Recombinant Human Interferon lambda-3 (IFNL3) (Active)

What is Interferon lambda-3 (IFNL3) and how does it differ from other type III interferons?

Interferon lambda-3 (IFNL3), formerly known as IL-28B, is a member of the type III interferon family which also includes IFNL1 (IL-29) and IFNL2 (IL-28A). These cytokines are class II cytokine receptor ligands that are distantly related to members of the IL-10 family and type I IFN family . IFNL3 demonstrates significant potency differences compared to other type III interferons:

• IFNL3 possesses the highest specific activity among human IFNL subtypes

• It exhibits approximately 2-fold higher activity than IFNL1

• It shows approximately 16-fold higher activity than IFNL2

• Human IFNL3 shares 94% amino acid identity with IFNL2 and 69% with IFNL1

The mature human IFNL3 protein consists of 175 amino acids (lacking N-glycosylation sites) and has a molecular weight of approximately 19.6 kDa .

What cellular receptors recognize IFNL3 and how is signaling initiated?

IFNL3 signals through a distinct heterodimeric receptor complex that differs from type I interferon receptors:

• The receptor is composed of IL-10 receptor β (IL-10Rβ) and the unique IFNL receptor α (IL-28Rα, also known as IFNL-R1)

• Unlike type I interferon receptors which are expressed on virtually all cells, the type III interferon receptor shows limited expression

• Receptor engagement leads to Jak-STAT signaling pathway activation

• The signaling results in phosphorylation of STATs and formation of the IFN-stimulated regulatory factor 3 (ISGF-3) transcription factor complex

• This pathway ultimately induces interferon-stimulated genes (ISGs) that mediate antiviral activity

Recent research indicates that human immune cells express both membrane-bound IFNL-R1 (mIFNL-R1) and soluble IFNL-R1 (sIFNL-R1) variants, with the soluble form potentially inhibiting ISG induction by IFNL3 .

How should recombinant human IFNL3 be properly reconstituted and stored for optimal activity?

Proper handling of recombinant IFNL3 is critical for maintaining its biological activity:

For carrier-containing formulations, the protein is typically supplied frozen in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) . The carrier protein enhances stability, increases shelf-life, and allows storage at more dilute concentrations compared to carrier-free versions .

What bioassays are suitable for determining IFNL3 biological activity?

Several bioassay systems have been validated for measuring IFNL3 activity:

• Cytopathic effect inhibition assay: The standard method uses human lung carcinoma A549 cells challenged with encephalomyocarditis virus (EMCV), with an EC50 of approximately 1 U/ml

• Antiviral protection assays: Using various viruses including HSV-2, reovirus, Sendai virus, or influenza virus

• Reporter assays: Using ISRE (Interferon-Stimulated Response Element)-driven luciferase reporters to measure pathway activation

• ISG induction measurement: Quantification of interferon-stimulated gene expression by RT-qPCR

When comparing activities between different IFNL proteins, it's important to note that IFNL3 has been shown to be more potent than other subtypes, which should be considered when designing dose-response experiments .

How does IFNL3 expression and activity differ among human immune cell populations?

Recent research has revealed differential IFNL3 responsiveness across human immune cell subsets, challenging previous mouse model findings:

This differential responsiveness can be explained by:

• Various expression levels of membrane-bound IFNL-R1

• Different ratios of soluble versus membrane-bound IFNL-R1

• T-cell receptor stimulation specifically upregulates membrane-bound IFNL-R1 expression in CD4+ T cells, enhancing antiviral gene induction

These findings indicate that IFNL3 directly interacts with human adaptive immune cells, unlike what has been previously observed in mouse models, suggesting potential applications for both mucosal and blood-borne viral infection interventions .

What are the genetic variants of IFNL3 and their association with disease outcomes?

Genetic variants near the IFNL3 gene have significant associations with disease outcomes:

• HCV infection: SNPs around IFNL3 strongly associate with both spontaneous clearance of HCV and response to interferon-based therapy

• COVID-19: Recent studies suggest that reduced IFNL1 and IFNL2, but not IFNL3, are associated with worse outcomes in COVID-19 patients

Several potentially causal SNPs have been identified:

The functional SNPs appear to influence transcription and post-transcriptional events that may lead to increased IFNL3 expression in individuals carrying the protective alleles .

How do expression systems affect the yield and biological activity of recombinant IFNL3?

Different expression systems produce recombinant IFNL3 with varying yields and activities:

When using E. coli expression systems, researchers have developed optimization strategies:

• IPTG concentration, temperature, and incubation time affect protein expression levels

• Inclusion body approach with 6His-tag facilitates purification

• Specific refolding methods have been developed for effective recovery of active protein

Studies comparing commercially available preparations with laboratory-produced IFNL3 have found that optimized production methods can yield superior activity .

What are the differences in IFNL3 activity between human and mouse models?

Species-specific differences in IFNL3 activity and receptor distribution have important implications for research:

• Mouse and human IFNL3 exhibit some species specificity, although much less than observed with type I interferons

• In humans, IFNL3 has broader effects on immune cells than previously reported in mouse models

• Human adaptive immune cells (B cells, CD8+ T cells) directly respond to IFNL3, while the response pattern differs in mice

• Mouse IFNL3 (produced from Asp20-Val193, with an N-terminal Met) is commercially available for comparative studies

These species differences are crucial to consider when:

• Designing animal studies

• Extrapolating results from mouse to human systems

• Developing therapeutic applications based on IFNL3 biology

What methodological approaches can be used to study IFNL3-induced transcriptional responses?

Several techniques are available for investigating IFNL3-induced gene expression:

Recent transcriptome analyses have identified distinct patterns of gene expression:

• In M1 macrophages, 1123 genes were significantly affected by IFNL3 treatment

• In M2 macrophages, over 2300 genes were significantly affected by IFNL3

• IFNL3 and IFNL4 can induce identical responses in some cell lines, depending exclusively on canonical signaling

When designing experiments to study IFNL3-induced transcriptional responses, researchers should consider cell type-specific effects and the temporal dynamics of the response.

How do storage conditions and freeze-thaw cycles affect IFNL3 stability and activity?

Maintaining IFNL3 activity requires careful attention to storage conditions:

• Storage at -70°C or below is recommended for retention of full activity

• Repeated freeze-thaw cycles significantly reduce biological activity

• For long-term storage, aliquoting the reconstituted protein is advisable

• Carrier proteins (like BSA) enhance stability during storage and freeze-thaw cycles

When conducting experiments with IFNL3, researchers should:

• Thaw aliquots quickly at 37°C and keep on ice once thawed

• Use manual defrost freezers to avoid temperature fluctuations

• Consider fresh reconstitution for critical experiments requiring maximum activity

What are the optimal concentrations of IFNL3 for different experimental applications?

Effective IFNL3 concentrations vary by application and cell type:

Important considerations for concentration determination:

• Epithelial cells typically require lower concentrations than immune cells

• IFNL3 is approximately 2-fold more potent than IFNL1 and 16-fold more potent than IFNL2

• Response thresholds may vary based on receptor expression levels

• Dose-response curves should be established for each experimental system

How can researchers distinguish between IFNL3-specific effects and those mediated by other interferons?

Distinguishing IFNL3-specific effects requires careful experimental design:

• Receptor knockout/knockdown approaches:

  • IFNL-R1 knockout cells respond to type I but not type III interferons

  • IL-10R2 knockout affects both IL-10 family and type III interferon signaling

• Neutralizing antibodies:

  • Anti-IFNL-R1 antibodies specifically block type III interferon signaling

  • Anti-IFNL3 antibodies can block specific IFNL3 effects

• Comparative studies:

  • Side-by-side comparison with type I interferons and other IFNL family members

  • Analysis of cell types with differential receptor expression patterns

• Genetic approaches:

  • CRISPR/Cas9 modification of receptor components

  • Expression of dominant-negative receptor variants

Experimental validation of IFNL3 specificity is particularly important when studying complex biological systems where multiple interferon pathways may be active simultaneously.

What are emerging applications for IFNL3 in therapeutic development?

Several promising therapeutic applications for IFNL3 are under investigation:

• Viral infections:

  • Unlike type I interferons, IFNL3's receptor has limited distribution, potentially reducing systemic side effects

  • May be particularly valuable for infections at mucosal surfaces where IFNL receptor expression is enriched

• Immune modulation:

  • Evidence suggests IFNL3 can influence T-cell responses and antibody production

  • May be useful for enhancing vaccine responses or modulating autoimmunity

• Cancer immunotherapy:

  • Potential to enhance anti-tumor immune responses

  • May synergize with existing immunotherapeutic approaches

Recent COVID-19 research suggests type III interferons may play protective roles in early infection stages, highlighting the need for further investigation of therapeutic applications during viral pandemics .

What technical challenges remain in measuring IFNL3 production and activity in biological samples?

Despite advances in IFNL3 research, several technical challenges persist:

• Detection sensitivity:

  • Low physiological concentrations in many biological samples

  • Need for highly sensitive ELISAs or bioassays

• Specificity issues:

  • Cross-reactivity between different IFNL family members in some assays

  • Some commercial antibodies may not distinguish between IFNL2 and IFNL3

• Standardization:

  • Lack of universally accepted international standards for activity measurements

  • Variability between different commercial preparations

• Receptor variant detection:

  • Difficulty distinguishing between membrane-bound and soluble receptor forms

  • Limited availability of specific antibodies for receptor isoforms