 for IL-2 secretion is 5-30 ng/mL of recombinant TNFSF9

When designing experiments, researchers should consider that TNFSF9's effects may differ depending on the phase of immune response, as it acts later than CD28 co-stimulation in T cell responses . Additionally, both membrane-bound and soluble forms of TNFSF9 (26 kDa) retain bioactivity and may have distinct effects .

How does TNFSF9 interact with its receptor TNFRSF9 at the molecular level?

The interaction between TNFSF9 and its receptor TNFRSF9 involves specific molecular binding events that trigger downstream signaling cascades critical for immune cell activation. Key aspects of this interaction include:

• Binding Kinetics and Affinity:

  • When recombinant TNFRSF9 is coated at 100 ng/mL, TNFSF9 binds with an ED50 of 1-6 ng/mL

  • This high-affinity interaction enables sensitive responses to relatively low concentrations of ligand

• Structural Requirements:

  • The extracellular domain of TNFSF9 contains the receptor-binding region

  • In recombinant systems, the region from amino acids 71-254 (for human TNFSF9) maintains biological activity

  • Proper folding is essential for activity, with proper disulfide bond formation crucial for maintaining the active conformation

• Receptor Expression and Distribution:

  • TNFRSF9 (4-1BB/CD137) is expressed on activated CD4+, CD8+, memory CD8+, NKT, and regulatory T cells

  • It is also found on myeloid and mast cell progenitors, dendritic cells, mast cells, and bacterially infected osteoblasts

  • This distribution pattern defines the cell types responsive to TNFSF9 signaling

• Functional Consequences of Binding:

  • TNFSF9-TNFRSF9 binding transduces a co-stimulatory signal that promotes T cell proliferation, activation, and survival

  • It supports the survival and responsiveness of memory T cells during viral infection

  • Unlike CD28 co-stimulation which is important for initial T cell expansion, TNFSF9-TNFRSF9 signaling acts later in the immune response

Understanding these molecular interactions is crucial for designing experiments to study TNFSF9 function and for developing therapeutic approaches targeting this pathway.

What signaling pathways does TNFSF9 activate in different immune cell populations?

TNFSF9 activates distinct signaling pathways in different immune cell populations, leading to context-dependent functional outcomes. These pathway activations have significant implications for experimental design and interpretation:

• T Cells:

  • Primary Pathways: NF-κB, JNK, and p38 MAPK signaling cascades

  • Functional Outcomes: Enhanced proliferation, cytokine production (particularly IL-2), and survival

  • Temporal Considerations: TNFSF9 signaling is particularly important during later phases of T cell responses, unlike CD28 which acts early

• Monocytes and Macrophages:

  • Through Reverse Signaling: TNFSF9 can transmit signals back through the ligand-expressing cell

  • Effects: Induces production of inflammatory cytokines in monocytes

  • In Macrophages: TNFSF9 associates with TLR4 and enhances inflammatory cytokine production in response to TLR4 ligation

• Dendritic Cells:

  • Expression Patterns: Found on activated dendritic cells

  • Role: Influences maturation and antigen presentation capabilities

• Myeloid Progenitors:

  • Regulatory Function: TNFSF9 expression on early myeloid progenitor cells limits the development of dendritic cells, monocytes, and B cells

  • Developmental Impact: Shapes the composition of the immune cell repertoire

These diverse signaling events highlight the need for cell-type-specific experimental approaches when studying TNFSF9 function. Researchers should carefully consider which cell populations and readouts are most relevant to their specific research questions about TNFSF9 biology.

What are common challenges in working with recombinant TNFSF9 and how can they be addressed?

Working with recombinant TNFSF9 presents several technical challenges that researchers should anticipate and address through careful experimental design:

• Protein Stability Issues:

  • Challenge: Recombinant TNFSF9 may lose activity during storage and freeze-thaw cycles

  • Solution: Add 5-50% glycerol as a cryoprotectant for long-term storage and prepare single-use aliquots to avoid repeated freeze-thaw cycles

  • Recommendation: Store working aliquots at 4°C for up to one week

• Carrier Protein Interference:

  • Challenge: BSA carrier in standard preparations may interfere with certain assays

  • Solution: Use carrier-free preparations (typically stabilized with trehalose instead of BSA) for applications where carrier might interfere

  • Applications: Carrier-free is preferred for binding assays, certain cell culture systems, and in vivo studies

• Reconstitution Difficulties:

  • Challenge: Incomplete solubilization of lyophilized protein

  • Solution: Briefly centrifuge the vial prior to opening, reconstitute in appropriate buffer (typically PBS), and ensure complete dissolution before use

  • Verification: Consider confirming protein concentration after reconstitution

• Activity Variations Between Preparations:

  • Challenge: Functional activity may vary between lots or preparations

  • Solution: Validate each new lot with functional assays such as binding to TNFRSF9 or T cell co-stimulation assays

  • Expected Values: ED50 for binding to TNFRSF9 should be 1-6 ng/mL; for T cell co-stimulation (IL-2 secretion) should be 5-30 ng/mL

• Endotoxin Contamination:

  • Challenge: Endotoxin can activate immune cells independently of TNFSF9

  • Solution: Use preparations certified for low endotoxin levels (<1.0 EU/μg)

  • Testing: Consider additional endotoxin testing for critical experiments

By addressing these common challenges, researchers can maximize the reliability and reproducibility of experiments involving recombinant TNFSF9.

How can researchers differentiate between the effects of membrane-bound and soluble TNFSF9?

Differentiating between the effects of membrane-bound and soluble TNFSF9 requires specialized experimental approaches that can isolate and compare these distinct forms:

• Expression System Selection:

  • For Membrane-bound TNFSF9: Use cell-based expression systems that anchor the full-length protein in the cell membrane

  • For Soluble TNFSF9: Use either recombinant protein preparations (typically 26 kDa) or culture supernatants from cells expressing the naturally cleaved form

• Experimental Separation Strategies:

  • Physical Separation: Use transwell systems to separate TNFSF9-expressing cells from target cells while allowing soluble factors to diffuse

  • Molecular Engineering: Create mutant forms that cannot be cleaved from the membrane or that are constitutively secreted

  • Selective Inhibition: Use metalloprotease inhibitors to prevent cleavage of membrane-bound TNFSF9

• Functional Comparison Methodologies:

  • Direct Comparison: Set up parallel experiments with recombinant soluble TNFSF9 versus fixed TNFSF9-expressing cells

  • Dose-Response Analysis: Compare concentration-dependent effects of soluble TNFSF9 with density-dependent effects of membrane-bound TNFSF9

  • Temporal Dynamics: Assess differences in signaling kinetics and durability between the two forms

• Analytical Considerations:

  • Western Blotting: Detect distinct molecular weight forms (approximately 50 kDa for membrane-bound versus 26 kDa for soluble)

  • Flow Cytometry: Quantify surface-expressed versus secreted TNFSF9 using specific antibodies

  • ELISA: Measure soluble TNFSF9 concentration in culture supernatants or biological fluids

Understanding the differential effects of these forms is critical as the 26 kDa soluble form released from activated cells retains bioactivity but may have distinct functional properties compared to the membrane-bound form .