Recombinant Human Tumor necrosis factor ligand superfamily member 18 protein (TNFSF18), partial (Active)

What is TNFSF18 and what are its alternative names in scientific literature?

TNFSF18, also known as Glucocorticoid-Induced TNF-Related Ligand (GITRL), is a type II transmembrane glycoprotein belonging to the tumor necrosis factor (TNF) superfamily. In scientific literature, it's also referred to as Activation-inducible TNF-related ligand (AITRL), TL6, and hGITRL in human contexts . The protein functions as a cytokine that binds to TNFRSF18/AITR/GITR and plays significant roles in regulating T-cell responses . The human TNFSF18 gene is located on chromosome 1q23 and encodes a protein with a molecular weight of approximately 22,724 Da .

What is the structural composition of human TNFSF18?

Human TNFSF18/GITR Ligand has a characteristic structure consisting of:

• A 50 amino acid cytoplasmic domain

• A 21 amino acid transmembrane segment

• A 128 amino acid extracellular domain (ECD)

Within the extracellular domain, human GITR Ligand shares 56% amino acid sequence identity with mouse GITR Ligand and 60% with rat GITR Ligand . Functionally, the protein forms homotrimers which is typical of the TNF superfamily proteins . The protein contains the characteristic TNF homology domain (THD) that is involved in both the formation of ligand trimers and in ligand-receptor interaction specificity .

What cell types express TNFSF18 naturally?

TNFSF18 demonstrates a specific cellular expression pattern that is relevant to its immunological functions. It is predominantly expressed on:

• Antigen-presenting cells (APCs)

• B cells

• Dendritic cells (DCs)

• Macrophages

• Endothelial cells

• CD4-CD8- double negative thymic precursors

• Neurons

• Cells in the eye

Expression of TNFSF18 is not static but can be transiently upregulated in response to proinflammatory stimulation, suggesting its role in inflammatory responses . This dynamic expression pattern is important to consider when designing experiments involving inflammatory conditions or when using TNFSF18 as a marker for certain immune states.

What is the relationship between TNFSF18 and TNFRSF18 (GITR)?

TNFSF18 functions as the specific ligand for TNFRSF18, also known as Glucocorticoid-Induced TNF Receptor Family Related Protein (GITR) . GITR is constitutively expressed at high levels on regulatory T cells (Tregs) and at lower levels on resting CD25-CD4+ T cells, though expression increases markedly following T cell activation . The receptor is also found on other activated immune cells including NK cells and neutrophils .

The interaction between TNFSF18 and GITR leads to several immunological outcomes:

• Costimulation of T cell activation and proliferation

• Promotion of cytokine production

• Enhanced expression of activation antigens

• In murine models, abrogation of Treg-mediated suppression (though this effect appears species-specific and may not occur in humans)

How do recombinant forms of TNFSF18 differ in their biological activity based on expression systems?

Different expression systems yield recombinant TNFSF18 proteins with varying biological activities, which is critical knowledge for experimental design. Based on available data, there are notable differences:

The enhanced activity observed with certain recombinant forms suggests that oligomerization or specific conformational changes may influence receptor binding and downstream signaling. When selecting a recombinant TNFSF18 for research, investigators should consider whether their experimental questions require baseline or enhanced activity profiles .

What are the signaling pathways activated by TNFSF18-GITR interaction and how can they be measured?

The TNFSF18-GITR interaction activates multiple downstream signaling pathways that are crucial for its immunomodulatory functions:

• NF-κB Pathway: TNFSF18 binding to GITR mediates activation of the NF-κB transcription factor . This can be measured through:

  • Nuclear translocation assays of NF-κB subunits

  • Luciferase reporter assays for NF-κB activity

  • Phosphorylation status of IκB proteins

• STAT1 Phosphorylation: TNFSF18 triggers increased phosphorylation of STAT1 . Measurement approaches include:

  • Western blotting for phospho-STAT1

  • Flow cytometry using phospho-specific antibodies

  • Immunofluorescence microscopy to detect nuclear translocation

• Adhesion Molecule Expression: TNFSF18 upregulates expression of VCAM1 and ICAM1 . This can be assessed by:

  • Flow cytometry of surface expression

  • qRT-PCR for transcript levels

  • Functional adhesion assays

When designing experiments to evaluate these pathways, time-course analyses are recommended as different pathways may activate with distinct kinetics following TNFSF18-GITR engagement.

How do species-specific differences in TNFSF18 function impact translational research?

Species-specific differences in TNFSF18 function present significant challenges for translational research:

What are optimal reconstitution and storage conditions for recombinant TNFSF18 protein?

Proper handling of recombinant TNFSF18 is critical for maintaining its biological activity. Based on manufacturer recommendations:

Reconstitution Protocol:

• Reconstitute lyophilized TNFSF18 at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin .

• Allow the protein to dissolve completely by gentle agitation.

• Aliquot to minimize freeze-thaw cycles.

Storage Conditions:

• Ship at ambient temperature

• Upon receipt, store immediately at recommended temperature

• Use a manual defrost freezer and avoid repeated freeze-thaw cycles

• For long-term storage, keep at -20°C or -80°C depending on formulation

Stability Considerations:

• Working stock solutions are typically stable for 1 month at 2-8°C

• Carrier-free formulations may have different stability profiles than those containing carriers like BSA

• Monitor protein activity periodically if stored for extended periods

How can TNFSF18 protein activity be validated in functional assays?

Validating the biological activity of recombinant TNFSF18 is essential before using it in complex experimental systems. Several functional assays can be employed:

• T Cell Proliferation Assay:

  • Co-culture purified T cells with recombinant TNFSF18 in the presence of suboptimal anti-CD3 stimulation

  • Measure proliferation via 3H-thymidine incorporation or CFSE dilution

  • Expected result: Enhanced proliferation compared to anti-CD3 alone

  • Effective concentration (EC50): Typically 4-20 ng/mL

• NF-κB Activation Assay:

  • Use reporter cell lines expressing GITR and an NF-κB responsive element driving luciferase expression

  • Treat with varying concentrations of TNFSF18

  • Measure luciferase activity after 6-24 hours

  • Expected result: Dose-dependent increase in reporter activity

• Cytokine Production:

  • Stimulate primary T cells or NK cells with TNFSF18

  • Measure cytokine production (e.g., IFN-γ, IL-2) by ELISA or flow cytometry

  • Expected result: Enhanced cytokine production in TNFSF18-treated cultures

• Treg Functional Assay (species-specific considerations required):

  • In mouse systems: Co-culture Tregs, effector T cells, and TNFSF18

  • Assess ability of TNFSF18 to reverse Treg-mediated suppression

  • Note: This effect may not be reproducible in human systems

What are the best methods for detecting endogenous and recombinant TNFSF18 in experimental samples?

Detection of TNFSF18 requires different approaches depending on the experimental context:

• Protein Detection Methods:

  • ELISA: Sandwich ELISA kits are commercially available with detection ranges of 78-5000 pg/mL and sensitivity around 20 pg/mL

  • Western Blotting: Use reducing conditions; molecular weight approximately 22.7 kDa

  • Flow Cytometry: Surface staining for membrane-bound form

  • Immunohistochemistry/Immunofluorescence: For tissue localization studies

• mRNA Detection:

  • qRT-PCR: Design primers specific to human TNFSF18 (avoid regions with high homology to other TNF family members)

  • RNA-Seq: For transcriptome-wide analysis of TNFSF18 expression in different cell populations

  • In situ hybridization: For spatial expression analysis in tissues

• Special Considerations:

  • When detecting recombinant tagged proteins, antibodies against the tag (e.g., His, HA) can be used

  • For detecting both membrane-bound and soluble forms, choose antibodies recognizing the extracellular domain

  • When working with primary human samples, account for potential polymorphisms that might affect antibody binding

How should researchers interpret seemingly contradictory data about TNFSF18 function in different experimental models?

Contradictory findings regarding TNFSF18 function are common in the literature and can arise from several factors:

• Species-Specific Differences:

  • The most notable contradiction involves Treg suppression, which is abrogated by TNFSF18-GITR interaction in mice but not in humans

  • Methodological approach: Always clearly specify the species origin of both the TNFSF18 protein and target cells in publications

  • Interpretation strategy: Consider evolutionary differences in immune regulation between species when comparing results

• Context-Dependent Signaling:

  • TNFSF18 can induce different outcomes depending on:

    • Cell activation state (resting vs. activated)

    • Cell type (T cells vs. APCs)

    • Presence of other costimulatory signals

  • Methodological approach: Include appropriate controls for cell activation state and document all experimental conditions thoroughly

• Membrane-Bound vs. Soluble Forms:

  • Membrane-bound TNFSF18 may signal differently than recombinant soluble forms

  • Interpretation strategy: Consider whether observed effects might differ between natural membrane-bound TNFSF18 and recombinant soluble forms

• Standardized Analysis Framework:

  • When comparing contradictory results, create a table documenting:

    • Species/cell origin

    • TNFSF18 form used (membrane vs. soluble, tagged vs. untagged)

    • Readout systems

    • Experimental conditions

  • This systematic approach helps identify variables that might explain seemingly contradictory outcomes

What critical controls should be included when studying TNFSF18-mediated effects in immune cells?

Robust experimental design for TNFSF18 research requires specific controls:

• Protein-Specific Controls:

  • Heat-inactivated TNFSF18 (to control for non-specific protein effects)

  • Isotype-matched control protein (for tagged recombinant proteins)

  • Blocking antibodies against TNFSF18 or GITR (to confirm specificity of observed effects)

• Cell-Specific Controls:

  • GITR-deficient cells (to confirm receptor dependency)

  • Comparison of effects on different T cell subsets (CD4+ vs. CD8+, naive vs. memory)

  • Inclusion of relevant APCs when studying T cell responses

• Activation Controls:

  • Submaximal TCR stimulation conditions (where TNFSF18 costimulatory effects are most evident)

  • Positive controls using established costimulatory molecules (e.g., anti-CD28)

  • Time-course experiments to capture both early and late events

• Signaling Pathway Controls:

  • Specific pathway inhibitors (e.g., NF-κB inhibitors) to confirm involvement of hypothesized mechanisms

  • Phosphorylation state analysis of multiple pathway components

How can recombinant TNFSF18 be utilized to manipulate regulatory T cell function in experimental systems?

Recombinant TNFSF18 offers multiple approaches to investigate and manipulate Treg function, though with important species-specific considerations:

• Mouse Models (where TNFSF18-GITR interaction abrogates Treg suppression):

  • In vitro applications:

    • Addition of recombinant TNFSF18 to Treg suppression assays (expected outcome: reduced suppression)

    • Pretreatment of isolated Tregs before functional assays

    • Dose titration to establish threshold effects

  • In vivo applications:

    • Administration of recombinant TNFSF18 or agonistic anti-GITR antibodies

    • Development of TNFSF18-expressing cell-based therapies

    • TNFSF18-Fc fusion proteins with extended half-life

• Human Systems (where direct abrogation of Treg suppression is not observed):

  • Focus on TNFSF18's costimulatory effects on effector T cells

  • Investigate whether indirect mechanisms might influence Treg/Teff balance

  • Examine effects on Treg stability and phenotype rather than suppressive function

• Methodological Recommendations:

  • Always include parallel mouse and human experiments when studying Treg effects

  • Thoroughly characterize Treg phenotype (FOXP3, CD25, GITR expression) before and after TNFSF18 treatment

  • Assess multiple suppression mechanisms (cytokine production, metabolic inhibition, direct contact)

What is the potential role of TNFSF18 in inflammation and autoimmunity research?

TNFSF18 has significant implications for inflammation and autoimmunity research due to its immunoregulatory properties:

• Endothelial Cell Interactions:

  • TNFSF18 is important for interactions between activated T lymphocytes and endothelial cells

  • It triggers increased phosphorylation of STAT1 and upregulates expression of VCAM1 and ICAM1

  • These effects promote leukocyte adhesion to endothelial cells and regulate migration of monocytes from the splenic reservoir to sites of inflammation

  • Research application: Study TNFSF18 as a potential target for modulating vascular inflammation

• Reverse Signaling in APCs:

  • TNFSF18-GITR interaction induces reverse signaling in GITR Ligand-expressing dendritic cells

  • This suppresses cellular activation through the same pathway induced by the immunosuppressant dexamethasone

  • Research application: Investigate TNFSF18 as a natural regulator of DC function in inflammation

• Autoimmune Disease Models:

  • Dysregulation of TNFSF18 has been implicated in various autoimmune diseases

  • Research applications include:

    • Studying TNFSF18 expression patterns in autoimmune disease tissues

    • Correlating TNFSF18 levels with disease severity or treatment response

    • Developing targeted therapies that modulate the TNFSF18-GITR axis

What are the current limitations in TNFSF18 research and how might they be addressed?

Several significant limitations exist in current TNFSF18 research:

• Species-Specific Differences:

  • The functional divergence between human and mouse TNFSF18-GITR systems complicates translational research

  • Solution approach: Development of humanized mouse models specifically for studying the human TNFSF18-GITR axis

• Structural Insights:

  • Limited high-resolution structural data on TNFSF18-GITR complexes hampers structure-based drug design

  • Solution approach: Prioritize crystallography or cryo-EM studies of the complex to guide development of more specific modulators

• Physiological Relevance:

  • Most studies use recombinant soluble TNFSF18, which may not fully recapitulate the biology of membrane-bound forms

  • Solution approach: Develop systems that present TNFSF18 in membrane-bound format (e.g., cell-based assays, liposome-bound protein)

• Context-Dependent Function:

  • TNFSF18 effects are highly dependent on the broader immunological context

  • Solution approach: More comprehensive systems biology approaches that examine TNFSF18 function in concert with other immunoregulatory pathways