Recombinant Human Interleukin-21 protein (IL21) (Active)

What is the structural composition of recombinant human IL-21?

Recombinant human IL-21 is typically comprised of amino acids Gln32-Ser162 with an N-terminal Met. This composition corresponds to the mature form of human IL-21. The protein exhibits distinct bands under reducing and non-reducing conditions when resolved with SDS-PAGE - approximately 16.4 kDa under reducing conditions and 14.8 kDa under non-reducing conditions . The structural blueprint of IL-21 has been determined by X-ray crystallography, revealing its interaction with IL-21R and γc in a ternary signaling complex, which provides insights into its biological function .

What are the primary biological activities of IL-21?

IL-21 demonstrates diverse biological activities through specific interactions with immune cells:

• T cell modulation: Promotes proliferation of CD8+ T cells and enhances their cytolytic activity.

• Cytokine production: Stimulates interferon-gamma (IFN-γ) secretion by natural killer cells and CD8+ T cells.

• Inhibitory receptor regulation: Down-regulates expression of inhibitory receptors such as programmed death 1 (PD-1) and T-cell immunoglobulin domain and mucin domain 3 (TIM-3) on CD8+ T cells .

• Effector function enhancement: Increases production of granzyme B and CD107a in antigen-specific CD8+ T cells .

The specific activity of recombinant human IL-21 is typically >1.00 × 10^6 units/mg, calibrated against internal reference standards .

How does IL-21 signal through its receptor complex?

IL-21 signals through a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common gamma chain (γc):

• The structure determined by X-ray crystallography at 2.8 Å resolution reveals that IL-21 forms a ternary complex with IL-21R and γc.

• Cryo-electron microscopy at 3.7 Å resolution has shown that this complex can form dimers of trimeric complexes.

• The IL-21–γc interface is particularly important for signal modulation and can be targeted to create IL-21 analogs with altered activity profiles .

After receptor binding, IL-21 primarily activates the JAK-STAT pathway, leading to phosphorylation of STAT proteins, particularly STAT3, which regulates gene expression in target cells .

What are the optimal experimental conditions for using IL-21 in cell culture systems?

For effective use of IL-21 in experimental systems:

• Concentration range: The effective dose (ED50) typically ranges from 5.00-50.0 ng/mL for B9 mouse hybridoma cell proliferation assays, and ≤8 ng/mL for enhancing IFN-gamma secretion in NK-92 human natural killer lymphoma cells .

• Treatment duration: For in vitro studies examining CD8+ T cell responses, cultures are typically maintained for 7-10 days with periodic IL-21 supplementation.

• Combination with other cytokines: IL-21 shows synergistic effects when combined with IL-7 or IL-15, particularly for CD8+ T cell expansion and functional enhancement .

• Cell types: Different cell types require different IL-21 concentrations; B cells, T cells, and NK cells all respond to IL-21 but with varying sensitivity.

How can researchers measure IL-21 biological activity in experimental systems?

Several validated assays can be used to measure IL-21 activity:

• Proliferation assays: B9 mouse hybridoma cell proliferation is commonly used, with an ED50 of 5.00-50.0 ng/mL indicating active protein .

• Cytokine secretion assays: Measuring IFN-gamma secretion from NK-92 cells, with an ED50 of ≤8 ng/mL .

• Flow cytometry: Detecting upregulation of CD25 expression on NK cells and CD8+ T cells following IL-21 exposure .

• RT-PCR: Measuring increased mRNA expression of IFN-gamma, perforin, and granzyme B in CD8+ T and NK cells .

• ELISA: Quantifying IL-21 levels in biological samples such as gingival crevicular fluid (GCF), with significant differences observed between health and disease states .

What methods can be used to analyze IL-21 levels in clinical samples?

For clinical research applications:

• ELISA: Enzyme-linked immunosorbent assay is the gold standard for measuring IL-21 concentration in fluids such as serum, plasma, or gingival crevicular fluid.

• Correlation analysis: Statistical methods such as Pearson's correlation can be used to assess relationships between IL-21 levels and clinical parameters, as demonstrated in periodontal disease studies .

Table: Comparison of IL-21 levels in gingival crevicular fluid among different patient groups

*Values derived from mean differences reported in the study

How does IL-21 influence antiviral immune responses in chronic viral infections?

IL-21 plays a critical role in sustaining effective antiviral immunity, particularly in chronic infections:

• CD8+ T cell reinvigoration: IL-21 promotes the proliferative capacity of virus-specific CD8+ T cells that would otherwise become exhausted during chronic infection.

• Inhibitory receptor modulation: IL-21 significantly down-regulates expression of PD-1 and TIM-3 on virus-specific CD8+ T cells, which helps restore their functionality .

• Effector function enhancement: Treatment with IL-21 boosts production of IFN-γ, granzyme B, and CD107a in virus-specific CD8+ T cells, enhancing their cytolytic activity against infected cells .

• Viral suppression: In HBV models, IL-21 enhances the ability of CD8+ T cells to suppress viral replication through both cytolytic and non-cytolytic mechanisms. Supernatants from IL-21-treated CD8+ T cells markedly reduced HBV replicative intermediates, HBsAg, HBeAg, and HBV DNA in HepG2.2.15 cells .

What is the significance of IL-21 receptor knockout models in understanding IL-21 function?

IL-21 receptor knockout (IL-21R-KO) models provide valuable insights into IL-21 biology:

• Impaired viral clearance: IL-21R-KO mice with HBV expression show significantly higher levels of serum HBsAg compared to wild-type mice, with 5 of 11 IL-21R-KO mice remaining HBsAg-positive at 28 days post-injection .

• Defective antibody responses: IL-21R-KO mice demonstrate an inability to produce hepatitis B surface antibodies (HBsAb) .

• Reduced T cell responses: IL-21R-KO mice exhibit strikingly reduced IFN-γ production by HBV-specific CD8+ T cells compared to wild-type mice .

• Normal CD8+ T cell numbers: Despite having normal numbers of circulating CD8+ T cells, IL-21R-KO mice show significantly reduced primary cytotoxic T lymphocyte (CTL) responses to viral antigens, indicating IL-21's role in antigen-specific clonal expansion rather than general CD8+ T cell development .

How can structure-guided design be used to create IL-21 analogs with altered functionality?

Recent structural studies have enabled the development of IL-21 variants with modified activities:

• Structural blueprint: X-ray crystallography and cryo-EM structures of the IL-21–IL-21R–γc ternary signaling complex provide a template for rational protein engineering .

• Interface targeting: By introducing substitutions at the IL-21–γc interface, researchers have created IL-21 analogs that act as partial agonists .

• Differential signaling: These modified IL-21 proteins modulate downstream activation of signaling molecules including pS6, pSTAT3, and pSTAT1 to varying degrees .

• Cell-type specificity: IL-21 analogs exhibit differential activity on T and B cell subsets, allowing for more targeted manipulation of immune responses .

• Functional outcomes: In human tonsil organoid models, these analogs can modulate antibody production, offering potential strategies for tunable manipulation of humoral immunity .

What has been learned from clinical trials using recombinant human IL-21?

Clinical investigations of recombinant human IL-21 (rIL-21) have provided valuable insights:

• Metastatic melanoma treatment: A phase II study of rIL-21 in patients with metastatic melanoma demonstrated clinical responses, including one confirmed complete response and one confirmed partial response, both in patients with lung metastases .

• Dosing regimen: The clinical trial administered rIL-21 at 30 μg/kg/dose by intravenous bolus injection in 8-week cycles (5 dosing days followed by 9 days of rest for 6 weeks and then 2 weeks off treatment) .

• Safety profile: Treatment was generally well tolerated, suggesting a manageable safety profile for clinical applications .

• Biomarker changes: Treatment resulted in increases in serum soluble CD25, frequencies of CD25+ NK and CD8+ T cells, and mRNA for IFN-γ, perforin, and granzyme B in CD8+ T and NK cells, providing pharmacodynamic markers of biological activity .

How is IL-21 associated with inflammatory and autoimmune diseases?

IL-21 plays complex roles in inflammation and autoimmunity:

• Dual inflammatory roles: IL-21 has both anti- and pro-inflammatory functions associated with chronic inflammation. It can cause tissue destruction by increasing pro-inflammatory cytokines while also curbing the activity of certain immune cells that have anti-inflammatory roles .

• Periodontal disease: IL-21 levels are significantly elevated in the gingival crevicular fluid of patients with generalized chronic periodontitis and generalized aggressive periodontitis compared to healthy controls .

• Correlation with disease severity: In periodontal disease, IL-21 levels show strong positive correlations with clinical parameters such as periodontal probing depth (r=0.966 in GAP, r=0.924 in GCP) and clinical attachment level (r=0.933 in GAP, r=0.963 in GCP) .

• Autoimmune risk: The potent effects of IL-21 on immune activation make it a potential contributor to autoimmune pathology, which presents challenges for its clinical application despite its beneficial effects in viral infections and cancer .

What strategies are being developed to harness IL-21 biology for therapeutic applications?

Several approaches are being explored to leverage IL-21's biological activities for therapy:

• Structure-based engineering: Using the structural understanding of IL-21 signaling to design variants with more targeted activities .

• Partial agonists: Development of IL-21 analogs that selectively activate certain downstream pathways while minimizing others, potentially reducing off-target effects .

• Combination therapies: Exploring synergistic effects when combining IL-21 with other immunomodulatory agents, similar to the synergy observed with IL-7 or IL-15 in experimental settings .

• Cell-type targeting: Creating IL-21 variants that preferentially act on specific immune cell subsets to achieve more precise immunomodulation .

• Biomarker-guided therapy: Using IL-21-responsive biomarkers like soluble CD25 and phosphorylated STAT proteins to monitor and optimize therapeutic interventions .