Recombinant Human Tumor necrosis factor ligand superfamily member 10 (TNFSF10), partial (Active)

What is TNFSF10 and what are its primary functions in human biology?

TNFSF10, also known as TNF-related apoptosis-inducing ligand (TRAIL), Apo-2 ligand, or CD253, is a cytokine belonging to the tumor necrosis factor (TNF) ligand family. It functions as a homotrimeric type II transmembrane ligand that preferentially induces apoptosis in transformed and tumor cells while sparing normal cells .

Beyond its well-established role in apoptosis, recent evidence indicates TNFSF10 plays significant roles in:

• Regulating antitumor immunity in cancer cells and the tumor microenvironment (TME)

• Promoting granzyme B expression in cytotoxic T-cells

• Potentially stimulating proliferation of regulatory T cells and M2 macrophages

• Modulating immune cell function and proliferation

TNFSF10 interacts with several members of the TNF receptor superfamily, including TNFRSF10A/TRAILR1, TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3, TNFRSF10D/TRAILR4, and possibly TNFRSF11B/OPG . Its apoptotic activity may be modulated by decoy receptors that cannot induce apoptosis.

How should recombinant TNFSF10 be stored and reconstituted for optimal activity?

For optimal storage and reconstitution of recombinant TNFSF10:

Storage conditions:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

• For short-term use (up to one week), reconstituted protein can be stored at 4°C

• For long-term storage, keep reconstituted protein at -20°C or -80°C

Reconstitution protocol:

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

• Reconstitute in sterile water or appropriate buffer (commonly PBS or 25mM Tris, 150mM NaCl, pH 7.5)

• Gently mix by inverting the vial several times, avoid vigorous vortexing

• Allow to stand for 5-10 minutes at room temperature before aliquoting

• Filter through a 0.22μm filter if sterility is required for cell culture applications

Note that active recombinant TNFSF10 typically has an ED50 < 40 ng/mL, as measured by cell growth inhibitory assay using RPMI-8226 cells, corresponding to a specific activity of > 2.5 × 10^4 units/mg .

What are the key differences between full-length and partial recombinant TNFSF10?

The differences between full-length and partial (commonly AA 114-281) recombinant TNFSF10 have important implications for research applications:

Full-length TNFSF10 (1-281):

• Contains the cytoplasmic domain (AA 1-18), transmembrane domain (AA 18-28), and extracellular domain (AA 28-281)

• More closely resembles native protein conformation

• Often used for studying membrane-bound TRAIL functions

• May provide context for studying interactions with the cellular membrane

Partial/soluble TNFSF10 (typically AA 114-281):

• Contains only the receptor-binding domain

• Higher specific activity in apoptosis assays

• Better protein yield and stability in recombinant expression systems

• Lower tendency to aggregate compared to full-length protein

• Most commonly used form for biological activity studies

Research has shown that the partial recombinant form (AA 114-281) retains full biological activity for inducing apoptosis through death receptors while offering superior stability characteristics . This explains why many commercial sources provide the partial active form rather than the full-length protein.

How should TNFSF10-induced apoptosis assays be designed and what controls are necessary?

When designing TNFSF10-induced apoptosis assays, consider the following methodology:

Experimental design:

• Cell selection: Use established TRAIL-sensitive (e.g., RPMI-8226, Jurkat) and TRAIL-resistant cell lines (e.g., some primary cells) as controls

• Dose response: Test concentrations ranging from 1-1000 ng/mL of recombinant TNFSF10

• Time course: Evaluate apoptosis at multiple timepoints (typically 4, 8, 12, 24 hours)

• Combination studies: Consider testing TNFSF10 with sensitizing agents like proteasome inhibitors or chemotherapeutic drugs

Essential controls:

• Untreated cells (negative control)

• Known apoptosis inducer (e.g., staurosporine) as positive control

• Heat-inactivated TNFSF10 (specificity control)

• Pan-caspase inhibitor (e.g., Z-VAD-FMK) to confirm caspase-dependent mechanism

Measurement methods (use at least two):

• Annexin V/PI staining with flow cytometry

• Caspase 3/7 activity assays

• DNA fragmentation analysis

• PARP cleavage detection by western blot

• Mitochondrial membrane potential assessment

Data should be presented showing both dose-response and time-course relationships, with statistical analysis of at least three independent experiments.

What methods are most appropriate for studying TNFSF10 expression at the mRNA and protein levels?

For comprehensive analysis of TNFSF10 expression:

mRNA expression analysis:

• qRT-PCR: Use TaqMan or SYBR Green assays with properly validated primers

  • For TaqMan: Hs00921974_m1 probe is validated for human TNFSF10

  • Normalize to validated reference genes (e.g., RNA18S, GAPDH, ACTB)

  • Calculate fold change using the ΔΔCT method

• RNA-Seq: For genome-wide expression context

  • Analyze TNFSF10 in context of pathway activation

  • Correlate with expression of other immune genes

Protein expression analysis:

• Western blot: For semi-quantitative analysis

  • Recommended antibodies: anti-TNFSF10 (Cell Signaling #3219, 1:300 dilution)

  • Use tubulin (Cell Signaling #2128) or β-actin (Abcam #ab8227) as loading controls

  • Employ fluorescent secondary antibodies for quantification

• Flow cytometry: For cell surface TNFSF10

  • Distinguishes between membrane-bound and intracellular forms

  • Use non-permeabilizing conditions for surface detection

• ELISA: For soluble TNFSF10 in supernatants/serum

  • Commercial kits available with detection limits ~2-5 pg/mL

When analyzing TNFSF10 expression, it's critical to account for both membrane-bound and soluble forms, as metalloprotease-mediated shedding can affect detection . Additionally, distinguishing between basal expression and induction (e.g., by IFNs) provides important context for understanding regulatory mechanisms.

How does TNFSF10 contribute to antiviral immune responses, and how is this distinct from its apoptotic function?

TNFSF10's role in antiviral immunity is distinct from yet complementary to its apoptotic function:

TNFSF10 in antiviral responses:

• Expression is strongly induced by type I interferons (IFN-α, IFN-β) but minimally by TNF-α, despite being a TNF family member

• Forms part of the innate immune response to viral infection

• Correlates highly with expression of classical antiviral genes (DDX58/RIG-I, IFIH1/MDA-5, OAS1)

• Contributes to poly(I:C)-induced apoptosis (mimicking viral RNA sensing)

Experimental evidence demonstrates that:

• IFN-β dramatically increases TNFSF10 expression in triple-negative breast cancer (TNBC) cells

• TNFSF10 expression correlates significantly with antiviral gene expression

• TNFSF10 knockout reduces poly(I:C)-induced apoptosis

• TNFSF10 plays an essential role in antiviral immunity-induced apoptosis, partly through type I IFN signaling

This dual role positions TNFSF10 at the intersection of tumor cell apoptosis and antiviral immunity, potentially explaining why its expression correlates with CD274 (PD-L1), a therapeutic target in cancer immunotherapy .

Researchers should design experiments that specifically distinguish between direct apoptotic effects and indirect immune-mediated effects when studying TNFSF10 function in complex biological systems.

What mechanisms explain TNFSF10 resistance in cancer cells and how can they be experimentally overcome?

Cancer cells employ multiple mechanisms to evade TNFSF10-induced apoptosis:

Resistance mechanisms and experimental countermeasures:

Research demonstrates that protective autophagy is activated in response to TNFSF10 treatment, as evidenced by:

• Conversion of MAP1LC3B-I to MAP1LC3B-II

• Reduction of SQSTM1/p62 expression levels

• Further elevation of MAP1LC3B-II when combining TNFSF10 with chloroquine

To overcome resistance experimentally:

• Perform combination treatments with sensitizing agents

• Target multiple resistance mechanisms simultaneously

• Use genetic approaches (CRISPR/siRNA) to identify specific resistance factors

• Consider the tumor microenvironment context, as TNFSF10 affects immune cell infiltration

When designing sensitization experiments, include appropriate controls to distinguish between enhanced TRAIL receptor signaling versus alternative apoptotic mechanisms.

How does genetic variation in TNFSF10 affect its expression and function, particularly in cancer?

Genetic variations in TNFSF10 significantly impact its expression and function:

SNP rs13074711 in TNBC:

• Located in a region with enhancer marks and open chromatin structure

• Regulates TNFSF10 expression in triple-negative breast cancer (TNBC) cells

• Expression correlates with SNP genotype:

  • CC genotype: highest TNFSF10 expression

  • TT genotype: lowest TNFSF10 expression

  • TC genotype: intermediate expression

• SNP modulates c-MYB binding activity at the enhancer motif

Association with racial disparities:

• TNFSF10 expression is consistently lower in African American (AA) cancer patients compared to European American (EA) patients

• Statistically significant lower expression observed in colon and kidney cancers of AA patients

• Potentially contributes to resistance to TNFSF10-driven apoptosis of cancer cells in AA patients

Other functional SNPs:
Three SNPs within the TNFSF10 gene have been associated with T4 effector memory lymphocyte radiosensitivity :

These same SNPs have been linked to risk and/or treatment outcomes in ovarian and breast cancer .

For experimental investigation of TNFSF10 genetic variants:

• Use site-directed mutagenesis to recreate variants in expression systems

• Employ CRISPR-Cas9 to introduce specific SNPs into model cell lines

• Perform allele-specific expression analyses in heterozygous samples

• Conduct reporter assays with wildtype versus variant enhancer sequences

How can TNFSF10's dual role in apoptosis and immune modulation be leveraged for cancer immunotherapy?

TNFSF10's dual functionality presents unique opportunities for cancer immunotherapy:

Apoptotic and immune functions:

• Direct tumor cell killing through death receptor signaling

• Modulation of tumor microenvironment and immune cell infiltration

• Correlation with expression of CD274 (PD-L1), suggesting potential synergy with checkpoint inhibitors

Therapeutic strategies leveraging TNFSF10:

• Combination with immune checkpoint inhibitors:

  • TNFSF10 influences tumor-infiltrating lymphocytes (TILs)

  • TNFSF10-knockout tumors showed significantly reduced CD4+ T-cell infiltration

  • Tumors with high TIL density respond better to immune checkpoint inhibitors

• Enhancement of antiviral immune signaling:

  • Activation of type I IFN pathways increases TNFSF10 expression

  • Antiviral innate immune responses promote antitumor immunity

  • Poly(I:C) or similar TLR agonists could enhance TNFSF10-mediated effects

• TNFSF10-based combination therapies:

  • Target both direct apoptosis and immune activation

  • Combine with sensitizing agents to overcome resistance mechanisms

  • Consider genetic background (e.g., rs13074711 genotype) for personalized approaches

Research models for studying these approaches should incorporate:

• Immunocompetent animal models (avoid athymic nude or NOD/SCID mice)

• Patient-derived xenografts in humanized mouse models

• Ex vivo tumor slice cultures that preserve tumor microenvironment

• Multi-parametric analysis of immune infiltrates following treatment

The 4T1 syngeneic mouse model has been successfully used to determine effects of TNFSF10-knockout on T-cell infiltration, demonstrating TNFSF10's role in regulating immune cell infiltration in the tumor microenvironment .

What methodological approaches best assess TNFSF10's effects on tumor microenvironment and immune cell infiltration?

To comprehensively evaluate TNFSF10's impact on the tumor microenvironment and immune infiltration:

Experimental approaches:

• Immunocompetent mouse models:

  • Use syngeneic models (e.g., 4T1 breast cancer model)

  • Create TNFSF10-knockout lines using CRISPR-Cas9

  • Compare wild-type vs. knockout tumors for immune infiltration

  • Essential to avoid immune-deficient models that cannot exhibit actual tumor microenvironment

• Multi-parameter flow cytometry:

  • Analyze tumor-infiltrating lymphocytes (TILs) including:

    • CD4+ T cells (helper, regulatory)

    • CD8+ T cells (cytotoxic)

    • NK cells

    • Myeloid populations (MDSCs, TAMs)

  • Include functional markers (activation, exhaustion, cytotoxicity)

• Spatial analysis:

  • Multiplex immunohistochemistry to preserve spatial relationships

  • Quantify immune cell density and distribution

  • Assess proximity of immune cells to tumor cells

  • Digital spatial profiling for high-dimensional analysis

• Transcriptomic approaches:

  • Bulk RNA-seq to identify pathway activation

  • Single-cell RNA-seq to define cellular heterogeneity

  • Correlate TNFSF10 expression with immune signatures

Key findings from research:

• TNFSF10-knockout significantly reduced tumor-infiltrating CD4+ T cells compared to wild-type tumors

• Expression of TNFSF10 correlates with antiviral gene expression rather than inflammatory or apoptosis-related genes

• Expression correlates with CD274 (PD-L1), suggesting potential impact on checkpoint inhibitor response

When designing such studies, researchers should:

• Include time-course analyses to capture dynamic changes

• Compare multiple tumor models with varying baseline immune infiltration

• Consider both membrane-bound and soluble forms of TNFSF10

• Incorporate relevant genetic variations (e.g., rs13074711) that affect TNFSF10 expression

How do different expression systems affect the quality and activity of recombinant TNFSF10?

The choice of expression system significantly impacts recombinant TNFSF10 quality and activity:

Comparison of expression systems:

Critical quality attributes to assess:

• Oligomeric state: Native TNFSF10 functions as a homotrimer; recombinant protein should maintain this state

• Endotoxin levels: Must be <0.2 EU/μg for cell culture applications, determined by LAL method

• Specific activity: ED50 < 40 ng/mL in RPMI-8226 cell growth inhibition assay, corresponding to >2.5 × 10^4 units/mg

• Purity: >95% as determined by SDS-PAGE and HPLC/SEC analysis

What technical challenges exist in distinguishing membrane-bound versus soluble TNFSF10 in experimental systems?

Distinguishing between membrane-bound TNFSF10 (mTRAIL) and soluble TNFSF10 (sTRAIL) presents specific technical challenges that researchers must address:

Methodological approaches to differentiate forms:

• Flow cytometry:

  • Non-permeabilized cells detect only membrane-bound form

  • Comparison of permeabilized versus non-permeabilized samples differentiates total versus surface TNFSF10

  • Use anti-TNFSF10 antibodies that recognize extracellular domain

• ELISA:

  • For culture supernatants/biological fluids to quantify sTRAIL

  • Important to generate standard curves with recombinant protein matching the form being measured

• Western blot:

  • Different molecular weights: full-length (~32-33 kDa) versus soluble (~24-28 kDa)

  • Membrane fractionation protocols to separate cellular compartments

• Metalloprotease inhibition:

  • 1,10-phenanthroline treatment blocks TNFSF10 shedding

  • Increased level of mTRAIL after treatment indicates active shedding

Research findings on form-specific functions:

• mTRAIL but not sTRAIL regulates radiation-induced apoptosis of T4EM lymphocytes

• Adding recombinant human soluble TRAIL (rh-sTRAIL) has inhibiting effect on radiation-induced apoptosis

• Higher cell concentrations reduce apoptosis, suggesting cell density affects mTRAIL-mediated effects

Experimental design considerations:

• Include metalloprotease inhibitors to prevent shedding when studying mTRAIL

• Account for cell density effects in functional assays

• Use genetic approaches (mutation of cleavage sites) to create non-cleavable variants

• Consider paracrine versus autocrine signaling in co-culture systems

Understanding the differential biology of membrane versus soluble TNFSF10 is particularly important given their potentially opposing effects in certain experimental contexts, as demonstrated in radiation-induced apoptosis studies .

How can researchers address the challenges of investigating TNFSF10 in early-life respiratory infections and subsequent chronic lung disease?

Investigating TNFSF10's role in early-life respiratory infections and chronic lung disease requires specialized approaches:

Experimental models and challenges:

• Neonatal mouse models:

  • TRAIL-deficient neonatal mice reveal TRAIL's role in infection-induced pathology

  • Neutralizing antibodies against TRAIL and its receptors in wild-type mice provide comparative data

  • Critical timing considerations for infection and analysis (developmental windows)

• Assessment parameters:

  • Histopathology: inflammation, mucus hypersecretion

  • Alveolar structure: morphometric analysis of alveolar enlargement

  • Pulmonary function: compliance, resistance measurements

  • Immunological markers: immune cell infiltration, cytokine profiling

• Technical challenges:

  • Small sample size from neonatal specimens

  • Difficulty distinguishing developmental versus pathological changes

  • Need for age-appropriate controls at each timepoint

  • Long-term follow-up required to assess chronic effects

Key research findings:

• TRAIL promotes infection-induced histopathology, inflammation, and mucus hypersecretion

• Contributes to subsequent alveolar enlargement and impaired lung function

• May offer therapeutic target for early-life respiratory infections and associated chronic lung disease

Methodological recommendations:

• Use longitudinal study designs with matched controls

• Employ both genetic (knockout) and pharmacological (neutralizing antibodies) approaches

• Consider sex as a biological variable in analysis

• Incorporate clinically relevant respiratory pathogens (Chlamydia, RSV)

• Use micro-sampling techniques to maximize data from limited specimens

• Correlate animal model findings with human clinical samples when available

This research area highlights the complex dual nature of TNFSF10 in both protection against infection and contribution to pathological processes, requiring careful experimental design to dissect these opposing functions.