Recombinant Mouse Interleukin-2 protein (Il2) (Active)

What is the molecular structure of recombinant mouse IL-2?

Recombinant mouse IL-2 is a 17.2 kDa O-glycosylated four alpha-helix bundle cytokine comprising amino acid residues Ala21-Gln169, sometimes with an additional N-terminal methionine depending on the expression system . The protein typically contains 149 amino acid residues in its mature form and shares 56% amino acid sequence identity with human IL-2 and 73% with rat IL-2 . When analyzed by SDS-PAGE under reducing conditions, recombinant mouse IL-2 appears as a band at approximately 19 kDa .

How should recombinant mouse IL-2 be reconstituted for optimal stability?

For carrier-containing formulations (with BSA), reconstitute the lyophilized protein at 100-200 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin . For carrier-free formulations, reconstitute at 100-200 μg/mL in sterile deionized water . Upon initial thawing, aliquot the reconstituted protein into polypropylene microtubes and store at -80°C to minimize freeze-thaw cycles . Alternatively, dilute in a sterile neutral buffer containing 0.5-10 mg/mL carrier protein (such as human or bovine serum albumin) before aliquoting and storing at -80°C . For long-term storage, the protein concentration should not be less than 10 μg/mL .

What is the biological activity of recombinant mouse IL-2 and how is it measured?

The biological activity of recombinant mouse IL-2 is typically measured using the CTLL-2 mouse cytotoxic T cell line proliferation assay . The ED50 (effective dose for 50% maximal response) ranges from 0.1-0.4 ng/mL for high-quality preparations . An activity range of 0.1-1.0 × 10^9 units/mg has been reported, with a unit defined as the amount needed to stimulate a half-maximal response at cytokine saturation . Investigators should titrate the recombinant protein in their specific experimental systems as activity may vary between different assays and cellular contexts .

How can recombinant mouse IL-2 be used in ELISA applications?

Recombinant mouse IL-2 serves as an excellent quantitative standard for IL-2-specific sandwich ELISAs . For optimal results, prepare doubling dilutions of the mouse IL-2 standard from approximately 2,000 to 15 pg/mL for each ELISA plate to generate linear standard curves . When designing an ELISA, the purified JES6-1A12 antibody can be used as a capture antibody, with biotinylated clone JES6-5H4 as the detection antibody . This ELISA configuration is primarily recommended for measuring IL-2 in experimental cell culture systems rather than in serum or plasma samples, for which specialized ELISA kits are available .

What considerations are important when using recombinant mouse IL-2 in T cell culture systems?

When using recombinant mouse IL-2 for T cell culture, several factors must be considered for optimal results. First, titrate the protein to determine the optimal concentration for your specific cell type and experimental purpose (typically in the range of 0.1-20 ng/mL) . For regulatory T cell (Treg) expansion, higher concentrations may be required than for effector T cell maintenance . Second, ensure carrier proteins in your IL-2 preparation do not interfere with your experimental readouts by pre-screening for toxicity, endotoxin levels, or blocking activity . Third, implement a consistent supplementation schedule, as IL-2 can be consumed rapidly in culture . Finally, consider the antagonistic relationship between IL-2 and IL-17, as IL-2 inhibits the development of Th17 polarized cells while promoting Treg expansion .

How can recombinant mouse IL-2 be used in immunological infection models?

Recombinant IL-2 can be administered therapeutically or prophylactically in experimental infection models . For therapeutic applications in chronic respiratory infection models, subcutaneous administration of 0.2-20 μg per mouse daily for 7-14 days has shown dose-dependent reduction in bacterial counts in the lungs . For prophylactic use, administration for 7 days before infection enhances bacterial clearance from the lungs after aerosol exposure . The immunomodulatory effects appear to be independent of natural killer cell activation, as the therapeutic effects are not abolished by anti-asialo GM1 antibody treatment . These protocols demonstrate that IL-2 can enhance host defense mechanisms against pathogens through multiple immune pathways that don't necessarily require specific antigen recognition .

How does mouse IL-2 interact with its receptor complex to induce signaling?

Mouse IL-2 binds to a receptor complex consisting of three subunits present on the cell surface in varying preformed complexes . The 55 kDa IL-2Rα (CD25) is specific for IL-2 and binds with low affinity . The 75 kDa IL-2Rβ (CD122), which is also a component of the IL-15 receptor, binds IL-2 with intermediate affinity . The 64 kDa common gamma chain (γc/IL-2Rγ, CD132), shared with receptors for IL-4, IL-7, IL-9, IL-15, and IL-21, does not independently interact with IL-2 . Signal transduction occurs through both IL-2Rβ and γc upon ligand binding . This hierarchical assembly of the receptor complex allows for different cellular responses depending on the expression levels of each receptor component, which varies among immune cell subsets and activation states.

What are the key immunological functions of mouse IL-2?

Mouse IL-2 exerts diverse immunological functions primarily through autocrine and paracrine activity on T cells . It drives resting T cells to proliferate and induces IL-2 and IL-2Rα synthesis in a positive feedback loop . IL-2 contributes to T cell homeostasis by promoting Fas-induced death of naïve CD4+ T cells while sparing activated CD4+ memory lymphocytes . A critical function of IL-2 is in the expansion and maintenance of regulatory T cells (Tregs), which are essential for preventing autoimmunity . Conversely, IL-2 inhibits the development of Th17 polarized cells, demonstrating its complex role in balancing immune responses . These seemingly contradictory functions (promoting both effector and regulatory T cell responses) highlight IL-2's central position in immune homeostasis and its potential as both an immune activator and regulator depending on the context .

How does mouse IL-2 differ from human IL-2 in structure and function?

Mouse IL-2 shares 56% amino acid sequence identity with human IL-2 . Despite this moderate sequence homology, mouse and human IL-2 exhibit cross-species activity, allowing human IL-2 to be used in mouse models and vice versa, albeit with potentially different potencies . Mouse IL-2 shows strain-specific heterogeneity in an N-terminal region containing a poly-glutamine stretch, which is not present in human IL-2 . This difference may affect protein stability and receptor binding kinetics between species. While the core biological functions remain similar across species (T cell proliferation, Treg expansion, etc.), subtle differences in receptor binding affinity and downstream signaling pathways may exist, which researchers should consider when extrapolating findings between mouse models and human applications.

How can mouse IL-2 variants be engineered to selectively target specific immune cell populations?

Engineering mouse IL-2 variants involves strategic modifications to the protein structure to alter receptor binding preferences and pharmacokinetics. Researchers have developed several approaches:

• Mutation of key contact residues: Altering amino acids at the interface between IL-2 and IL-2Rα (CD25) can create variants with reduced affinity for Tregs (which express high levels of CD25) while maintaining activation of effector T cells and NK cells that express IL-2Rβ and γc .

• PEGylation strategies: Site-specific PEGylation near the IL-2Rα binding site can sterically hinder interaction with CD25 while preserving IL-2Rβ/γc signaling, thus shifting the balance from Treg to effector T cell activation.

• Fusion proteins: Creating fusion proteins of IL-2 with antibodies or other targeting moieties can direct the cytokine to specific tissues or cell types, reducing systemic side effects.

These engineering approaches require careful characterization of receptor binding kinetics, signaling pathway activation, and functional outcomes in various immune cell subsets to ensure the desired biological activity is achieved.

What are the considerations for using mouse IL-2 in combination with other cytokines or immunomodulators?

When using mouse IL-2 in combination with other cytokines or immunomodulators, researchers must consider several complex interactions:

• Receptor competition: IL-2 shares the common gamma chain (γc) receptor component with IL-4, IL-7, IL-9, IL-15, and IL-21, potentially leading to competition for this signaling subunit .

• Signaling pathway cross-talk: IL-2 primarily signals through JAK1/3 and STAT5, but cross-talk with other pathways activated by additional cytokines may lead to synergistic or antagonistic effects.

• Temporal considerations: The timing of IL-2 administration relative to other immunomodulators can significantly impact outcomes. For example, early IL-2 exposure may promote Th1 differentiation, while delayed administration might preferentially expand existing Tregs.

• Dose-dependent interactions: The ratio of IL-2 to other cytokines can determine the immunological outcome. Low-dose IL-2 tends to preferentially expand Tregs due to their higher CD25 expression, while higher doses activate a broader range of immune cells .

• Target cell susceptibility: Different immune cell populations vary in their responsiveness to IL-2 based on their receptor expression profile, activation state, and differentiation stage, which can be further modulated by other cytokines.

Systematic titration experiments and time-course analyses are essential to determine optimal combinations for specific research objectives.

How do different formulations of recombinant mouse IL-2 impact experimental reproducibility?

Different formulations of recombinant mouse IL-2 can significantly impact experimental reproducibility through several mechanisms:

• Expression systems: E. coli-derived mouse IL-2 lacks glycosylation, while mammalian cell-expressed IL-2 contains O-glycosylation, potentially affecting protein stability and bioactivity .

• Carrier proteins: The presence of carrier proteins like BSA enhances stability and shelf-life but may interfere with certain applications . For applications where BSA may interfere, carrier-free formulations are recommended, though these may have different stability profiles .

• Buffer composition: Variations in buffer components, pH, and stabilizing agents between different commercial preparations can affect protein conformation and activity.

• Endotoxin contamination: Endotoxin levels (ideally ≤0.1 ng/μg of mouse IL-2) can vary between preparations and significantly impact immune cell responses, potentially confounding experimental results .

• Batch-to-batch variation: Even within the same product line, batch-to-batch variations in bioactivity may occur, necessitating internal standardization.

To ensure reproducibility, researchers should consistently use the same formulation throughout a study, validate each new batch using bioactivity assays (e.g., CTLL-2 proliferation), and thoroughly document the exact product specifications in publications .

What strategies can resolve decreased bioactivity of recombinant mouse IL-2 in long-term storage?

To address decreased bioactivity of recombinant mouse IL-2 during long-term storage, implement these strategies:

• Optimal initial aliquoting: Upon first thawing, immediately divide the stock into single-use aliquots in polypropylene microtubes and store at -80°C to minimize freeze-thaw cycles .

• Carrier protein addition: For long-term storage, ensure a carrier protein concentration of 0.5-10 mg/mL (e.g., human or bovine serum albumin) and maintain IL-2 concentration above 10 μg/mL .

• Storage buffer optimization: For carrier-containing formulations, use sterile PBS with at least 0.1% human or bovine serum albumin . For carrier-free formulations, store in sterile neutral buffer after reconstitution .

• Temperature stability: Use a manual defrost freezer at -80°C and avoid repeated freeze-thaw cycles . If partial thawing occurs during storage, the protein should be re-validated before experimental use.

• Activity monitoring: Periodically test the bioactivity using standardized assays like CTLL-2 proliferation to track any decline in potency over time and adjust dosing accordingly .

• Reconstitution practice: Always reconstitute lyophilized protein using recommended concentrations (100-200 μg/mL) and buffers to ensure proper solubilization and maintenance of tertiary structure .

If significant activity loss is detected despite these measures, fresh recombinant protein should be obtained and the new batch cross-calibrated with the previous lot to maintain experimental consistency.

What are the potential causes and solutions for variability in mouse IL-2 bioassay results?

Variability in mouse IL-2 bioassay results can stem from multiple factors that require specific solutions:

• Cell line condition: The CTLL-2 indicator cell line's responsiveness can vary with passage number and culture conditions . Solution: Maintain standardized culture protocols and use cells within a specific passage range. Periodically validate the cell line's responsiveness using a reference IL-2 standard.

• Assay protocol variations: Minor differences in incubation times, cell densities, or detection methods can impact results . Solution: Establish detailed standard operating procedures for each step of the bioassay and implement strict quality control measures.

• Interference from carrier proteins: BSA or other carriers in IL-2 preparations may affect cellular responses . Solution: Pre-screen carrier proteins for potential effects in your experimental system and use carrier-free preparations when necessary.

• Endotoxin contamination: Even low levels of endotoxin can stimulate immune cells and confound IL-2 bioactivity measurements . Solution: Confirm endotoxin levels are ≤0.1 ng/μg of mouse IL-2 and consider including polymyxin B in bioassays to neutralize potential endotoxin effects.

• Receptor saturation: At high IL-2 concentrations, receptor saturation can occur, causing deviation from dose-linearity . Solution: Ensure your standard curve includes multiple points within the linear range of the assay (typically ED50 of 0.1-0.4 ng/mL for CTLL-2 cells).

• Statistical analysis approach: Different curve-fitting methods for ED50 calculation can yield varying results. Solution: Apply consistent statistical methods and include internal standards in each assay to normalize between experiments.

How can researchers optimize recombinant mouse IL-2 dosing for in vivo experimental models?

Optimizing recombinant mouse IL-2 dosing for in vivo models requires a systematic approach considering multiple variables:

• Dose-response relationships: Establish dose-response curves by testing a wide range of doses (e.g., 0.2-20 μg per mouse daily) to identify both minimum effective and potential toxicity thresholds . Different immunological outcomes may require different dosing regimens - for example, Treg expansion may occur at lower doses than required for effector T cell activation.

• Administration route considerations: Different routes (subcutaneous, intraperitoneal, intravenous) affect pharmacokinetics and tissue distribution. Subcutaneous administration has been shown effective for respiratory infection models at doses of 0.2-20 μg daily , but optimal routes may vary by disease model.

• Treatment duration optimization: Test various treatment durations to determine minimum effective periods. In respiratory infection models, 7-14 days of daily administration has shown efficacy, with longer treatment periods (14 days) being effective even at lower doses (0.2 μg/day) .

• Timing relative to disease induction: For therapeutic applications, initiate treatment after disease establishment (e.g., 2 weeks post-infection in respiratory models) . For prophylactic use, administration for 7 days before challenge has shown efficacy .

• Monitoring parameters selection: Choose appropriate readouts based on expected mechanisms (bacterial clearance, cell population changes, cytokine profiles, etc.). In respiratory infection models, bacterial counts in lungs, monocyte/lymphocyte counts in peripheral blood, and agglutinin titers in serum provide comprehensive evaluation .

• Combination with blocking antibodies: To dissect mechanisms, combine IL-2 administration with blocking antibodies against specific immune cell populations. For example, anti-asialo GM1 antibody can block NK cells to determine their contribution to IL-2 effects .

This systematic optimization approach ensures reproducible and mechanistically informative in vivo experiments with recombinant mouse IL-2.