EBI3 Recombinant Monoclonal Antibody

What is EBI3 and what are its main biological functions?

EBI3 is a secreted glycoprotein of the hematopoietin receptor family that plays critical regulatory roles in immune responses. It dimerizes with p28 and p35 subunits of IL-12 to form the composite cytokines IL-27 and IL-35, respectively. EBI3 is involved in multiple immunoregulatory functions, including:

• Regulation of Th2-type immune responses and development of Th2-mediated tissue inflammation

• Control of iNKT cell function

• Negative regulation of Th17 cell differentiation (via IL-27)

• Inhibition of delayed-type hypersensitivity responses by suppressing IL-17 production and inducing IL-10 hyperproduction

• Control of tumor metastasis via lung CD8+ T cells

• Regulation of allergic airway inflammation through modulation of Th2 cytokines

In recent research, EBI3 has been found to play an unexpected role in mediating IL-6 trans-signaling, which has significant implications for inflammatory processes .

What cell types express EBI3 and under what conditions?

EBI3 is widely expressed across multiple cell types, with expression patterns that vary depending on activation state and environmental conditions:

• Dendritic cells: Expression is transcriptionally regulated by TLR signaling via MyD88 and NF-kappaB during innate immune responses

• Human neutrophils: Express and release EBI3 when activated by TLR8 agonists, with or without IFNγ

• Natural killer (NK) cells: Both human and mouse NK cells express EBI3 after stimulation. Human NK cells specifically express EBI3 after NKG2D or IL-12 plus IL-18 stimulation

• Keratinocytes: Psoriatic keratinocytes have been shown to produce EBI3-containing cytokines

The expression timing is also significant - in mouse NK cells during MCMV infection, EBI3 protein expression is a late activation event, appearing after early activation markers like IFNγ production and CD69 expression .

How does EBI3 interact with other cytokine subunits, and what complexes does it form?

EBI3 functions primarily through dimerization with other cytokine subunits to form heterodimeric cytokines:

• EBI3 + p28 → IL-27: An early product of activated antigen-presenting cells produced upon TLR ligation that negatively regulates Th17 cell differentiation

• EBI3 + p35 → IL-35: Forms a composite cytokine shown to activate homodimers of the signal transducing subunit of gp130

• EBI3 + IL-6: Recent research has demonstrated that EBI3 can bind IL-6, forming a complex that mediates IL-6 trans-signaling, albeit less efficiently than soluble IL-6Rα. This interaction was confirmed through co-immunoprecipitation experiments and surface plasmon resonance (SPR)

These interactions demonstrate EBI3's versatility in immune regulation through the formation of different cytokine complexes with distinct biological activities.

What is the mechanism of EBI3-mediated IL-6 trans-signaling and how does it differ from classical IL-6 signaling?

EBI3 has been recently discovered to mediate IL-6 trans-signaling through direct binding to IL-6. This mechanism involves:

• Formation of an EBI3·IL-6 complex that can activate cells expressing the signal-transducing receptor component gp130, even in the absence of membrane-bound IL-6Rα

• Activation of the JAK/STAT signaling pathway, specifically inducing STAT3 phosphorylation

• A time-dependent effect: while classical IL-6 signaling induces STAT3 phosphorylation within 15 minutes, EBI3-mediated trans-signaling requires longer incubation periods (overnight incubation)

• Unlike classical IL-6 signaling that is blocked by anti-IL-6Rα antibodies, EBI3-induced STAT3 phosphorylation is not affected by these antibodies

This trans-signaling mechanism expands the range of cells that can respond to IL-6 beyond those expressing membrane-bound IL-6Rα. Experimentally, this can be demonstrated using the IL-6-dependent B9 mouse plasmacytoma cell line, where EBI3 induces proliferation that can be inhibited by anti-IL-6 antibodies or anti-IL-6·IL-6Rα complex antibodies, confirming the involvement of IL-6 in this process .

How does EBI3 contribute to the regulation of NK cell responses during viral infections?

EBI3 plays a crucial role in regulating NK cell responses during viral infections, particularly cytomegalovirus:

• Both human and mouse NK cells express EBI3 and its receptor gp130 after stimulation

• During mouse cytomegalovirus (MCMV) infection, EBI3 expression in NK cells is a late activation event

• EBI3 affects the establishment of MCMV latency, as demonstrated in EBI3-deficient mice

• MCMV-infected EBI3-deficient mice exhibit multiple immunological changes compared to wild-type mice:

  • Decreased IL-10 production by NK cells

  • Enhanced dendritic cell maturation

  • Increased activation of CD8+ T cells

  • Significantly reduced viral loads in salivary glands and oral lavage

These findings suggest that EBI3 functions as an immunoregulatory molecule that promotes viral persistence by modulating both innate and adaptive immune responses. Mechanistically, EBI3 appears to induce IL-10 production in NK cells, which creates an immunosuppressive environment favorable for viral persistence .

What are the contradictions in EBI3 function across different disease models and how might these be reconciled?

EBI3 exhibits seemingly contradictory functions across different disease models:

These contradictions might be reconciled by considering:

• Context-dependent interactions: EBI3 forms different complexes (IL-27, IL-35, EBI3-IL-6) depending on the local cytokine milieu

• Differential timing: EBI3 expression is a late activation event in some contexts, allowing different outcomes at different disease stages

• Cell-specific effects: EBI3 affects various cell types (NK cells, dendritic cells, T cells) differently

• Dose-dependent effects: Concentration variations might trigger different signaling pathways

These observations suggest that therapeutic approaches targeting EBI3 would need to be tailored to specific disease contexts and carefully timed to achieve desired outcomes.

What are the optimal detection methods for studying EBI3 expression and function in different experimental systems?

For optimal detection of EBI3 in various experimental systems, researchers should consider these methodological approaches:

Flow Cytometry:

• For intracellular staining, cells should be fixed with paraformaldehyde and permeabilized with saponin

• This technique was successfully used to detect IL-27/IL-35 EBI3 Subunit in KG-1 human cell line treated with IL-18/IL-1F4

• When analyzing STAT3 activation, use FITC-labeled anti-phospho-STAT3 (Tyr-705) antibodies for detection

Western Blotting:

• For detection of secreted EBI3, concentrate cell culture supernatants using centrifugal filters

• EBI3 can be immunoprecipitated using specific anti-EBI3 antibodies followed by detection with HRP-conjugated secondary antibodies

• For studying EBI3-IL-6 interactions, co-immunoprecipitation using His-tagged EBI3 and detection with appropriate antibodies can be employed

Functional Assays:

• The B9 mouse plasmacytoma cell line provides a reliable system to assess EBI3 biological activity

• Proliferation can be measured using fluorometric assays like AlamarBlue after 72-hour incubation

• For inhibition studies, include IL-6 or gp130 targeting antibodies (5 μg/ml) or anti-IL6·IL-6Rα complex antibodies (10 μg/ml)

In vivo Models:

• EBI3-deficient mice provide valuable tools for studying EBI3 functions in disease models

• Monitor viral loads in appropriate tissues (e.g., salivary glands for MCMV)

• Assess immune cell activation markers and cytokine production profiles

How can researchers differentiate between the effects of EBI3 alone versus its heterodimeric cytokines (IL-27 and IL-35) in experimental settings?

Differentiating between EBI3 alone and its heterodimeric forms requires careful experimental design:

Recombinant Protein Approaches:

• Compare responses to purified EBI3 alone versus recombinant IL-27 (EBI3+p28) or IL-35 (EBI3+p35)

• Control for potential contamination or interaction with endogenous p28 or p35 by using cells known not to express these subunits (verify by RT-PCR)

Receptor Blocking Strategy:

• Use receptor-specific blocking antibodies:

  • Anti-IL-6Rα mAbs block IL-6 but not EBI3-induced effects

  • Anti-gp130 antibodies block both IL-6 and EBI3-mediated responses

  • IL-27Rα-specific antibodies block IL-27 but not EBI3 or IL-35 effects

Genetic Approaches:

• Compare phenotypes between EBI3-deficient, p28-deficient, and p35-deficient mice or cells

• Use siRNA knockdown of specific subunits to create cells deficient in just one component

• Employ cell lines expressing only certain receptor components (e.g., Ba/F3-gp130 cells)

Time-Course Experiments:

• Monitor early versus late responses, as EBI3 expression is often a late activation event

• Assess STAT3 phosphorylation at both early (15 min) and late (16 h) timepoints

These approaches enable researchers to dissect the specific contributions of EBI3 versus its heterodimeric partners in complex immune responses.

What considerations should be made when designing experiments to study EBI3's role in IL-6 trans-signaling?

When investigating EBI3's role in IL-6 trans-signaling, researchers should consider:

Controls and Validation:

• Include appropriate controls to distinguish classical IL-6 signaling from trans-signaling:

  • Use soluble IL-6Rα + IL-6 as a positive control for trans-signaling

  • Include blocking anti-IL-6 antibodies to confirm IL-6 dependency

  • Use cell lines expressing only gp130 without IL-6Rα (e.g., Ba/F3-gp130) to specifically study trans-signaling

Concentration and Timing:

• Use EBI3 concentrations ranging from 0.5 to 2 μg/ml, as these have been shown to induce STAT3 phosphorylation

• Include both short (15 min) and long (16+ h) incubation periods, as EBI3-mediated effects often require longer incubation times

• Serum- and cytokine-starve cells for 16 h before experiments to reduce background signaling

Detection Systems:

• For studying STAT3 activation, use both flow cytometry and Western blotting methods

• When working with primary cells, include cell-type specific markers (e.g., CD4 for T cells) to identify responding populations

• Consider using reporter cell lines expressing luciferase under STAT3-responsive promoters for enhanced sensitivity

Binding Confirmation:

• Verify EBI3-IL-6 binding using:

  • Co-immunoprecipitation with tagged proteins

  • Surface plasmon resonance (SPR) to assess binding kinetics

  • Stable transfectants expressing individual components or combinations

These methodological considerations will help ensure robust and interpretable results when investigating this newly discovered function of EBI3.

What are the therapeutic implications of EBI3's dual roles in immune regulation for autoimmune and inflammatory diseases?

The complex roles of EBI3 in immune regulation suggest several therapeutic considerations:

This dual nature of EBI3 highlights the importance of comprehensive understanding of its mechanisms before clinical translation. The therapeutic potential may depend on tilting the balance toward anti-inflammatory functions (via IL-27/IL-35) while blocking pro-inflammatory activities (via IL-6 trans-signaling).

How might EBI3 expression patterns be utilized as biomarkers in viral infections and inflammatory conditions?

EBI3 expression patterns show potential as biomarkers in several contexts:

• In viral infections like cytomegalovirus, elevated EBI3 expression in NK cells appears to correlate with viral persistence and establishment of latency

• EBI3-deficient mice show significantly diminished viral loads in salivary glands and oral lavage during MCMV infection, suggesting that EBI3 levels might predict viral control

• The timing of EBI3 expression (late activation event) in NK cells during viral infections provides a potential window for monitoring disease progression

• In inflammatory conditions, levels of EBI3 relative to its binding partners (p28, p35, IL-6) might reflect the predominant inflammatory pathway activated (IL-27, IL-35, or IL-6 trans-signaling)

Importantly, interpreting EBI3 as a biomarker requires consideration of the entire cytokine network, as its biological activity depends on the availability of binding partners and the expression of relevant receptors on target cells.

What emerging technologies could advance our understanding of EBI3's complex roles in immune regulation?

Several emerging technologies could enhance EBI3 research:

Single-Cell Analysis:

• Single-cell RNA sequencing could reveal cell-specific expression patterns of EBI3 and its binding partners during immune responses

• Mass cytometry (CyTOF) would allow simultaneous detection of EBI3, its binding partners, and downstream signaling molecules at the single-cell level

Advanced Imaging:

• Intravital microscopy could track EBI3-expressing cells in vivo during immune responses

• Proximity ligation assays might visualize EBI3 interactions with binding partners in situ

Structural Biology:

• Cryo-electron microscopy of EBI3-containing complexes would provide insights into the structural basis of their diverse functions

• Hydrogen-deuterium exchange mass spectrometry could map interaction interfaces between EBI3 and its various binding partners

Systems Biology:

• Network analysis integrating transcriptomic, proteomic, and functional data could help reconcile the seemingly contradictory functions of EBI3 across different disease models

• Mathematical modeling of cytokine networks including EBI3 might predict context-dependent outcomes of therapeutic interventions

These technologies would help address the complexity of EBI3 biology by providing more comprehensive, contextual, and mechanistic insights.

What strategies could optimize the use of EBI3 recombinant monoclonal antibodies in research applications?

To optimize EBI3 recombinant monoclonal antibodies for research:

Epitope Mapping and Selection:

• Develop antibodies targeting different epitopes to distinguish various functional domains of EBI3

• Select antibodies that specifically block or not block interaction with different binding partners (p28, p35, IL-6)

Validation Across Applications:

• Thoroughly validate antibodies for specific applications (Western blot, flow cytometry, immunoprecipitation, functional blocking)

• Test across multiple cell types and species to ensure consistent performance

Combination Approaches:

• Use EBI3 antibodies in combination with antibodies against binding partners or receptors

• Develop bi-specific antibodies that can simultaneously target EBI3 and a binding partner or receptor

Novel Formats:

• Explore nanobody or single-chain variable fragment (scFv) formats for improved tissue penetration

• Develop reporter-coupled antibodies for real-time monitoring of EBI3 expression in live cells or tissues

These strategies would enhance the utility of EBI3 recombinant monoclonal antibodies as both research tools and potential therapeutic agents.