Recombinant Mouse Interleukin-21 protein (Il21) (Active)

What are the fundamental biological activities of recombinant mouse IL-21?

Recombinant mouse IL-21 exhibits multiple immunomodulatory functions that differ in some aspects from human IL-21. It enhances proliferation and activation of CD8+ T cells, enhances natural killer (NK) cell activity, and costimulates anti-CD40-driven B-cell proliferation in mice. Unlike human NK cells that can be sustained by IL-21 alone, murine NK cells cannot be sustained by IL-21 without other cytokines present. Recombinant mouse IL-21 also plays critical roles in regulating both adaptive and immune responses with documented antiviral and antitumor activities across multiple experimental systems .

How does mouse IL-21 signal through its receptor system?

Mouse IL-21 signals through a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common gamma chain (γc) receptor. Upon binding, IL-21 activates the JAK-STAT signaling pathway, primarily STAT3, but also STAT1 and STAT5. This activation leads to transcription of target genes that regulate immune cell proliferation, differentiation, and effector functions. The signaling cascade is crucial for IL-21's role in T cell, B cell, and NK cell responses. Experimental data shows that engineered variants like 21h10 generate significantly prolonged STAT signaling in vivo compared to native IL-21, contributing to their enhanced antitumor activity .

What are the recommended storage and stability considerations for recombinant mouse IL-21?

For optimal stability of recombinant mouse IL-21:

• Store lyophilized protein at -20°C to -80°C

• After reconstitution, prepare aliquots to avoid repeated freeze-thaw cycles

• Short-term storage (1-2 weeks) at 4°C is possible after reconstitution in sterile buffer containing carrier protein (0.1% BSA or HSA)

• Working solutions should be prepared fresh before experiments

• Stability studies show that engineered IL-21 variants like 21h10 have augmented stability compared to native IL-21, which is an important consideration when designing experiments requiring prolonged cytokine activity

What dosing parameters should be considered when using recombinant mouse IL-21 in tumor models?

When designing experiments using recombinant mouse IL-21 in tumor models, researchers should consider:

• Dose-response relationship: Typically, doses between 5-50 μg/kg have shown efficacy in murine models, with 30 μg/kg being commonly used based on clinical translation studies

• Administration schedule: Five-day treatment cycles followed by rest periods have demonstrated efficacy in clinical settings and should be considered for mouse models

• Route of administration: Intravenous, intraperitoneal, subcutaneous, and intratumoral routes show different biodistribution profiles

• Potential toxicities: Be aware of flu-like symptoms, cytopenias, hypophosphatemia, and increased hepatic enzymes as possible adverse effects

• Combination approaches: Consider TNFα blockade to mitigate systemic toxicity without compromising antitumor efficacy

For engineered variants such as 21h10, significantly lower doses may be effective due to enhanced potency and stability compared to native IL-21 .

How should researchers design experiments to assess IL-21's effects on different immune cell populations in vitro?

To assess IL-21's effects on different immune cell populations in vitro:

Include time-course analysis (24h, 48h, 72h, 5d) and dose-response studies to fully characterize IL-21's effects. Flow cytometry should be used to analyze surface receptor expression, intracellular cytokine production, and proliferation markers .

What considerations are important when designing in vivo experiments with recombinant mouse IL-21 in autoimmune disease models?

When designing in vivo experiments using recombinant mouse IL-21 in autoimmune disease models:

• Model selection: For autoimmune uveitis studies, the B10.RIII mice with IRBP 161-180 peptide emulsified with Complete Freund's Adjuvant provides a reliable EAU model

• Control groups: Include recovery phase mice (5-6 weeks post-immunization) and normal controls (CFA only) for comparison

• Tissue analysis: Examine both draining lymph nodes (DLN) and spleen for IL-21 and IL-21R expression

• Assessment methods:

  • RT-PCR for mRNA expression of IL-21 and IL-21R

  • Flow cytometry for cellular expression of IL-21R on CD4+ and CD8+ T cells

  • ELISA for IL-17, IL-21, and other cytokine production

• Experimental timeline: Monitor disease progression through clinical observation and histopathologic evaluation

• Mechanistic studies: Consider IL-21 blockade or genetic ablation studies to confirm causal relationships

These considerations enable comprehensive assessment of IL-21's role in autoimmune pathogenesis and potential intervention strategies .

How does mouse IL-21 influence the tumor microenvironment and what markers should be evaluated?

Mouse IL-21 exerts complex effects on the tumor microenvironment (TME) that researchers should assess through multiple parameters:

• T cell populations:

  • IL-21 induces highly cytotoxic antitumor T cells from clonotypes with varying affinities for endogenous tumor antigens

  • 21h10 (IL-21 mimic) robustly expands low-affinity cytotoxic T cells and drives high expression of IFN-γ and granzyme B

  • Increases the frequency of IFN-γ+ Th1 cells while reducing Foxp3+ Tregs

• Critical TME markers to evaluate:

  • PD-1 and Tim-3 expression on CD8+ T cells (IL-21 selectively expands PD-1intTim-3- CD8+ functional T cells)

  • Effector molecule expression (granzyme B, perforin, IFN-γ)

  • Memory phenotype markers (CD44, CD62L, KLRG1, CD127)

  • Regulatory T cell infiltration (Foxp3+ cells)

• Spatial distribution analysis:

  • Use multiplex immunohistochemistry to assess cellular proximity and interactions

  • Evaluate tumor-infiltrating lymphocyte density in tumor core versus periphery

These analyses provide comprehensive understanding of how IL-21 reshapes the immunological landscape within tumors, particularly important when evaluating engineered IL-21 variants with enhanced potency .

What are the key differences between native mouse IL-21 and engineered IL-21 variants in cancer models?

Engineered IL-21 variants demonstrate several important differences compared to native mouse IL-21 in cancer models:

Researchers should consider these differences when selecting the appropriate IL-21 variant for their specific research question and tumor model. The engineered variants offer superior antitumor activity but may require additional considerations for toxicity management .

How should researchers optimize combination therapy approaches with mouse IL-21?

Optimizing combination therapy approaches with mouse IL-21 requires systematic evaluation of several parameters:

• Checkpoint inhibitor combinations:

  • IL-21 with anti-PD-1/PD-L1: Has demonstrated dramatic tumor volume reduction and enhanced survival

  • IL-21 with anti-CTLA-4: Induced complete tumor clearance in some mouse models

  • The Erb-IL-21 fusion protein combined with anti-PD-L1 significantly reduced tumor volume while combination with anti-CTLA-4 induced tumor clearance in all mice tested

• Timing and sequencing:

  • IL-21 administration prior to checkpoint blockade may prime T cells for enhanced responses

  • Concurrent administration may provide synergistic effects

  • Sequential administration should be tested systematically

• Dose optimization:

  • Reduced IL-21 doses may be effective in combination settings

  • Test multiple dose ratios to identify optimal therapeutic window

  • Monitor for potential synergistic toxicities

• Mechanistic assessments:

  • Evaluate changes in tumor-infiltrating lymphocyte composition

  • Measure PD-1 expression dynamics on tumor-reactive T cells

  • Assess memory T cell formation for long-term responses

• Alternative combinations:

  • IL-21 with PI3Kδ inhibitors prevented virus uptake by macrophages and significantly inhibited tumor growth

  • IL-21-armed oncolytic viruses demonstrated superior efficacy in several cancer models

  • IL-21 with CAR-T cell therapy showed synergistic effects

These approaches should be systematically tested in the appropriate tumor models with comprehensive immune monitoring to identify optimal combination strategies .

How can researchers address variability in mouse IL-21 responsiveness across different experimental models?

Variability in mouse IL-21 responsiveness across different experimental models can be addressed through several methodological approaches:

• Standardize IL-21 source and quality:

  • Use recombinant protein from a consistent supplier

  • Validate biological activity of each lot using established bioassays

  • Consider preparing a large single batch for long-term studies

• Account for strain-specific differences:

  • B10.RIII mice show distinct IL-21 response patterns in autoimmune uveitis models

  • C57BL/6 background strains may exhibit different baseline IL-21R expression

  • Document strain-specific baseline cytokine profiles before intervention

• Control for environmental variables:

  • Standardize housing conditions (SPF vs. conventional)

  • Monitor for underlying infections that might affect cytokine responses

  • Consider gut microbiome influences on cytokine signaling

• Technical recommendations:

  • Include internal standards in each experiment

  • Use flow cytometry to quantify IL-21R expression on target cell populations

  • Perform dose-response studies to identify optimal concentration ranges

  • Include time-course analyses to capture dynamic responses

• Statistical approaches:

  • Use appropriate power calculations based on preliminary data variability

  • Consider mixed-effects models to account for inter-animal and inter-experimental variation

  • Report detailed methodological parameters to enhance reproducibility

By implementing these strategies, researchers can minimize variability and improve the reliability and interpretability of IL-21-based experiments .

What techniques should be used to accurately assess IL-21-dependent signaling in mouse models?

To accurately assess IL-21-dependent signaling in mouse models, researchers should employ a comprehensive toolkit of complementary techniques:

• Phospho-flow cytometry:

  • Directly measures phosphorylation of STAT1, STAT3, and STAT5 in specific cell populations

  • Enables single-cell resolution analysis of signaling dynamics

  • Can be combined with surface marker staining to identify responding subpopulations

  • Optimal time points: 15-30 minutes for peak phosphorylation, with additional points at 1-24 hours

• Western blotting:

  • Quantifies total signaling protein levels and phosphorylation states

  • Useful for tissues where flow cytometry is challenging

  • Include both early (5-30 min) and late (1-24h) time points to capture signaling dynamics

• Transcriptional profiling:

  • RT-PCR for key IL-21 target genes (including IL-21R itself, which shows upregulation in response to IL-21)

  • RNA-seq for comprehensive signaling pathway analysis

  • Consider single-cell RNA-seq to capture heterogeneity in responses

• Functional readouts:

  • Cytokine production (ELISA, intracellular cytokine staining)

  • Proliferation assays (CFSE dilution, Ki-67 staining)

  • Cytotoxicity assays for NK and CD8+ T cells

• In vivo reporter systems:

  • STAT3 reporter mice to visualize IL-21 signaling in tissue contexts

  • Consider utilizing conditional knockout models to validate signaling pathways

Engineered IL-21 variants like 21h10 generate significantly prolonged STAT signaling compared to native IL-21, requiring extended time-course analyses to fully characterize their signaling profile .

How should researchers reconcile contradictory results between mouse and human IL-21 studies?

When encountering contradictory results between mouse and human IL-21 studies, researchers should:

• Recognize established species differences:

  • IL-21 alone cannot sustain survival of murine NK cells but can sustain human NK cells

  • IL-21 effects on receptor expression differ between species (e.g., NKG2A, CD25, CD86, CD69 upregulation patterns)

  • Explicitly acknowledge these differences in experimental design and interpretation

• Methodological approaches to reconcile contradictions:

  • Perform parallel experiments with both mouse and human cells using identical protocols

  • Use humanized mouse models when studying human-specific effects

  • Consider cross-species reactive engineered variants (like 21h10) that work in both systems

  • Validate key findings from mouse models using human ex vivo systems like patient-derived organotypic tumor spheroids (PDOTS)

• Experimental design considerations:

  • Use concentration ranges appropriate for each species

  • Account for differences in receptor expression and affinity

  • Consider differences in downstream signaling pathway activation

• Reporting guidelines:

  • Clearly state the species origin of IL-21 used

  • Document differences in experimental conditions between human and mouse studies

  • Avoid overgeneralizing findings from one species to another without validation

• Translational implications:

  • Successful clinical trials have used 30 μg/kg rIL-21 with evidence of antitumor activity

  • Mouse model results showing pathway-specific effects may still be translatable even if magnitude or kinetics differ

  • Focus on consistent mechanistic findings across species rather than absolute values

By systematically addressing these considerations, researchers can better understand the translational relevance of their findings and avoid misinterpretation of contradictory results .

What considerations are important when translating mouse IL-21 findings to human clinical applications?

When translating mouse IL-21 findings to human clinical applications, researchers should consider:

• Species-specific biological differences:

  • Mouse IL-21 cannot sustain murine NK cell survival alone, while human IL-21 can sustain human NK cell survival

  • Receptor expression patterns and signaling kinetics may differ between species

  • Dosing and administration requirements may not directly scale between mouse and human systems

• Pharmacological considerations:

  • Human equivalent doses should be calculated using appropriate allometric scaling

  • Phase I clinical trials have established 30 μg/kg as a well-tolerated dose in humans with melanoma and renal cell carcinoma

  • Side effect profiles observed in mice (flu-like symptoms, cytopenias, etc.) should be monitored in human applications

• Biomarker selection for clinical monitoring:

  • Track STAT3 phosphorylation in peripheral blood lymphocytes

  • Monitor NK cell and CD8+ T cell activation markers

  • Assess target engagement through IL-21R occupancy assays

  • Consider tumor biopsies to confirm immune infiltration patterns

• Combination therapy approaches:

  • Checkpoint inhibitor combinations showing synergy in mice may require validation in human ex vivo systems

  • Timing and sequencing of combinations may differ between species

• Patient selection strategies:

  • Consider tumor types with demonstrated IL-21 responsiveness in mouse models

  • Evaluate baseline IL-21R expression in patient tumors

  • Assess immune infiltration status as a potential predictor of response

Clinical trials with recombinant IL-21 have demonstrated safety, tolerability, and early evidence of efficacy in melanoma and renal cell carcinoma patients, validating key findings from mouse models despite species differences .

How do engineered IL-21 variants compare in terms of biological activity and potential applications?

Engineered IL-21 variants demonstrate diverse properties and applications:

Each variant offers specific advantages depending on the target disease, route of administration, and desired immunological effect. The 21h10 IL-21 mimic shows particular promise with its human/mouse cross-reactivity, high stability and potency, and ability to potentiate low-affinity antitumor responses .

What methodological approaches should be used to study IL-21's effects on low-affinity vs. high-affinity T cell responses?

To effectively study IL-21's differential effects on low-affinity versus high-affinity T cell responses, researchers should implement the following methodological approaches:

• Experimental systems:

  • TCR transgenic models with known affinity variants (e.g., OT-I/OT-III for SIINFEKL with varying affinities)

  • Peptide variants with altered MHC binding or TCR contact residues

  • Single-cell sequencing of tumor-infiltrating T cells to correlate TCR sequence with functional output

• Affinity measurement techniques:

  • Surface plasmon resonance (SPR) to quantify TCR-pMHC binding kinetics

  • Tetramer decay assays to measure off-rates of TCR-pMHC interactions

  • Functional avidity assays using peptide titrations for activation thresholds

• Analytical approaches:

  • Flow cytometry with tetramer staining at different concentrations

  • Intracellular cytokine production in response to varying antigen concentrations

  • Proliferation indices at different stimulation strengths

• Critical parameters to assess:

  • IFN-γ and granzyme B expression levels correlate with IL-21's enhancement of low-affinity responses

  • Expansion rates under limiting antigen conditions

  • Cytotoxic potential against targets expressing varying antigen densities

  • Memory formation and recall response quality

• In vivo validation:

  • Adoptive transfer of mixed high/low affinity T cell populations

  • Competition assays tracking relative expansion of clones with different affinities

  • Tumor models with heterogeneous antigen expression

The engineered IL-21 mimic 21h10 has demonstrated superior capacity to induce highly cytotoxic antitumor T cells from clonotypes with a range of affinities for endogenous tumor antigens, robustly expanding low-affinity cytotoxic T cells while driving high expression of IFN-γ and granzyme B compared to native IL-21. These properties make it particularly valuable for studying and potentially exploiting low-affinity T cell responses in the context of tumor immunology .