IL35 Human

What is the structure and composition of human IL-35?

Human IL-35 is a heterodimeric cytokine belonging to the IL-12 family, composed of two distinct subunits: IL-12 p35 (also called IL-12A) and Epstein-Barr virus-induced gene 3 (EBI3). The IL-12 p35 subunit is synthesized as a 219 amino acid precursor protein with a 22 amino acid signal sequence and a 197 amino acid mature region . The EBI3 subunit is synthesized as a 229 amino acid precursor protein containing a 20 amino acid signal sequence and a 209 amino acid mature region . These two subunits combine to form the functional IL-35 heterodimer. IL-35 shares the p35 subunit with IL-12 and the EBI3 subunit with IL-27, creating a complex interrelationship within the IL-12 cytokine family . This shared subunit composition has important implications for both detection methodologies and functional studies, as researchers must carefully distinguish IL-35 from other IL-12 family members in experimental settings.

How does human IL-35 expression differ from mouse IL-35?

The expression patterns of IL-35 differ significantly between humans and mice, which has important implications for translational research. In mice, both IL-35 subunits (IL-12A and EBI3) are constitutively expressed in regulatory T cells (Tregs), allowing for immediate secretion of the mature IL-35 cytokine . In contrast, human Tregs constitutively express only the IL-12 p35 subunit, while EBI3 expression must be induced upon immune activation . This fundamental difference means that non-stimulated human Tregs do not secrete IL-35, unlike their mouse counterparts. Additionally, studies have shown that in tissue distribution patterns, mouse blood, bone marrow, liver, and thymus tissues express both subunits of IL-35 in a "ready to go" status, while none of the examined human tissues exhibited this pattern . Human and mouse IL-35 share 58% and 62% sequence homology in their IL-12 p35 and EBI3 subunits, respectively . These species-specific differences necessitate caution when extrapolating findings from mouse models to human conditions in IL-35 research.

What are the main functions of IL-35 in human immune regulation?

IL-35 functions primarily as an immunosuppressive cytokine with multiple regulatory effects on immune cell populations. In both humans and mice, IL-35 has been demonstrated to suppress effector T cell proliferation, inhibit Th17 cell development, and promote the conversion of conventional T cells into regulatory T cells (termed iTr35 cells) . IL-35 can also convert B cells into regulatory B cells, expanding its immunosuppressive network . Within the tumor microenvironment, IL-35 derived from Tregs can induce CD4+ and CD8+ T cell exhaustion, reducing their effector functionality and impairing the generation of cytotoxic T lymphocytes (CTLs) . IL-35 does not directly suppress CTLs but rather downregulates the costimulatory molecule CD28 on immature CD8+ T cells, interfering with their differentiation into anti-tumor CTLs . Additionally, IL-35 can disrupt Th1 cell activation, thereby suppressing differentiated anti-tumor CTL activity . IL-35 also influences dendritic cell (DC) function, suppressing lipopolysaccharide-triggered DC maturation and influencing their production of other regulatory cytokines like IL-10 and TGF-β . These diverse immunoregulatory properties position IL-35 as a critical mediator in controlling inflammatory responses, maintaining immune tolerance, and potentially contributing to tumor immune evasion.

What cell types produce IL-35 in humans?

• Microvascular endothelial cells - when stimulated with proinflammatory cytokines such as TNF-α, IFN-γ, and IL-1β

• Aortic smooth muscle cells - following inflammatory stimulation

• Epithelial cells - particularly when stimulated with TNF-α which induces both IL-12A and EBI3 subunits

• Immature dendritic cells - especially following LPS stimulation

• Placental trophoblasts - which constitutively express and secrete IL-35

• Tolerogenic dendritic cells (TolDCs) - which express both IL-12p35 and EBI3 subunits

• Certain tumor cells - which may produce IL-35 as an immune evasion mechanism

Importantly, the upregulation of IL-35 in non-T cells follows a "double-gated" control mechanism, requiring two different signals to induce both subunits. For example, in epithelial cells, while TNF-α can induce both subunits, IFN-γ only upregulates IL-12A but not EBI3, though TNF-α and IFN-γ can synergistically enhance the expression of both .

How are the IL-35 subunits regulated at the transcriptional and post-transcriptional levels?

The regulation of IL-35 subunits involves complex transcriptional and post-transcriptional mechanisms that explain its inducible rather than constitutive expression pattern. Several key regulatory mechanisms have been identified:

Transcriptional regulation:

• NF-κB transcription factor has higher binding frequencies in the promoter regions of IL-35 subunits compared to other anti-inflammatory cytokines, suggesting a strong responsiveness to inflammatory signals

• Alternative promoter usage contributes to the differential expression of IL-35 subunits in various cell types and conditions

• Epigenetic mechanisms, particularly DNA methylation status, influence IL-35 expression, with hypomethylation associated with higher IL-35 expression

Post-transcriptional regulation:

• AU-rich elements (AREs) in the 3' untranslated regions (UTRs) of IL-35 subunit transcripts enable rapid mRNA degradation, contributing to the non-constitutive expression pattern

• MicroRNAs differentially target the two IL-35 subunits, providing another layer of post-transcriptional control

• Alternative splicing affects the structure and expression of IL-35 subunits, adding complexity to their regulation

The dual control requirements for expressing both subunits create a tightly regulated system where IL-35 production is specifically induced in inflammatory contexts rather than being constitutively present like TGF-β . This regulatory pattern positions IL-35 as a responsive anti-inflammatory cytokine rather than a housekeeping one, allowing for context-specific immunosuppression.

What is the receptor complex for IL-35 and how does signaling occur?

IL-35 exhibits unique receptor binding flexibility, engaging multiple receptor configurations to initiate signaling. IL-35 can bind to three distinct receptor complexes: the homodimeric IL-12Rβ2 receptor, the homodimeric gp130 receptor, and the heterodimeric IL-12Rβ2/gp130 receptor . This receptor binding versatility distinguishes IL-35 from other IL-12 family cytokines that typically utilize one specific receptor complex.

The signaling mechanisms activated by IL-35 binding include:

• In cells expressing both IL-12Rβ2 and gp130, IL-35 binding to the heterodimeric receptor activates both STAT1 and STAT4 signaling pathways

• In cells expressing only IL-12Rβ2 homodimers, IL-35 activates only STAT4

• In cells expressing only gp130 homodimers, IL-35 activates only STAT1

This unique pattern enables IL-35 to exert immunoregulatory effects across diverse cell populations with different receptor expression profiles. Notably, the receptors for IL-35 (IL-12Rβ2 and gp130) are constitutively expressed in cardiovascular and other tissues even when IL-35 itself is not expressed . This suggests that tissues are primed to respond to IL-35 when it's induced during inflammatory conditions, allowing for rapid immunoregulation when needed. The signaling pathways activated by IL-35 ultimately lead to suppression of T cell proliferation, inhibition of effector cytokine production, and induction of regulatory phenotypes in target cells.

What methodologies are most effective for detecting IL-35 in human samples?

Detecting IL-35 in human samples presents unique challenges due to its heterodimeric nature and shared subunits with other IL-12 family cytokines. Researchers should consider the following methodological approaches:

For protein detection:

• Co-immunoprecipitation followed by Western blotting - This approach can verify the association of p35 and EBI3 subunits, confirming the presence of the heterodimer rather than individual subunits . Use antibodies specific to each subunit and perform reciprocal immunoprecipitation to confirm interaction.

• Sandwich ELISA - Custom designed ELISAs using capture antibodies against one subunit and detection antibodies against the other can specifically detect the heterodimer. Commercial ELISA kits should be validated for specificity against other IL-12 family members.

• Proximity ligation assay (PLA) - This technique detects protein-protein interactions when the target proteins are in close proximity (≤40 nm), making it valuable for confirming the presence of the IL-35 heterodimer in tissue sections.

For mRNA detection:

• Dual fluorescence in situ hybridization (FISH) - This allows simultaneous detection of both IL-12A and EBI3 transcripts within the same cell.

• Quantitative RT-PCR - While measuring individual subunit transcripts doesn't confirm heterodimer formation, coordinated upregulation of both subunits suggests potential IL-35 production. Design primers specific to full-length transcripts to avoid splice variants.

• Single-cell RNA sequencing - This approach identifies cells co-expressing both IL-35 subunits and can reveal novel cellular sources in complex tissue environments.

When conducting IL-35 studies, researchers should always assess both subunits simultaneously and utilize complementary methods to verify results. Additionally, including appropriate controls (such as recombinant IL-35, IL-12, and IL-27) is crucial for confirming assay specificity, particularly given the shared subunit composition across the IL-12 cytokine family.

How can researchers differentiate between IL-35 and other IL-12 family cytokines in experimental settings?

Differentiating IL-35 from other IL-12 family members (particularly IL-12 and IL-27) requires strategic experimental approaches due to shared subunits. Researchers should implement the following methodological strategies:

• Subunit-specific functional analysis:

  • Use neutralizing antibodies against specific subunits to determine which heterodimer is responsible for observed effects

  • Employ siRNA or CRISPR-Cas9 to selectively knock down individual subunits and observe functional consequences

• Receptor engagement analysis:

  • IL-35 uniquely signals through gp130/IL-12Rβ2 heterodimers or homodimers of either receptor

  • IL-12 signals through IL-12Rβ1/IL-12Rβ2

  • IL-27 signals through gp130/WSX-1

  • Blocking specific receptor components can help identify which cytokine is mediating observed effects

• Downstream signaling evaluation:

  • Measure activation patterns of STAT proteins (STAT1, STAT3, STAT4)

  • IL-35 primarily activates STAT1 and STAT4, creating a distinct signaling signature

  • Analysis of phospho-STAT patterns after cytokine stimulation can distinguish which IL-12 family member is active

• Functional readouts:

  • IL-35 uniquely induces regulatory T and B cells (iTr35 and iBreg)

  • IL-12 promotes Th1 differentiation and IFN-γ production

  • IL-27 has mixed pro- and anti-inflammatory effects

  • Measuring these distinct functional outcomes helps differentiate cytokine activities

• Molecular sizing:

  • Use gel filtration chromatography to separate heterodimers based on molecular weight before immunodetection

  • This physically separates different IL-12 family cytokines prior to analysis

When publishing research on IL-35, researchers should explicitly detail the methods used to distinguish IL-35 from other IL-12 family members and include appropriate controls demonstrating specificity of detection or functional attribution.

What are the challenges in studying IL-35 function in human tissues compared to mouse models?

Studying IL-35 function in human tissues presents several methodological challenges distinct from mouse models, requiring researchers to adapt their experimental approaches:

• Expression pattern discrepancies:

  • Mouse Tregs constitutively express IL-35, while human Tregs require stimulation to express both subunits and secrete IL-35

  • Mouse tissues (blood, bone marrow, liver, thymus) have "ready to go" IL-35 expression, while human tissues lack constitutive expression of both subunits

  • These differences necessitate stimulation protocols when studying human samples

• Tissue accessibility limitations:

  • Human tissue samples are typically limited in quantity and availability

  • Mouse studies can utilize genetically modified models (knockout/knockin) for IL-35 or its subunits, which isn't possible in humans

  • Researchers must rely on ex vivo stimulation of human samples or humanized mouse models

• Heterogeneity considerations:

  • Human samples exhibit greater genetic and environmental heterogeneity than inbred mouse strains

  • This variability necessitates larger sample sizes and stratification strategies for human studies

  • Patient history, medication status, and comorbidities can influence IL-35 expression and function

• Technical detection challenges:

  • Lower expression levels in humans may require more sensitive detection methods

  • Confirming functional IL-35 heterodimer formation (rather than individual subunits) is essential

  • Cross-reactivity with other IL-12 family cytokines requires rigorous specificity controls

• Validation approaches for human studies:

  • Utilize primary human cells for in vitro studies rather than cell lines when possible

  • Employ patient-derived xenografts or humanized mouse models

  • Complement with immunohistochemistry of human tissue samples from relevant disease states

  • Use recombinant human IL-35 in functional assays rather than extrapolating from mouse IL-35

Researchers should explicitly acknowledge these species differences when designing experiments and interpreting results. Combining approaches that bridge mouse and human systems (such as parallel experiments in both species or using humanized mouse models) can help address these challenges.

How does IL-35 contribute to tumor immune evasion and what experimental approaches can assess this?

IL-35 plays multifaceted roles in tumor immune evasion through several mechanisms that can be experimentally evaluated:

• T cell suppression mechanisms:

  • IL-35 induces exhaustion in CD4+ and CD8+ T cells within tumors, reducing their effector functionality

  • Downregulation of the costimulatory molecule CD28 on immature CD8+ T cells prevents their differentiation into anti-tumor CTLs

  • Disruption of Th1 cell activation suppresses differentiated anti-tumor CTL activity

  Experimental approaches:

  • Flow cytometric analysis of exhaustion markers (PD-1, Tim-3, CTLA-4) on tumor-infiltrating lymphocytes with/without IL-35 neutralization

  • Mixed lymphocyte-tumor cell reactions with IL-35 blockade to measure T cell cytotoxicity recovery

  • In vivo tumor models comparing growth rates and lymphocyte infiltration with IL-35 pathway disruption

• Dendritic cell modulation:

  • IL-35 can convert immunogenic DCs into tolerogenic DCs (TolDCs)

  • IL-35+ DCs enrich P35 and EBI3 expression and reduce T cell infiltration in tumors

  • IL-35 induces arginase-1 (Arg1) expression in DCs, functioning as an immune checkpoint mechanism

  Experimental approaches:

  • Phenotypic characterization of tumor-associated DCs with/without IL-35 blockade

  • T cell stimulation assays using DCs isolated from IL-35-rich vs. IL-35-depleted tumor environments

  • Metabolic profiling of arginase activity in tumor-associated DCs following IL-35 manipulation

• Angiogenesis promotion:

  • IL-35 induces expression of CD31 and vascular endothelial growth factor (VEGF), promoting angiogenesis

  • IL-35 facilitates tumor cell endothelial adhesion and transendothelial migration via ICAM1

  • IL-35 increases monocyte recruitment through the IL-35-CXCL5 axis, promoting angiogenesis

  Experimental approaches:

  • Microvessel density quantification in tumors with IL-35 modulation

  • Endothelial tube formation assays using conditioned media from IL-35-treated tumor cells

  • In vivo imaging of tumor vasculature following anti-IL-35 therapy

  • Chemokine/cytokine profiling of tumor microenvironment with/without IL-35 blockade

• Regulatory cell induction:

  • IL-35 promotes conversion of conventional T cells into iTr35 regulatory cells

  • These induced regulatory cells further suppress anti-tumor immunity

  Experimental approaches:

  • Lineage tracing experiments to track conversion of conventional T cells to regulatory phenotypes

  • Adoptive transfer studies with IL-35-deficient vs. IL-35-competent immune cells

  • Single-cell RNA sequencing to identify regulatory cell induction in tumor microenvironment

Comprehensive evaluation of IL-35 in tumor immunity should combine these approaches in both in vitro models and in vivo systems, ideally including patient-derived samples to validate translational relevance.

What methodological approaches are recommended for studying IL-35 induction in non-T cells?

Studying IL-35 induction in non-T cells requires specialized methodological approaches to capture the dual-subunit regulation and context-dependent expression:

• Cell type-specific stimulation protocols:

  • For endothelial cells: TNF-α, IFN-γ, and IL-1β have been demonstrated to induce IL-35 subunit expression

  • For smooth muscle cells: Proinflammatory cytokine cocktails including TNF-α

  • For epithelial cells: TNF-α induces both subunits, while IFN-γ only upregulates IL-12A

  • For dendritic cells: LPS stimulation markedly upregulates IL-35

  Methodological considerations:

  • Include time-course experiments (6, 12, 24, 48 hours) to capture optimal induction kinetics

  • Test both individual cytokines and combinations to identify synergistic effects

  • Include positive controls (e.g., known IL-35-producing cells) in parallel experiments

• Subunit-specific detection strategies:

  • Quantitative RT-PCR for both IL-12A and EBI3 transcripts simultaneously

  • Immunocytochemistry with dual staining for both subunits

  • Proximity ligation assays to confirm heterodimer formation within cells

  • ELISA of culture supernatants to detect secreted IL-35

  Critical considerations:

  • Always measure both subunits, as the "double-gated" control requires both for functional IL-35

  • Include appropriate negative controls (unstimulated cells) and positive controls

  • Verify protein-level expression and secretion, not just mRNA upregulation

• Functional validation approaches:

  • Collect conditioned media from stimulated non-T cells and test for T cell suppressive activity

  • Use siRNA knockdown of either subunit to confirm specificity of observed effects

  • Include recombinant IL-35 as a positive control for functional assays

  • Employ neutralizing antibodies against IL-35 or its receptor components to confirm specificity

• Epigenetic regulation analysis:

  • Assess promoter methylation status of IL-35 subunit genes before and after stimulation

  • Perform chromatin immunoprecipitation (ChIP) assays to evaluate NF-κB binding to IL-35 subunit promoters

  • Investigate histone modifications at IL-35 loci following stimulation

• Post-transcriptional regulation studies:

  • Analyze mRNA stability through actinomycin D chase experiments

  • Investigate the role of AU-rich elements in 3'UTRs using reporter constructs

  • Assess potential microRNA regulation through target prediction and validation experiments

When reporting results, researchers should clearly distinguish between expression of individual subunits versus confirmed heterodimer formation and secretion, as this distinction is critical for functional IL-35 activity.

How can researchers manipulate IL-35 expression or signaling in experimental settings?

Researchers have several strategic options for manipulating IL-35 expression or signaling in experimental settings, each with specific advantages for different research questions:

• Genetic manipulation approaches:

  • CRISPR-Cas9 knockout of EBI3 or IL-12A in cell lines or primary cells

  • siRNA or shRNA knockdown for transient reduction of subunit expression

  • Overexpression systems using viral vectors containing IL-12A and EBI3 coding sequences

  • Inducible expression systems (e.g., Tet-On) for temporal control of IL-35 expression

  Implementation considerations:

  • Target either or both subunits depending on experimental goals

  • For complete IL-35 functional disruption, EBI3 knockout may be preferable as it less impacts other cytokines

  • Validate knockdown/overexpression at both mRNA and protein levels

  • Consider potential compensatory mechanisms in long-term knockout models

• Protein-level manipulation:

  • Recombinant IL-35 administration (Fc-chimera proteins are commercially available)

  • Neutralizing antibodies against IL-35 heterodimer or individual subunits

  • Soluble receptor components (IL-12Rβ2-Fc or gp130-Fc fusion proteins) as decoy receptors

  • Receptor-blocking antibodies targeting IL-12Rβ2 or gp130

  Methodological considerations:

  • Use concentration gradients to establish dose-response relationships

  • Include appropriate controls (isotype antibodies, irrelevant recombinant proteins)

  • Time-course experiments to determine optimal treatment durations

  • Validate functional neutralization through reporter assays

• Signaling pathway modulation:

  • JAK inhibitors to block downstream signaling

  • STAT1/STAT4 inhibitors to interfere with IL-35-specific transcriptional responses

  • Small molecule inhibitors targeting specific components of IL-35 signaling cascade

  Important considerations:

  • These approaches affect multiple pathways beyond IL-35 signaling

  • Use targeted inhibitors in combination with IL-35 stimulation to establish specificity

  • Include appropriate vehicle controls and concentration gradients

• Epigenetic manipulation strategies:

  • DNA methyltransferase inhibitors (e.g., 5-azacytidine) to modulate IL-35 subunit promoter methylation

  • Histone deacetylase inhibitors to alter chromatin accessibility at IL-35 loci

  Methodological notes:

  • These approaches have broad genomic effects beyond IL-35

  • Confirm direct effects on IL-35 expression through ChIP or bisulfite sequencing

  • Use targeted approaches (like CRISPR-dCas9 epigenetic editors) for greater specificity

• Animal model approaches:

  • EBI3 or IL-12A knockout mice

  • IL-35 receptor component knockout mice

  • Transgenic mice overexpressing IL-35 in specific tissues

  • Neutralizing antibody administration in vivo

  Critical considerations:

  • Remember species differences in IL-35 biology when designing studies

  • Include appropriate littermate controls

  • Consider potential developmental compensation in germline knockout models

  • Use conditional knockout systems for tissue-specific or temporal control

For all manipulation approaches, researchers should include comprehensive validation steps and appropriate controls to confirm specificity and efficacy of the intervention.

What are the current contradictions in IL-35 research data and how might they be resolved?

Several important contradictions and knowledge gaps exist in IL-35 research that require methodological resolution:

To advance the field, researchers should design experiments specifically aimed at resolving these contradictions, use multiple complementary methodologies, and explicitly address limitations of previous studies in their experimental design and reporting.

How might IL-35 and its signaling pathway be targeted in experimental therapeutic approaches?

IL-35 represents a promising therapeutic target for various conditions, including cancer immunotherapy and autoimmune diseases. Several experimental approaches for targeting IL-35 include:

• Cancer immunotherapy strategies:

  • Neutralizing IL-35 monoclonal antibodies to enhance anti-tumor immunity

  • Dual-specific antibodies targeting IL-35 and immune checkpoint molecules

  • Small molecule inhibitors of IL-35 signaling components

  • Genetic modification of tumor-infiltrating lymphocytes to resist IL-35 suppression

  Experimental design considerations:

  • Test in combination with established immunotherapies like PD-1/PD-L1 blockade

  • Evaluate effects on tumor microenvironment, particularly on CD8+ T cell exhaustion

  • Monitor potential conversion of effector T cells to regulatory phenotypes

  • Assess changes in arginase-1 expression as a downstream mediator

  • Evaluate impacts on tumor angiogenesis through VEGF and CD31 expression

• Autoimmune disease therapeutic applications:

  • Recombinant IL-35 administration to suppress pathogenic inflammation

  • Gene therapy approaches to induce IL-35 expression in affected tissues

  • Cell-based therapies using IL-35-expressing regulatory cells

  • Small molecule enhancers of endogenous IL-35 production

  Methodological approaches:

  • Establish dose-response relationships for recombinant IL-35

  • Determine optimal delivery routes and timing for therapeutic efficacy

  • Develop biomarkers for monitoring IL-35 pathway activation

  • Compare efficacy to established anti-inflammatory therapies

• Targeted delivery systems:

  • Nanoparticle-encapsulated IL-35 or IL-35 inhibitors for tissue-specific delivery

  • Engineered cell-based delivery systems (e.g., mesenchymal stem cells overexpressing IL-35)

  • Viral vector-mediated local expression of IL-35 or its inhibitors

  • Tissue-specific promoter-driven expression systems

  Experimental design elements:

  • Validate tissue-specific targeting using reporter systems

  • Assess biodistribution and pharmacokinetics

  • Monitor off-target effects in distant tissues

  • Compare systemic versus local delivery approaches

• Biomarker development for personalized approaches:

  • Serum IL-35 levels as predictive biomarkers for immunotherapy response

  • Genetic polymorphisms in IL-35 pathway components as stratification markers

  • Tissue expression patterns of IL-35 receptors to predict responsiveness

  • IL-35-induced gene signatures as companion diagnostics

  Methodological considerations:

  • Standardize detection methods for consistent biomarker measurement

  • Validate in diverse patient cohorts

  • Correlate with clinical outcomes in retrospective and prospective studies

  • Perform receiver operating characteristic analyses to establish clinical utility

• Combinatorial therapeutic approaches:

  • IL-35 pathway modulation plus conventional therapies

  • Sequential treatment protocols (timing IL-35 intervention for optimal efficacy)

  • Multi-target approaches addressing IL-35 alongside other immunoregulatory pathways

  Experimental design elements:

  • Use factorial experimental designs to identify synergistic combinations

  • Establish optimal sequencing through time-course experiments

  • Monitor immune parameters and functional outcomes simultaneously

  • Assess potential antagonistic combinations to avoid in clinical translation

When developing IL-35-targeted experimental therapeutics, researchers should incorporate comprehensive safety assessments, consider species differences in IL-35 biology , and include appropriate control groups to establish specificity and efficacy of the intervention.