GO:0070745 interleukin-35 complex: Components, Assembly and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0070745 (interleukin-35 complex) is a secreted heterodimeric cytokine complex composed of the IL12A-encoded p35 subunit and the EBI3 subunit.
• IL-35 is a member of the IL-12 cytokine family, which also includes IL-12, IL-23, and IL-27, all sharing alpha and beta subunit architectures.
• IL-35 signals through a heterodimeric receptor composed of IL12Rβ2 and gp130, activating STAT1 and STAT4 transcription factors.
• IL-35 is produced by regulatory T cells and suppresses effector T cell proliferation, contributing to immune tolerance.
• Dysregulated IL-35 expression is implicated in autoimmune diseases, atherosclerosis, myocardial infarction, acute lung injury, and immune thrombocytopenia.
• CRISPR-based knockout, knock-in, and overexpression models enable causal interrogation of IL12A and EBI3 in immune regulation and disease.
Description
The interleukin-35 (IL-35) complex, annotated as GO:0070745, is a secreted heterodimeric protein complex belonging to the IL-12 cytokine family. It is composed of two distinct subunits: the p35 alpha subunit encoded by the IL12A gene and the EBI3 beta subunit encoded by the EBI3 gene. Unlike IL-12, which shares the p35 subunit with IL-35, IL-35 is unique in pairing p35 with EBI3 rather than p40. This distinct subunit composition confers unique functional properties, including potent immunosuppressive activity primarily attributed to regulatory T cells. Since its identification as a novel cytokine of the IL-12 family, IL-35 has emerged as a critical mediator of immune tolerance and homeostasis. It is secreted into the extracellular space where it engages a heterodimeric receptor composed of IL12Rβ2 and gp130, leading to activation of STAT1 and STAT4 signaling pathways. This signaling cascade is essential for its immunoregulatory functions, including suppression of effector T cell proliferation and promotion of regulatory T cell expansion. Research on IL-35 has expanded beyond basic immunology into diverse pathological contexts. Dysregulated IL-35 expression has been associated with autoimmune diseases, cardiovascular disorders, and inflammatory conditions. Understanding the molecular composition, assembly, and regulation of the IL-35 complex is therefore essential for developing targeted therapeutic strategies and for interpreting its role in disease pathogenesis.
interleukin-35 complex At A Glance
| GO ID | GO:0070745 |
|---|---|
| GO term | interleukin-35 complex |
| Ontology | cellular_component |
| Synonym | EBI3, IL12A, IL-35 complex, p35 |
| Major function | Secreted heterodimeric cytokine complex that suppresses effector T cell responses and promotes immune tolerance |
| Subunit composition | IL12A-encoded p35 subunit and EBI3 subunit |
| Cellular localization | Secreted into the extracellular space |
| Signaling receptor | IL12Rβ2 and gp130 heterodimer |
| Downstream effectors | STAT1 and STAT4 transcription factors |
What Is GO:0070745?
GO:0070745, the interleukin-35 complex, is a secreted protein complex that consists of two subunits: the interleukin-12 alpha subunit (p35), which is the product of the IL12A gene, and the EBI3 subunit. This heterodimeric complex is released into the extracellular space where it functions as an immunomodulatory cytokine.
Why Is interleukin-35 complex Important in Cell Biology?
The interleukin-35 complex is critically important because it represents a key immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity. Its unique subunit composition distinguishes it from other IL-12 family members, and its ability to suppress effector T cell responses makes it a central player in immune tolerance. Dysregulation of IL-35 has been implicated in a wide range of human diseases, including autoimmune disorders, cardiovascular diseases, and inflammatory conditions. Understanding the biology of the IL-35 complex is therefore essential for researchers investigating immune regulation, developing immunotherapies, and identifying novel therapeutic targets.
• IL-35 is a potent immunosuppressive cytokine that inhibits effector T cell proliferation and function.
• It is preferentially produced by regulatory T cells and contributes to their suppressive capacity.
• IL-35 signaling through IL12Rβ2/gp130 activates STAT1 and STAT4, linking it to JAK-STAT pathways.
• Dysregulated IL-35 expression is associated with autoimmune diseases such as immune thrombocytopenia.
• IL-35 plays a protective role in atherosclerosis by reducing inflammation.
• IL-35 promotes macrophage survival and improves wound healing after myocardial infarction in mice.
• IL-35 reduces inflammation in acute lung injury through inhibition of TLR4/NF-κB signaling.
• The IL-35 complex is a member of the IL-12 family, which includes cytokines targeted by therapeutics such as ustekinumab.
• IL-35 serves as a potential biomarker and therapeutic target in inflammatory and cardiovascular diseases.
• CRISPR-based models enable precise dissection of IL12A and EBI3 gene function in immune regulation.
Structure and Composition of interleukin-35 complex
IL12A-encoded p35 subunit
In simple terms: The p35 protein is one half of the IL-35 complex.
The p35 subunit is encoded by the IL12A gene and is a member of the IL-12 alpha subunit family. It is a four-helix bundle cytokine that shares structural homology with other IL-12 family alpha subunits. In the IL-35 complex, p35 is disulfide-linked to the EBI3 subunit to form the mature heterodimer.
EBI3 subunit
In simple terms: EBI3 is the other half of the IL-35 complex.
EBI3 (Epstein-Barr virus-induced gene 3) is a soluble cytokine receptor-like subunit encoded by the EBI3 gene. It belongs to the IL-12 beta subunit family and contains fibronectin type III domains. EBI3 pairs with p35 to form IL-35, and it also pairs with p28 to form IL-27, demonstrating its versatility in cytokine assembly.
Heterodimer assembly and secretion
In simple terms: The two subunits combine inside the cell and are then released outside.
The IL-35 complex is assembled through the non-covalent and disulfide-linked association of the p35 and EBI3 subunits within the endoplasmic reticulum and Golgi apparatus. The assembled heterodimer is then secreted into the extracellular space. This secretion is essential for its function as a soluble cytokine that can act on neighboring cells in a paracrine or autocrine manner.
Receptor complex and signaling
In simple terms: IL-35 binds to a receptor on target cells to send a signal.
The secreted IL-35 complex binds to a heterodimeric receptor composed of IL12Rβ2 and gp130. This receptor engagement leads to activation of JAK kinases, which phosphorylate STAT1 and STAT4 transcription factors. Activated STAT1 and STAT4 then translocate to the nucleus to regulate gene expression programs that mediate the immunosuppressive functions of IL-35.
Regulation of IL-35 complex expression
In simple terms: Cells control how much IL-35 they make.
Expression of the IL-35 subunits is regulated at the transcriptional level in a cell-type-specific manner. Regulatory T cells are a major source of IL-35, and its expression is induced upon activation. The balance between IL-35 and other IL-12 family cytokines is tightly controlled to maintain immune homeostasis.
Key Genes Involved in GO:0070745 interleukin-35 complex
The following genes and proteins are directly involved in the structure, regulation, and function of the interleukin-35 complex.
| Gene | Major Role | Research Relevance |
|---|---|---|
| IL12A | Encodes the p35 alpha subunit of IL-35 | Essential for IL-35 complex formation and secretion |
| EBI3 | Encodes the EBI3 beta subunit of IL-35 | Required for IL-35 heterodimer assembly and function |
| IL12RB2 | Encodes IL12Rβ2 receptor subunit | Mediates IL-35 signaling through STAT4 |
| IL6ST | Encodes gp130 receptor subunit | Mediates IL-35 signaling through STAT1 |
| STAT1 | Transcription factor activated by IL-35 signaling | Key downstream effector of IL-35-mediated immunosuppression |
| STAT4 | Transcription factor activated by IL-35 signaling | Key downstream effector of IL-35-mediated immunosuppression |
| JAK1 | Janus kinase associated with gp130 | Phosphorylates STAT proteins upon IL-35 receptor engagement |
| JAK2 | Janus kinase associated with IL12Rβ2 | Phosphorylates STAT proteins upon IL-35 receptor engagement |
| FOXP3 | Master transcription factor of regulatory T cells | Regulates IL-35 expression in regulatory T cells |
| IL12B | Encodes p40 subunit of IL-12 and IL-23 | Distinguishes IL-12/IL-23 from IL-35, which uses EBI3 instead |
| IL27 | Encodes p28 subunit that pairs with EBI3 | Shares EBI3 subunit with IL-35, highlighting subunit promiscuity |
| IL23A | Encodes p19 subunit of IL-23 | Related IL-12 family cytokine with distinct functions |
| TGFB1 | Immunosuppressive cytokine that induces IL-35 expression | Cooperates with IL-35 in regulatory T cell function |
| IL10 | Anti-inflammatory cytokine with overlapping functions | Often co-expressed with IL-35 in regulatory T cells |
| CD4 | Marker of helper T cells including regulatory T cells | Defines the major cell type producing IL-35 |
| CD25 | IL-2 receptor alpha chain, marker of regulatory T cells | Enriches for IL-35-producing regulatory T cells |
| TLR4 | Toll-like receptor 4 involved in innate immunity | IL-35 inhibits TLR4/NF-κB signaling in acute lung injury |
| NFKB1 | NF-κB transcription factor subunit | Inhibited by IL-35 in inflammatory contexts |
How Is interleukin-35 complex Regulated?
The expression and activity of the interleukin-35 complex are regulated at multiple levels. Transcription of IL12A and EBI3 is induced in regulatory T cells upon activation, and FOXP3 is a key transcription factor driving this expression. The assembly and secretion of the heterodimer are controlled by intracellular trafficking pathways. Extracellularly, IL-35 activity is modulated by receptor availability and downstream signaling components, including JAK kinases and STAT transcription factors. Additionally, IL-35 expression can be influenced by other cytokines such as TGF-β and IL-10, which are often co-expressed in regulatory T cells. The balance between IL-35 and pro-inflammatory cytokines determines the outcome of immune responses.
interleukin-35 complex and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| IL12A | Autoimmune diseases, immune thrombocytopenia | Il12a knockout mouse or human cell line |
| EBI3 | Inflammatory diseases, atherosclerosis | Ebi3 knockout mouse or CRISPR knockout cell line |
| IL12RB2 | Immune dysregulation, cardiovascular disease | Il12rb2 knockout mouse |
| IL6ST | Acute lung injury, inflammation | Il6st conditional knockout mouse |
| STAT1 | Inflammatory and autoimmune diseases | Stat1 knockout mouse or cell line |
IL-35 in autoimmune and inflammatory diseases
Dysregulated IL-35 expression has been observed in autoimmune conditions such as immune thrombocytopenia, where altered IL-35 levels may contribute to impaired immune tolerance. IL-35 is also implicated in other autoimmune and inflammatory diseases, and its immunosuppressive properties suggest a protective role in limiting excessive inflammation. Targeting IL-35 or its receptor components may offer therapeutic avenues for modulating autoimmune responses.
IL-35 in cardiovascular disease
IL-35 plays a protective role in atherosclerosis by reducing inflammation and inhibiting the development of atherosclerotic plaques. In myocardial infarction, IL-35 promotes macrophage survival and improves wound healing in mouse models. These findings suggest that IL-35 could be a therapeutic target for cardiovascular diseases characterized by inflammation.
IL-35 in acute lung injury
IL-35 reduces inflammation in acute lung injury through inhibition of the TLR4/NF-κB signaling pathway. This anti-inflammatory effect highlights the potential of IL-35 as a therapeutic agent for acute respiratory distress syndrome and other inflammatory lung conditions.
IL-35 and therapeutic targeting of IL-12 family cytokines
The IL-12 family, including IL-35, is a target for therapeutic intervention in immune-mediated diseases. Lessons from ustekinumab, which targets IL-12/23p40, demonstrate the clinical potential of modulating this cytokine family. However, the distinct subunit composition and functions of IL-35 require careful consideration when designing targeted therapies.
From interleukin-35 complex-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| Does IL12A loss abolish IL-35 production? | IL12A knockout cell line or mouse |
| Does EBI3 loss abolish IL-35 production? | EBI3 knockout cell line or mouse |
| Does a point mutation in IL12A affect heterodimer assembly? | Point-mutation knock-in cell line |
| Can tagged IL-35 be used for tracking secretion? | Tagged knock-in of IL12A or EBI3 |
| Does IL-35 overexpression suppress T cell proliferation? | IL12A/EBI3 overexpression cell line |
| Does IL-35 receptor mutation affect STAT signaling? | IL12RB2 or IL6ST point-mutation knock-in |
How to Study the interleukin-35 complex Process
| Method | What It Measures | Typical Application |
|---|---|---|
| CRISPR knockout | Loss of IL12A or EBI3 gene function | Assessing IL-35 requirement in immune suppression |
| Knock-in tagging | Endogenous IL-35 localization and secretion | Tracking IL-35 in live cells |
| Overexpression | Gain of IL-35 function | Producing recombinant IL-35 for assays |
| Co-immunoprecipitation | Protein-protein interactions | Identifying IL-35 receptor components |
| Western blot | Protein expression and phosphorylation | Detecting IL-35 subunits and STAT activation |
| ELISA | Secreted IL-35 levels | Quantifying IL-35 in culture supernatants or serum |
| Flow cytometry | Cell surface markers and intracellular cytokines | Identifying IL-35-producing regulatory T cells |
| RNA-seq | Transcriptional changes | Global gene expression upon IL-35 treatment |
CRISPR knockout for loss-of-function studies
CRISPR-Cas9 knockout of IL12A or EBI3 can be used to abolish IL-35 complex production in cell lines or primary cells. This approach enables researchers to assess the contribution of IL-35 to immune suppression, cytokine secretion, and disease models.
Knock-in and tagged knock-in for tracking and localization
Knock-in of epitope tags (e.g., FLAG, HA) into endogenous IL12A or EBI3 loci allows for detection, purification, and tracking of the IL-35 complex. This is particularly useful for studying assembly, secretion, and receptor binding.
Overexpression for gain-of-function studies
Overexpression of IL12A and EBI3 in cell lines can be used to produce recombinant IL-35 for functional assays, including T cell suppression assays and signaling studies. This approach helps determine whether increased IL-35 levels are sufficient to modulate immune responses.
Proteomics and interactomics
Mass spectrometry-based proteomics can identify IL-35 interacting partners and post-translational modifications. Co-immunoprecipitation followed by mass spectrometry can reveal the composition of the IL-35 receptor complex and downstream signaling molecules.
How CRISPR Can Be Used to Study GO:0070745 interleukin-35 complex
Knockout
CRISPR-Cas9 knockout of IL12A or EBI3 is used to completely abrogate IL-35 complex formation. This model is essential for determining whether IL-35 is required for regulatory T cell function and for studying its role in autoimmune and inflammatory diseases.
Point Mutation
Point mutations can be introduced into IL12A or EBI3 to disrupt specific residues involved in subunit interaction or receptor binding. Such models help dissect the structural requirements for IL-35 assembly and signaling.
Knock-in
Knock-in of reporter genes or epitope tags into the IL12A or EBI3 loci enables real-time tracking of IL-35 expression and secretion. This approach is valuable for studying the dynamics of IL-35 production in vivo.
Overexpression
CRISPR activation (CRISPRa) or lentiviral overexpression can be used to increase IL-35 levels in target cells. Overexpression models are useful for gain-of-function studies and for producing IL-35 for therapeutic applications.
How EDITGENE Supports interleukin-35 complex Research
Researchers studying interleukin-35 complex-related genes often need to determine whether a candidate gene is causally involved in IL-35 assembly, secretion, or signaling. EDITGENE provides comprehensive CRISPR-based services to enable precise genetic interrogation of IL12A, EBI3, and related pathway components.
Contact EDITGENE today to design your custom CRISPR model for interleukin-35 complex research.
Frequently Asked Questions About interleukin-35 complex
What is the interleukin-35 complex?
The interleukin-35 complex (GO:0070745) is a secreted heterodimeric cytokine composed of the IL12A-encoded p35 subunit and the EBI3 subunit.
What genes are involved in the interleukin-35 complex?
The interleukin-35 complex is encoded by the IL12A and EBI3 genes, which produce the p35 and EBI3 subunits, respectively.
What is the function of IL-35?
IL-35 functions as an immunosuppressive cytokine that inhibits effector T cell proliferation and promotes immune tolerance.
How does IL-35 signal?
IL-35 signals through a heterodimeric receptor composed of IL12Rβ2 and gp130, activating STAT1 and STAT4 transcription factors.
Which cells produce IL-35?
IL-35 is primarily produced by regulatory T cells, and its expression is induced upon activation.
What diseases are associated with IL-35?
IL-35 has been implicated in autoimmune diseases, atherosclerosis, myocardial infarction, acute lung injury, and immune thrombocytopenia.
Is IL-35 the same as IL-12?
No, IL-35 and IL-12 are distinct cytokines. IL-12 is composed of p35 and p40 subunits, while IL-35 is composed of p35 and EBI3 subunits.
What is the GO ID for interleukin-35 complex?
The Gene Ontology ID for interleukin-35 complex is GO:0070745.
How can I study IL-35 using CRISPR?
CRISPR knockout of IL12A or EBI3 can abolish IL-35 production, while knock-in and overexpression models enable tracking and gain-of-function studies.
What is the role of IL-35 in cardiovascular disease?
IL-35 plays a protective role in atherosclerosis and promotes macrophage survival and wound healing after myocardial infarction in mice.
Conclusion
The interleukin-35 complex (GO:0070745) is a unique immunosuppressive cytokine of the IL-12 family, composed of the IL12A-encoded p35 subunit and EBI3 subunit. Its secretion and signaling through IL12Rβ2/gp130 and STAT1/STAT4 pathways make it a critical regulator of immune tolerance and inflammation. Dysregulation of IL-35 is associated with a range of human diseases, including autoimmune disorders, cardiovascular disease, and acute lung injury. Continued research using advanced CRISPR models will further elucidate its mechanisms and therapeutic potential.
References
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- 4. Collison LW et al.. 2008. Interleukin-35: odd one out or part of the family?. Immunol Rev 226:248-62 PMID: 19161429
- 5. Lin J et al.. 2015. The role of interleukin 35 in atherosclerosis.. Curr Pharm Des 21(35):5151-9 PMID: 26530251
- 6. Jia D et al.. 2019. Interleukin-35 Promotes Macrophage Survival and Improves Wound Healing After Myocardial Infarction in Mice.. Circ Res 124(9):1323-1336 PMID: 30832557
- 7. Pan W et al.. 2020. Interleukin-35 reduces inflammation in acute lung injury through inhibiting TLR4/NF-κB signaling pathways.. Exp Ther Med 19(3):1695-1700 PMID: 32104222
- 8. Elliott M et al.. 2009. Ustekinumab: lessons learned from targeting interleukin-12/23p40 in immune-mediated diseases.. Ann N Y Acad Sci 1182:97-110 PMID: 20074279
