Recombinant Mouse Interleukin-7 protein (Il7) (Active)

What is Recombinant Mouse IL-7 protein and what are its structural characteristics?

Recombinant Mouse IL-7 is a 25 kDa cytokine of the hemopoietin family that plays crucial roles in lymphocyte differentiation, proliferation, and survival. Commercial preparations typically consist of the mature protein sequence (Glu26-Ile154) with an N-terminal methionine residue, expressed in E. coli systems. When analyzed by SDS-PAGE under reducing conditions, the protein appears as a single band at approximately 17 kDa . Mouse IL-7 shares approximately 88% amino acid sequence identity with rat IL-7 and 58-60% with human, equine, bovine, ovine, porcine, feline, and canine IL-7 . Both human and mouse IL-7 exhibit cross-species activity, allowing for some flexibility in experimental models .

How does IL-7 contribute to immune cell development and homeostasis?

IL-7 plays essential but species-specific roles in lymphocyte development and maintenance. In mice, IL-7 is critical for both T cell and B cell lineage development, while in humans, it is primarily required for T cell but not B cell development . For T cells, IL-7 contributes to the maintenance of both naïve and memory populations by promoting expression of the anti-apoptotic protein Bcl-2 . IL-7 is produced by stromal epithelial cells in primary and secondary lymphoid tissues, including the thymus, bone marrow, and intestines .

In B cell development, IL-7 is expressed prior to the appearance of surface IgM and functions in both mouse and human pro-B cells to suppress premature immunoglobulin light chain recombination during proliferative growth . IL-7 is also essential for optimal T cell-dendritic cell interaction and plays roles in natural killer (NK) cell development and homeostasis .

What are the optimal storage and handling procedures for Recombinant Mouse IL-7?

For optimal research outcomes with Recombinant Mouse IL-7, proper storage and handling are critical:

Reconstituted protein should be used within the same day for critical applications, and working dilutions should be prepared fresh before use . These precautions help maintain the biological activity of the protein throughout the experimental timeframe.

How is the biological activity of Recombinant Mouse IL-7 measured?

The biological activity of Recombinant Mouse IL-7 is typically measured through cell proliferation assays. The standard assay evaluates the stimulation of PHA-activated human peripheral blood lymphocytes, with an effective dose (ED50) typically in the range of 0.15-0.3 ng/mL . This cross-species activity provides a reliable bioassay for confirming protein functionality.

Alternative methods to assess IL-7 activity include:

• Phosphorylation of STAT5 in IL-7-responsive cell lines

• Survival assays with IL-7-dependent lymphocytes

• Quantification of IL-7-regulated genes such as Bcl-2

• Flow cytometric analysis of IL-7Rα downregulation after stimulation

These methods can be used to verify activity before proceeding with more complex experiments.

What are the differences between carrier-free and BSA-containing IL-7 preparations?

Researchers must choose between carrier-free and BSA-containing IL-7 preparations based on experimental requirements:

BSA-containing preparation:

• Contains bovine serum albumin as a carrier protein

• Offers enhanced protein stability and increased shelf-life

• Can be stored at more dilute concentrations

• Recommended for cell culture applications and as ELISA standards

• Reconstitution typically at 50 μg/mL in sterile PBS containing albumin

Carrier-free preparation:

• Does not contain BSA or other carrier proteins

• Recommended for applications where BSA might interfere

• Reconstitution typically at 50-100 μg/mL in sterile PBS

• May have slightly reduced stability compared to preparations with carriers

• Preferred for in vivo studies, mass spectrometry applications, or antibody generation

The choice between these preparations should be based on the specific requirements of the experimental system to ensure optimal results and prevent potential interference.

How do IL-7/anti-IL-7 mAb complexes enhance cytokine potency in vivo?

IL-7/anti-IL-7 mAb complexes dramatically enhance the in vivo potency of IL-7 through several mechanisms:

• FcRn-mediated half-life extension:

  • The Fc domain of the antibody engages the neonatal Fc receptor (FcRn)

  • This interaction protects the complex from degradation

  • FcRn expression in both hematopoietic and non-hematopoietic cells contributes to this effect

  • The extended half-life accounts for the majority of the enhanced activity

• Cytokine depot effect:

  • The Fab domain of the neutralizing antibody (M25) serves as a cytokine reservoir

  • This provides a steady release of active cytokine over time

  • Despite being a "neutralizing" antibody, M25 enhances in vivo effects

• Spatial localization:

  • IL-7/mAb complexes exert their effects primarily in T-cell zones of secondary lymphoid tissues

  • T cells require access to these zones to respond optimally to IL-7/mAb stimulation

  • Pertussis toxin treatment, which disrupts chemokine receptor signaling and lymphoid tissue homing, dramatically reduces responsiveness to IL-7/mAb complexes

This enhanced potency makes IL-7/anti-IL-7 mAb complexes a valuable tool for immunomodulation in research models, requiring significantly lower doses than uncomplexed IL-7.

How does IL-7 signaling work and what are the key receptor interactions?

IL-7 signaling operates through a specific receptor complex with well-characterized downstream events:

Receptor composition:

• IL-7 signals through a heterodimeric receptor consisting of IL-7 Receptor alpha subunit (IL-7Rα, CD127) and the common gamma chain (γc)

• The γc is also a subunit of receptors for IL-2, IL-4, IL-9, IL-15, and IL-21

Signaling cascade:

• Receptor engagement activates Janus kinases (JAKs), primarily JAK1 and JAK3

• JAK activation leads to STAT5 phosphorylation

• Phosphorylated STAT5 dimerizes and translocates to the nucleus

• Nuclear STAT5 regulates gene expression, including upregulation of survival factors like Bcl-2

Receptor regulation:

• IL-7Rα expression is dynamically regulated on lymphocytes

• It is expressed on double-negative (CD4-CD8-) and single-positive T cells

• IL-7 stimulation induces receptor downregulation, creating a negative feedback loop

• Regulatory T cells typically lack IL-7Rα expression

This signaling pathway is essential for lymphocyte development and homeostasis, with genetic defects in this pathway leading to severe combined immunodeficiency.

What role does FcRn play in the enhanced activity of IL-7/antibody complexes?

The neonatal Fc receptor (FcRn) plays a crucial role in the enhanced activity of IL-7/antibody complexes:

• Half-life extension mechanism:

  • FcRn selectively binds to the Fc portion of IgG antibodies in acidic endosomal compartments

  • This interaction rescues antibodies from lysosomal degradation

  • In FcRn-deficient mice, the serum persistence of anti-IL-7 mAb (M25) is greatly reduced

  • The potency of IL-7/M25 complexes is dramatically diminished in FcRn-deficient hosts

• Cellular distribution of FcRn effects:

  • Both hematopoietic and non-hematopoietic cell-expressed FcRn contribute to the full potency of IL-7/M25

  • Chimeric hosts with FcRn expression restricted to either compartment show intermediate effects

• Specificity of the FcRn effect:

  • The effect is specific to intact antibody complexes

  • IL-7 complexed with Fab fragments shows minimal enhancement

  • IL-7-Fc fusion proteins also exhibit enhanced potency compared to IL-7 alone, further confirming the importance of Fc-FcRn interactions

These findings highlight the critical role of FcRn in mediating the enhanced potency of cytokine-antibody complexes, providing insights for designing improved immunotherapeutic approaches.

What are the spatial requirements for T cells to respond to IL-7 stimulation?

T cells have specific spatial requirements to optimally respond to IL-7 stimulation, which is important for experimental design:

• T-cell zone localization:

  • T cells responding to endogenous IL-7 require access to T-cell zones in secondary lymphoid tissues

  • These zones are primary sites of IL-7 production by stromal epithelial cells

  • When treated with pertussis toxin (PTX) to disrupt CCR7-dependent homing to lymphoid tissues, T cells show dramatically reduced responsiveness to IL-7/anti-IL-7 mAb complexes

  • In contrast, treatment with FTY720, which traps T cells within lymphoid tissues, only slightly diminishes the response to IL-7/anti-IL-7 mAb stimulation

• Implications for experimental design:

  • Consider the anatomical distribution of target cells when administering IL-7

  • Ensure intact chemokine receptor signaling for optimal responses

  • Account for potential differences in response depending on tissue microenvironment

  • Recognize that systemic administration of IL-7 primarily stimulates cells within secondary lymphoid tissues

Understanding these spatial requirements is crucial for correctly interpreting results from IL-7 treatment studies and optimizing experimental protocols.

What are the optimal concentration ranges for different IL-7 experimental applications?

Optimal concentration ranges for IL-7 vary significantly by application type:

These ranges provide starting points that should be optimized for each specific experimental system through dose-response studies. The biological activity of commercial IL-7 preparations may vary between lots, necessitating standardization for critical experiments.

How should researchers design dose-response experiments with Recombinant Mouse IL-7?

Effective dose-response experiments with Recombinant Mouse IL-7 require systematic planning:

• Concentration selection:

  • Begin with a broad range spanning at least 3 orders of magnitude (e.g., 0.01-10 ng/mL)

  • Include concentrations above and below the reported ED50 (0.15-0.3 ng/mL)

  • Use half-log or quarter-log increments for precise curve fitting

• Experimental setup:

  • Include appropriate positive controls (e.g., known IL-7-responsive cells)

  • Include negative controls (untreated cells and IL-7Rα-blocking conditions)

  • Perform replicates (minimum triplicate) for each concentration

  • Consider time-course experiments at key concentrations

• Readout selection:

  • For proliferation: CFSE dilution, Ki-67 expression, or BrdU uptake

  • For survival: Annexin V/PI staining, caspase activation, or metabolic assays

  • For signaling: Phospho-STAT5, Bcl-2 induction, or pathway-specific gene expression

• Data analysis:

  • Calculate percentage of maximum response for each concentration

  • Fit data to sigmoidal dose-response curves

  • Determine EC50 values and compare across experimental conditions

  • Consider both potency (EC50) and efficacy (maximum response) in interpretations

This systematic approach ensures robust and reproducible dose-response characterization for IL-7 experiments.

What controls should be included in IL-7 stimulation experiments?

Comprehensive controls are essential for proper interpretation of IL-7 experiments:

Negative controls:

• Untreated/vehicle control: Cells with no IL-7 treatment

• Heat-inactivated IL-7: IL-7 heated at 95°C for 10 minutes to denature the protein

• IL-7Rα blocking: Anti-IL-7Rα antibody to block receptor binding

• Irrelevant cytokine control: A cytokine not expected to affect the readout

Positive controls:

• Known IL-7-responsive cell line (e.g., pre-B cell line)

• Alternative stimulus inducing similar response (e.g., IL-2 for T cell proliferation)

• Fresh vs. stored IL-7 comparison to check for activity retention

Technical controls:

• Carrier protein only control (e.g., BSA alone)

• Endotoxin control: Polymyxin B addition to rule out LPS contamination effects

• For IL-7/mAb complex experiments, include antibody-only controls

Validation controls:

• JAK inhibitor to confirm signaling pathway involvement

• STAT5 inhibitor to verify downstream effector requirements

• Bcl-2 inhibitor to confirm survival mechanism

These controls enable proper interpretation of IL-7-specific effects and help troubleshoot unexpected results.

How can researchers troubleshoot variable responses to IL-7 in experimental models?

Variability in responses to IL-7 can be addressed through systematic troubleshooting:

Common sources of variability:

• Receptor expression levels: IL-7Rα expression varies with activation state

• Competition for limiting IL-7: Endogenous lymphocytes may consume exogenous IL-7

• Target cell location: Access to secondary lymphoid tissues affects response magnitude

• IL-7 protein quality: Activity can diminish with improper storage or freeze-thaw cycles

Troubleshooting approaches:

• Confirm IL-7 activity using a standard bioassay (e.g., proliferation of PHA-activated PBMCs)

• Verify IL-7Rα expression on target cells by flow cytometry

• Test responses in lymphopenic models to reduce competition for IL-7

• Consider using IL-7/anti-IL-7 complexes for more consistent in vivo effects

• Ensure proper reconstitution and storage of IL-7 protein according to manufacturer guidelines

• Verify that pertussis toxin treatment doesn't interfere with IL-7 signaling when studying trafficking requirements

Systematic application of these approaches can improve reproducibility in IL-7-based experiments and resolve inconsistent results across studies.

How can IL-7 be used in immune reconstitution studies?

IL-7 is valuable in immune reconstitution studies due to its critical role in lymphocyte development and homeostasis:

Applications in immune reconstitution research:

• Accelerating T-cell recovery after lymphodepletion

• Enhancing thymic output and broadening T-cell receptor diversity

• Expanding naïve and memory T-cell populations

• Improving immune responses in immunocompromised hosts

Methodological approaches:

• Direct administration of recombinant IL-7 (typically 2-10 μg per mouse)

• Use of IL-7/anti-IL-7 mAb complexes for enhanced potency (typically 1.5 μg IL-7 + 7.5 μg mAb per injection)

• Administration of IL-7-Fc fusion proteins

• Multiple dosing strategies (daily, every other day, or twice weekly injections)

Evaluating reconstitution:

• Monitoring absolute lymphocyte counts in peripheral blood

• Assessing T-cell subset distribution (CD4/CD8 ratio, naïve/memory proportions)

• Measuring proliferation through Ki-67 expression or BrdU incorporation

• Analyzing T-cell receptor repertoire diversity

These approaches can be tailored to specific research questions regarding immune system recovery after various forms of immunodepletion.

What are the best methods to detect IL-7-induced signaling events?

Multiple complementary methods can detect IL-7-induced signaling events:

Protein phosphorylation detection:

• Flow cytometry for phospho-STAT5 (most direct and rapid readout)

• Western blotting for phospho-JAK1, phospho-JAK3, and phospho-STAT5

• Mass spectrometry-based phosphoproteomics for comprehensive pathway analysis

Transcriptional readouts:

• qRT-PCR for IL-7-regulated genes (Bcl-2, SOCS1, CISH)

• RNA-seq for genome-wide transcriptional changes

• Reporter assays with STAT5-responsive elements

Protein expression analysis:

• Flow cytometry for Bcl-2 family members

• Western blotting for survival proteins

• Proteomics for comprehensive protein changes

Receptor dynamics:

• Surface IL-7Rα expression (typically downregulated after stimulation)

• Receptor internalization assays

• Receptor recycling studies

Timing considerations:

• Phospho-STAT5: Peaks at 15-30 minutes post-stimulation

• Transcriptional changes: 1-6 hours

• Protein expression changes: 6-24 hours

• Functional outcomes: 24-72 hours

These approaches provide comprehensive assessment of IL-7 signaling at multiple levels, enabling detailed mechanistic studies.