IL 23 Human, His

What is IL-23 and what is its molecular structure?

The cytokine signals through the IL-23 receptor complex, which consists of the IL-23R subunit paired with IL-12Rβ1. This receptor complex primarily activates the STAT3 signaling pathway, though it can also trigger other downstream effectors depending on the cellular context .

What is the significance of His-tagged IL-23 in research applications?

His-tagged IL-23 is engineered with a polyhistidine tag, typically at the N-terminus of the IL-23 alpha subunit, while the IL-12 beta subunit may remain untagged . This configuration enables:

• Simplified purification: The His-tag allows for single-step affinity purification using immobilized metal affinity chromatography (IMAC)

• Orientation-specific coupling: For binding studies or functional assays where directional coupling is important

• Detection flexibility: The tag provides an epitope for antibody-based detection independent of the protein's native structure

• Experimental versatility: The His-tag can be used for pull-down assays to identify novel binding partners

In research settings, His-tagged IL-23 demonstrates binding affinity to IL-23R with equilibrium dissociation constants (Kd) of approximately 5.36-9.25 nM, as measured by surface plasmon resonance (SPR) and bio-layer interferometry (BLI) assays .

How can researchers verify the biological activity of recombinant IL-23?

Functional verification of recombinant IL-23 can be assessed through multiple complementary approaches:

• Receptor binding assays: Using immobilized IL-23R (typically as Fc fusion protein), with a linear detection range of 20-78 ng/mL for properly folded His-tagged IL-23

• STAT3 phosphorylation: Stimulation of cells expressing IL-23R should induce STAT3 phosphorylation, which can be detected by immunoblotting or flow cytometry

• Cytokine induction assays: Active IL-23 induces IFN-γ in Vδ2+ γδ T cells and MAIT cells, and uniquely induces IL-17A in MAIT cells but not other lymphocyte subsets

• Reconstitution experiments: Restoring IL-23 signaling in IL-23R-deficient cells should rescue specific functional defects characterized in patient cells

What are the distinct immunological functions of IL-23 in mycobacterial immunity?

IL-23 plays a crucial and non-redundant role in immunity against mycobacteria that extends beyond its classical association with Th17 responses. Recent research has demonstrated that:

• IL-23 is essential for both baseline and Mycobacterium-induced IFN-γ production by specific innate-like lymphocyte populations, particularly Vδ2+ γδ T cells and MAIT (Mucosal-Associated Invariant T) cells

• IL-23R deficiency leads to Mendelian Susceptibility to Mycobacterial Disease (MSMD) with high penetrance, revealing the critical importance of this pathway in antimycobacterial immunity

• Contrary to earlier paradigms that positioned IL-23 primarily as an IL-17 inducer, human IL-23 induces IL-17A only in MAIT cells, while it induces IFN-γ in multiple lymphocyte subsets

• The identification of patients with loss-of-function variants in IL23R who developed mycobacterial diseases demonstrates that human IL-23 is required for optimal IFN-γ-dependent immunity to mycobacteria

This represents a paradigm shift from the traditional Th1/Th17 dichotomy, where IL-12 was considered the signature inducer of IFN-γ and IL-23 the signature inducer of IL-17A/F .

How should researchers design experiments to study IL-23 signaling in different immune cell populations?

When designing experiments to investigate IL-23 signaling across immune cell subsets, researchers should consider:

Cell isolation strategies:

• Use magnetic bead separation or flow cytometry-based sorting to isolate specific lymphocyte subsets (Vδ2+ γδ T cells, MAIT cells, NK cells)

• Consider both peripheral blood mononuclear cells (PBMCs) and tissue-resident lymphocytes, as responses may differ

Stimulation protocols:

• Baseline assessment: Measure cytokine production without stimulation

• IL-23 stimulation: Titrate recombinant IL-23 (typically 10-100 ng/mL)

• Mycobacterial challenge: Use heat-killed or live attenuated mycobacteria (e.g., BCG)

• Combined stimulation: Assess synergy between IL-23 and other cytokines/PAMPs

Readouts:

• Intracellular cytokine staining for IFN-γ and IL-17A/F

• Phospho-flow cytometry for STAT3 and other signaling molecules

• Transcriptional profiling of stimulated vs. unstimulated cells

• Functional assays including mycobacterial growth inhibition

Controls:

• IL-23R-deficient cells as negative controls

• Parallel stimulation with IFN-α2b (which activates STAT3 independently of IL-23R)

• Cell type-matched controls from healthy donors

What are the immunological consequences of IL-23R deficiency in humans?

IL-23R deficiency manifests with specific immunological signatures that highlight the unique roles of IL-23 in human immunity:

Clinical presentations:

• All identified patients with homozygous loss-of-function IL-23R variants developed MSMD (Mendelian Susceptibility to Mycobacterial Disease)

• Only a subset (two of six reported patients) developed CMC (Chronic Mucocutaneous Candidiasis)

Cellular immunological features:

• Marked defect in IFN-γ production by:

  • Natural killer cells

  • Vδ2+ γδ T cells

  • MAIT cells

• Selective defect in IL-17A production only in MAIT cells, but not other cell types

• Normal responses to IFN-α2b and other cytokines, demonstrating specificity of the defect

Genetic insights:

• Complete penetrance for BCG disease in AR IL-23R deficiency

• Significant enrichment of homozygous predicted loss-of-function variants in MSMD patients compared to controls (p=5.7×10⁻⁴) and the general population (p=3.5×10⁻⁹)

These findings establish that IL-23 signaling is particularly critical for IFN-γ-dependent immunity to mycobacteria, while its role in IL-17-dependent immunity to Candida is more restricted and may be partly redundant with other pathways.

How can genetic and functional studies of IL-23R be designed to understand disease mechanisms?

To comprehensively investigate IL-23R function and disease associations, researchers should implement:

Genetic approaches:

• Screening for IL23R variants in patients with MSMD, TB, or CMC

• Analysis of population genetics metrics such as the Consensus-based measure of Negative Selection (CoNeS) score and Gene Damage Index (GDI)

• Assessment of the impact of variants using in silico prediction tools combined with functional validation

Functional validation methods:

• Overexpression systems:

  • Transfection of wild-type or mutant IL-23R in appropriate cell lines

  • Assessment of surface expression by flow cytometry

  • Evaluation of downstream signaling (STAT3 phosphorylation)

• Patient-derived cell studies:

  • Ex vivo stimulation of various lymphocyte subsets

  • Comparison of baseline and stimulation-induced cytokine production

  • Assessment of specific cell population frequencies

• Reconstitution experiments:

  • Introduction of wild-type IL-23R into patient-derived cells

  • Rescue of functional defects as proof of causality

Experimental controls:

• Parallel testing of common polymorphisms to distinguish disease-causing variants

• Inclusion of known neutral variants as negative controls

• Use of other cytokine stimulations (e.g., IFN-α2b) to verify signaling pathway integrity

What methodological considerations are important when working with His-tagged IL-23 in functional assays?

When utilizing His-tagged IL-23 for functional studies, researchers should consider several methodological aspects:

Protein quality considerations:

• Verify heterodimer integrity via reducing and non-reducing SDS-PAGE

• Confirm glycosylation status, as this influences bioactivity

• Use multi-angle light scattering (MALS) to assess molecular weight and aggregation

Buffer and storage optimization:

• Lyophilized protein is typically reconstituted in PBS (pH 7.4) with trehalose as a protectant

• For long-term storage, aliquot and store at -80°C to avoid freeze-thaw cycles

• Include carrier proteins for dilute solutions to prevent adsorption loss

Functional assay considerations:

• The His-tag may influence protein orientation in binding assays

• Equilibration time impacts measured binding affinities in SPR/BLI assays (typically 5.36-9.25 nM for IL-23R binding)

• For cell-based assays, pre-warm protein solutions to 37°C before addition to cells

Potential limitations:

• The His-tag might interfere with certain interaction interfaces

• Tag-dependent artifacts should be controlled for by comparing with tag-free versions

• Recombinant proteins may not fully recapitulate all post-translational modifications of native IL-23

How does IL-23 research inform our understanding of human mycobacterial immunity?

Research on IL-23 has substantially revised our understanding of antimycobacterial immunity in humans:

• Paradigm shift in cytokine functionality: IL-23 was traditionally associated with IL-17 responses, but human studies have revealed its critical role in IFN-γ-dependent antimycobacterial immunity

• Cell type-specific effects: IL-23 induces different responses in various lymphocyte populations:

  • IFN-γ production in multiple innate-like lymphocytes (NK cells, Vδ2+ γδ T cells, MAIT cells)

  • IL-17A production only in MAIT cells

• Clinical translational insights: IL-23R deficiency causes:

  • High penetrance for mycobacterial diseases (MSMD)

  • Lower penetrance for Candida infections (CMC)

• Mechanistic understanding: Provides evidence that:

  • Innate-like T cells are critical sources of early IFN-γ during mycobacterial infection

  • IL-23 signaling is required for both baseline and infection-induced IFN-γ immunity

These findings challenge previous assumptions about strict cytokine-response linearity and highlight the context-dependent effects of IL-23 in human immunity.

What are promising research directions for IL-23 in infectious disease immunology?

Future research on IL-23 should explore:

• Tissue-specific IL-23 functions:

  • Compare IL-23 effects in blood vs. tissue-resident lymphocytes

  • Investigate IL-23 sources and concentrations across different tissue microenvironments

• Temporal dynamics of IL-23 responses:

  • Characterize the kinetics of IL-23 production during infection

  • Determine how IL-23 coordinates with other cytokines in sequential immune responses

• Therapeutic applications:

  • Explore IL-23 supplementation as adjunctive therapy for mycobacterial infections

  • Investigate targeted IL-23 delivery to enhance local antimycobacterial responses

• Integration with other immunity pathways:

  • Examine interactions between IL-23 and other cytokine networks

  • Study compensatory mechanisms in IL-23R-deficient individuals

• Single-cell approaches:

  • Apply single-cell transcriptomics to identify IL-23-responsive cell populations

  • Characterize heterogeneity in IL-23 responsiveness within canonical lymphocyte subsets

This research would further refine our understanding of IL-23's multifaceted roles in human immunity and potentially inform novel therapeutic strategies for infectious diseases.