TNFSF8 Human, Sf9

What is the molecular structure and composition of TNFSF8 Human, Sf9?

TNFSF8 Human produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 181 amino acids (spanning positions 63-234) with a theoretical molecular mass of 20.7kDa. Due to glycosylation patterns in insect cells, the protein typically appears between 18-40kDa when analyzed by SDS-PAGE. The protein is expressed with a 6 amino acid His tag at the C-Terminus to facilitate purification and detection in experimental settings. This recombinant form maintains the core functional domains necessary for receptor binding while being optimized for solubility and stability in research applications .

The protein is supplied as a sterile filtered colorless solution formulated in Phosphate Buffered Saline (pH 7.4) with 10% glycerol as a cryoprotectant. This formulation helps maintain protein integrity during storage and handling procedures common in research settings .

How do TNFSF8 polymorphisms affect its biological function?

Genetic variations in TNFSF8 have been associated with significant differences in immune responses. Two key polymorphisms, rs927374 and rs2295800, which are in high linkage disequilibrium (r²=0.97), have been linked to variations in neutrophil counts following myocardial infarction. Specifically, for rs927374, GG homozygotes exhibited approximately 16% lower neutrophil counts compared to CC homozygotes (7.6±5.1 vs. 9.0±5.2) .

These associations remained significant after controlling for clinical characteristics, suggesting that genetic variation in TNFSF8 may influence inflammatory processes at a fundamental level. This has important implications for understanding individual variations in inflammatory responses and potentially predicting outcomes in conditions with inflammatory components .

What distinguishes TNFSF8 from other members of the TNF superfamily?

TNFSF8 has several distinctive characteristics compared to other TNF superfamily members:

• Receptor specificity: TNFSF8 binds primarily to CD30 (TNFRSF8), which has a relatively restricted expression pattern focused on activated lymphocytes and certain lymphoma cells.

• Pleiotropic effects: TNFSF8 demonstrates unusual functional duality, enhancing proliferation in some lymphoma cell lines while inducing cell death in others, setting it apart from many TNF family members with more consistent effects .

• Role in B cell function: TNFSF8 expressed on B cell surfaces plays an inhibitory role in modulating immunoglobulin class switching, a function not shared by most TNF family members .

• Pathophysiological relevance: TNFSF8 has a specific pathophysiologic role in Hodgkin's and certain non-Hodgkin's lymphomas, making it a potential therapeutic target for these malignancies .

What are optimal protocols for storage and handling of TNFSF8 Human, Sf9 in laboratory settings?

To maintain optimal bioactivity of TNFSF8 Human, Sf9, researchers should follow these evidence-based handling protocols:

• Short-term storage (≤4 weeks): Store at 4°C in the original formulation containing PBS (pH 7.4) and 10% glycerol.

• Long-term storage: Maintain at -20°C, with minimal freeze-thaw cycles. For extended storage periods, aliquoting the protein solution is strongly recommended to avoid repeated freeze-thaw cycles that can compromise structural integrity .

• Working conditions: When preparing experimental dilutions, use sterile buffers and preferably work on ice to minimize protein degradation.

• Stability testing: Before initiating critical experiments, verify protein activity using functional assays that measure CD30 binding or downstream signaling.

• Reconstitution after lyophilization: If working with lyophilized protein, reconstitute using sterile buffer and gentle agitation rather than vortexing, which can cause protein denaturation.

How can researchers effectively validate TNFSF8-CD30 interactions in experimental systems?

Multiple complementary approaches can be employed to validate TNFSF8-CD30 interactions in different experimental contexts:

• Flow cytometry binding assays: Utilize fluorescently-labeled TNFSF8 Human, Sf9 (directly labeled or detected via the His-tag) to measure binding to CD30+ cells. Specificity can be confirmed using blocking antibodies against either TNFSF8 or CD30.

• Functional validation: Measure biological outcomes of TNFSF8-CD30 interaction, such as NF-κB pathway activation, cell proliferation/death, or cytokine production in CD30+ cells. Comparison of responses in CD30+ and CD30- cells provides important biological validation.

• Co-immunoprecipitation: Isolate protein complexes from cells treated with TNFSF8 using antibodies against CD30 (or vice versa), followed by Western blot detection of the binding partner.

• Doublet formation assays: Similar to the approach used with CD27xEGFR bispecific antibodies, researchers can assess TNFSF8-mediated cell-cell interactions when one cell population expresses CD30 .

• Competition assays: Demonstrate that unlabeled TNFSF8 competes with labeled TNFSF8 for binding to CD30, confirming specificity of the interaction.

What methodological approaches can be used to study TNFSF8-mediated signal transduction?

Multiple complementary techniques can be employed to dissect TNFSF8-mediated signaling cascades:

• Phosphorylation dynamics:

  • Western blotting for phosphorylated signaling proteins (p-IκB, p-MAPK, p-JNK)

  • Phospho-flow cytometry for single-cell analysis of signaling activation

  • Phosphoproteomic analysis for comprehensive pathway mapping

• Transcriptional responses:

  • RNA-seq or qPCR arrays to identify genes regulated by TNFSF8-CD30 signaling

  • ChIP-seq to map transcription factor binding following pathway activation

  • Reporter assays using NF-κB-responsive promoters to quantify transcriptional activity

• Protein-protein interactions:

  • Proximity ligation assays to visualize CD30-associated signaling complexes

  • Mass spectrometry-based interactome analysis following TNFSF8 stimulation

  • FRET/BRET approaches to monitor dynamic protein interactions in living cells

• Genetic manipulation:

  • CRISPR-Cas9 knockout of specific signaling components to establish pathway dependencies

  • Overexpression of dominant-negative signaling proteins to block specific branches

  • Rescue experiments with wild-type vs. mutant signaling proteins

How does TNFSF8 contribute to lymphoma pathophysiology?

TNFSF8 has complex and sometimes contradictory roles in lymphoma biology:

• Differential growth regulation: TNFSF8 enhances proliferation in some lymphoma cell lines while inducing cell death in others. This context-dependent effect suggests that the tumor microenvironment or intrinsic cellular factors may determine whether TNFSF8-CD30 signaling promotes or inhibits lymphoma growth .

• Immune modulation: TNFSF8 expressed on B cell surfaces has an inhibitory role in modulating immunoglobulin class switching, which may contribute to altered humoral immunity in lymphoma patients .

• NF-κB pathway activation: In many CD30+ lymphomas, TNFSF8-CD30 signaling activates the NF-κB pathway, promoting cell survival and proliferation. This constitutive pathway activation represents a potential therapeutic vulnerability.

• Biomarker potential: CD30 expression is a hallmark of Hodgkin lymphoma and anaplastic large cell lymphoma, making the TNFSF8-CD30 axis relevant for both diagnostic and therapeutic applications in these malignancies .

Understanding these diverse mechanisms is crucial for developing targeted therapies that modulate the TNFSF8-CD30 axis in lymphoma treatment.

What role does TNFSF8 play in inflammatory responses and autoimmune conditions?

TNFSF8 has significant functions in regulating inflammatory processes:

• Neutrophil regulation: TNFSF8 polymorphisms (rs927374 and rs2295800) are associated with differential neutrophil counts following myocardial infarction, suggesting a role in neutrophil mobilization, survival, or function. Specifically, individuals with the GG genotype at rs927374 showed approximately 16% lower neutrophil counts compared to CC homozygotes .

• T cell function modulation: Through CD30 signaling, TNFSF8 can influence T cell proliferation, cytokine production, and survival, potentially affecting T cell-mediated inflammatory processes.

• Integration with other immune pathways: Recent analyses of CAR-T cell therapy-associated toxicities identified several TNF receptor superfamily proteins (including TNFRSF1A, TNFRSF1B, and CD40) as being dysregulated during severe immune reactions, suggesting complex interactions between these pathways during inflammatory responses .

These findings suggest that TNFSF8-CD30 signaling may represent a potential therapeutic target in inflammatory conditions characterized by dysregulated neutrophil responses or T cell hyperactivation.

How can TNFSF8 Human, Sf9 be utilized in developing novel therapeutic strategies?

TNFSF8 Human, Sf9 offers several applications for therapeutic development:

• Target validation: The recombinant protein can be used to validate CD30 as a therapeutic target by demonstrating functional outcomes of receptor engagement in different cell types.

• Screening platforms: TNFSF8 Human, Sf9 can be employed in high-throughput screening assays to identify small molecules or biologics that modulate CD30 signaling.

• Development of TNFSF8 variants: Structure-function studies using the recombinant protein can guide the engineering of modified versions with enhanced or selective activities for therapeutic applications.

• Bispecific antibody development: Similar to the CD27xEGFR approach, bispecific antibodies targeting CD30 alongside tumor antigens could be developed and initially tested using TNFSF8 Human, Sf9 as a competitor or control reagent .

• CAR-T cell optimization: Given the relevance of TNF receptor superfamily proteins in CAR-T toxicities, understanding TNFSF8-CD30 signaling may help optimize CAR-T cell therapies to reduce adverse events like cytokine release syndrome and neurotoxicity .

What systems biology approaches can elucidate TNFSF8 signaling networks?

Advanced systems biology approaches provide comprehensive insights into TNFSF8 signaling:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from cells treated with TNFSF8 Human, Sf9 can reveal the complete cellular response signature. Ultrasensitive assays like NULISA™, which can simultaneously quantify over 200 proteins, offer particular value for mapping complex signaling networks activated by TNFSF8 .

• Network analysis: Computational approaches can identify key nodes and regulatory hubs within TNFSF8-induced signaling networks. Physical protein association network analysis, as applied in CAR-T toxicity studies, can reveal how TNFSF8 signaling components interact with other pathways .

• Temporal dynamics analysis: Time-course experiments with high-resolution sampling can elucidate the sequence of signaling events following TNFSF8-CD30 engagement, distinguishing between rapid, intermediate, and delayed responses.

• Single-cell analyses: Single-cell RNA-seq and proteomics approaches can reveal heterogeneity in cellular responses to TNFSF8, identifying responder and non-responder subpopulations even within seemingly homogeneous cell types.

• Perturbation biology: Systematic genetic or pharmacological perturbation of signaling components, coupled with measurement of pathway outputs, can delineate the functional architecture of TNFSF8-CD30 signaling networks.

How does TNFSF8 interact with other TNF superfamily members in immune regulation?

TNFSF8 functions within a complex network of TNF superfamily members with intricate regulatory interactions:

• Co-regulation patterns: Analysis of protein expression in severe CAR-T cell therapy toxicity has revealed coordinated regulation of multiple TNF receptor superfamily proteins, including TNFRSF1A, TNFRSF1B, and CD40, suggesting interconnected regulatory mechanisms .

• Pathway convergence: TNFSF8-CD30 signaling activates NF-κB and MAPK pathways, which are also engaged by other TNF family members, creating potential for signal integration, amplification, or antagonism.

• Receptor cross-regulation: CD30 expression can be modulated by signaling through other TNF receptors, and conversely, CD30 activation may influence the expression or function of other TNFR family members.

• Temporal coordination: Different TNF family members may act sequentially during immune responses, with TNFSF8-CD30 interactions potentially playing specific roles during certain phases of the response.

• Connections with checkpoint pathways: Recent studies have identified associations between TNF receptor superfamily proteins and immune checkpoint molecules like CTLA4, PDCD1 (PD-1), LAG3, and CD274 (PD-L1) during severe immune reactions, suggesting complex regulatory networks involving both stimulatory and inhibitory pathways .

What are the challenges in translating TNFSF8 research to clinical applications?

Researchers face several complex challenges when attempting to translate TNFSF8 basic research into clinical applications:

• Contextual signaling outcomes: The ability of TNFSF8 to induce opposing effects (proliferation vs. cell death) in different contexts presents challenges for therapeutic development, requiring careful patient stratification and biomarker identification to predict responses.

• Redundancy within signaling pathways: The overlap between TNFSF8-CD30 signaling and other TNF family pathways may lead to compensatory mechanisms that limit therapeutic efficacy when targeting this pathway in isolation.

• Biomarker development: Identifying reliable biomarkers that predict TNFSF8-CD30 pathway activation or therapeutic response remains challenging. Advanced proteomics approaches like those used in CAR-T toxicity studies may help address this gap .

• Balancing efficacy and toxicity: Given the role of TNF receptor superfamily proteins in immune-related adverse events, as seen in CAR-T cell therapy, modulating TNFSF8-CD30 signaling requires careful optimization to achieve therapeutic benefits without exacerbating inflammatory toxicities .

• Target validation in diverse pathologies: While the TNFSF8-CD30 axis is well-established in certain lymphomas, its relevance in inflammatory or autoimmune conditions requires further validation before therapeutic development can proceed.

What controls are essential when studying TNFSF8 in experimental systems?

Rigorous experimental design requires multiple controls when studying TNFSF8:

• Specificity controls:

  • CD30-negative cells to confirm receptor dependence

  • Blocking antibodies against CD30 or TNFSF8 to verify specific interactions

  • Structurally related but functionally distinct TNF family members to demonstrate ligand specificity

• Activity verification:

  • Positive control cell lines with well-characterized responses to TNFSF8

  • Parallel assessment of known TNFSF8-induced signaling events (e.g., NF-κB activation)

  • Dose-response analyses to establish concentration-dependent effects

• Technical controls:

  • Heat-inactivated TNFSF8 to control for potential contaminants in protein preparations

  • Buffer-only controls to account for effects of the formulation components

  • Time-matched samples to control for temporal variations in cellular responses

• Genetic controls:

  • When studying polymorphisms, careful matching of demographic and clinical characteristics between genotype groups, as done in studies examining rs927374 and rs2295800

  • CRISPR-edited isogenic cell lines differing only in CD30 expression to isolate receptor-specific effects

How can researchers address variability in TNFSF8-mediated responses across different cell types?

To manage the inherent variability in cellular responses to TNFSF8, researchers should implement these methodological approaches:

• Comprehensive cell characterization:

  • Quantify CD30 expression levels across cell populations

  • Assess baseline activation state of relevant signaling pathways

  • Determine expression of potential co-receptors or regulatory molecules

• Mechanistic dissection:

  • Use pathway inhibitors to identify signaling components contributing to response heterogeneity

  • Compare transcriptional profiles of responder versus non-responder cells

  • Investigate post-translational modifications that might modulate receptor sensitivity

• Experimental design considerations:

  • Include multiple cell types/lines within the same experiment

  • Standardize culture conditions to minimize environmental variables

  • Use paired statistical analyses when comparing responses across cell types

• Advanced analytical approaches:

  • Single-cell analyses to detect and characterize response heterogeneity within populations

  • Machine learning algorithms to identify patterns predictive of TNFSF8 responsiveness

  • Systems biology modeling to integrate multiple parameters influencing response variability

What novel technologies are advancing TNFSF8 research?

Cutting-edge technologies are transforming TNFSF8 research:

• Advanced protein analysis:

  • Ultrasensitive multiplexed assays such as NULISA™, which can simultaneously quantify hundreds of proteins, allowing comprehensive analysis of how TNFSF8 signaling affects the broader immune proteome

  • Cryo-electron microscopy for high-resolution structural analysis of TNFSF8-CD30 complexes

• Genetic engineering tools:

  • CRISPR-Cas9 for precise genetic modifications to study TNFSF8 function

  • Base editors and prime editors for introducing specific polymorphic variants to study their functional effects

  • AAV-mediated gene delivery for in vivo modulation of TNFSF8 or CD30 expression

• Advanced imaging technologies:

  • Proximity ligation assays for visualizing protein interactions in situ

  • Lattice light-sheet microscopy for high-resolution, low-phototoxicity imaging of signaling dynamics

  • Intravital multiphoton microscopy for studying TNFSF8-CD30 interactions in living tissues

• Bispecific molecule development:

  • Similar to CD27xEGFR approaches, novel bispecific platforms targeting CD30 alongside tumor antigens or immune receptors offer promising therapeutic applications

  • Nanobody-based constructs for improved tissue penetration and novel binding properties

• Computational approaches:

  • Network analysis and pathway modeling tools for interpreting complex datasets

  • AI/ML algorithms for predicting TNFSF8-CD30 interaction outcomes

  • Molecular dynamics simulations for understanding ligand-receptor binding mechanisms

These technological advances are providing unprecedented insights into TNFSF8 biology and accelerating the development of therapeutic approaches targeting this signaling axis.