Recombinant Mouse Interleukin-7 (Il7) (Active)

What is the molecular structure of recombinant mouse IL-7?

Recombinant mouse IL-7 is a full-length protein spanning amino acids 26 to 154, with a molecular weight of approximately 17 kDa as determined by SDS-PAGE under reducing conditions . The protein features three disulfide bonds and belongs to the IL-7/IL-9 family . The primary sequence is: ECHIKDKEGKAYESVLMISIDELDKMTGTDSNCPNNEPNFFRKHVCDDTKEAAFLNRAARKLKQFLKMNISEEFNVHLLTVSQGTQTLVNCTSKEEEKNVKEQKKNDACFLKRLLREIKTCWNKILKGSI . When comparing across species, 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 .

What are the primary biological functions of mouse IL-7?

Mouse IL-7 functions as a hematopoietic cytokine that plays essential roles in the development, expansion, and survival of naive and memory T-cells and B-cells . It regulates the number of mature lymphocytes and maintains lymphoid homeostasis . Mechanistically, IL-7 exerts its biological effects through a receptor composed of the IL7RA subunit and the cytokine receptor common subunit gamma (CSF2RG) . This receptor binding activates various kinases including JAK1 or JAK3, depending on the cell type, subsequently propagating signals through several downstream pathways including the PI3K/Akt/mTOR or the JAK-STAT5 pathways . Additionally, IL-7 selectively promotes IL-17-producing γδ cells in both mice and humans .

How is recombinant mouse IL-7 produced for research applications?

Recombinant mouse IL-7 can be produced using different expression systems. Common production methods include expression in HEK 293 cells, which yields protein with >95% purity suitable for various applications including SDS-PAGE, functional studies, mass spectrometry, and HPLC . Alternative production methods include expression in E. coli systems . The choice of expression system may affect post-translational modifications and biological activity, with mammalian expression systems generally providing more physiologically relevant modifications. Upon production, the protein is typically formulated as a lyophilized powder from a 0.2 μm filtered solution in PBS, either with or without bovine serum albumin (BSA) as a carrier protein .

How should recombinant mouse IL-7 be reconstituted and stored for optimal activity?

For optimal reconstitution of lyophilized recombinant mouse IL-7, researchers should follow specific protocols depending on the formulation. For preparations containing BSA as a carrier protein, reconstitution at 50 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin is recommended . For carrier-free preparations, reconstitution at 50 μg/mL (for 5 μg vials) or 100 μg/mL (for 25 μg or larger vials) in sterile PBS is advised .

For storage, use a manual defrost freezer and avoid repeated freeze-thaw cycles to maintain protein integrity . While short-term storage at 2-8°C is acceptable for reconstituted protein, aliquoting and storing at -20°C to -80°C is recommended for long-term preservation of activity. Working dilutions should be prepared fresh for each experiment to ensure consistent biological activity.

What is the effective dose range of recombinant mouse IL-7 in common experimental systems?

The effective dose of recombinant mouse IL-7 varies by application and cell type. For stimulation of cell proliferation in PHA-activated human peripheral blood lymphocytes, the ED50 (effective dose for 50% maximum response) is 0.15-0.3 ng/mL . In mouse models studying IL-17-producing γδ cells, concentrations around 10 ng/mL have been used for in vitro cultures . For in vivo applications such as those studying glioblastoma models, higher doses (10 mg/kg) of long-acting IL-7 analogs like NT-I7 have been employed . Researchers should establish dose-response relationships specific to their experimental system, as sensitivity to IL-7 varies significantly between different cell populations.

Which cell types and tissues are most responsive to mouse IL-7 treatment?

IL-7 primarily affects lymphoid cell populations. The most responsive cells include:

• T lymphocytes, particularly:

  • Naive and memory T-cells

  • CD44hi γδ 27− T-cells (which show selective expansion with IL-7 treatment)

  • CD4+ and CD8+ T-cells

• B lymphocytes in developmental stages

• Lymphoid tissues showing significant responses include:

  • Thymus

  • Lymph nodes

  • Spleen

Notably, IL-7 treatment leads to differential expansion of cell subsets, with CD44hi γδ 27− cells showing much greater proliferation (>90% Ki67 positive after 4 days in IL-7) compared to γδ 27+ cells (only ~30% Ki67 positive) . This selective expansion correlates with the capacity for IL-17 production, with expanding cells accounting for almost all IL-17 production upon stimulation .

How does recombinant mouse IL-7 affect specific T-cell subpopulations in experimental settings?

Recombinant mouse IL-7 has distinct effects on different T-cell subpopulations, demonstrating remarkable specificity in its actions. When applied to lymphocyte cultures, IL-7 selectively promotes the expansion of CD44hi γδ 27− T-cells while CD44lo γδ 27+ T-cells show minimal proliferation . This selective expansion results in a three- to fourfold increase in absolute numbers of γδ 27− cells over 4 days, while γδ 27+ cell numbers decline by approximately 70% .

The expanded γδ 27− population shows enrichment for cells with IL-17–producing capacity, increasing from ~30% to ~70% of the γδ 27− subset . This correlates with increased expression of the transcription factor RORγt, a primary regulator of IL-17 production . In contrast, T-bet expression (associated with IFN-γ production) is not significantly affected by IL-7 treatment .

In vivo administration of recombinant IL-7 similarly increases absolute numbers of IL-17-competent cells in lymph nodes by >fivefold, compared with only two- to threefold increases in IFN-γ–competent cells . These findings demonstrate IL-7's potential for selective modulation of T-cell functional subsets.

What signaling pathways are activated by mouse IL-7, and how can they be experimentally monitored?

Mouse IL-7 activates several key signaling pathways that can be monitored using various experimental approaches:

• JAK-STAT Pathway:

  • IL-7 activates JAK1 or JAK3 (depending on cell type), leading to STAT5 phosphorylation

  • In γδ 27− cells, IL-7 selectively activates STAT3

  • Monitoring method: Western blot or flow cytometry using phospho-specific antibodies against p-STAT3 and p-STAT5

• PI3K/Akt/mTOR Pathway:

  • IL-7 activates the PI3K/Akt/mTOR pathway in responsive cells

  • Monitoring method: Western blot for phosphorylated Akt (Ser473) and downstream targets like p-S6

• Cell Cycle Progression:

  • IL-7 promotes cell division in responsive populations

  • Monitoring method: Ki67 staining (>90% of γδ 27− cells become Ki67+ after IL-7 treatment) or CFSE dilution assays

The differential activation of these pathways explains the selective effects of IL-7 on different cell populations. For example, γδ 27− cells express very low levels of SOCS3 (a STAT3 suppressor) compared to CD44lo γδ 27+ cells, which correlates with their selective response to IL-7 .

How does mouse IL-7 influence the tumor microenvironment in cancer models?

Research with long-acting IL-7 (NT-I7) in glioblastoma (GBM) mouse models demonstrates significant impacts on the tumor microenvironment. Treatment with NT-I7 (10 mg/kg) in combination with radiotherapy (RT) leads to:

• Enhanced anti-tumor immune responses:

  • Increased infiltration of cytotoxic CD8 T lymphocytes within tumors

  • Enhanced IFNγ production by infiltrating T cells

  • Decreased regulatory T cells (Tregs) in the tumor microenvironment

• Systemic immune modulation:

  • Mitigation of radiotherapy-related lymphopenia

  • Increased T lymphocytes in lymph nodes, thymus, and spleen

  • Enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumors

• Improved survival outcomes:

  • Significant increase in survival in orthotopic glioma-bearing mice

  • Effect abrogated by CD8 T cell depletion, demonstrating the necessity of these cells for IL-7's therapeutic effects

These findings suggest that IL-7-based therapies may enhance anti-tumor immunity through multiple mechanisms, potentially overcoming immunosuppression in the tumor microenvironment.

What are the critical factors for successful experimental design using recombinant mouse IL-7?

Successful experiments with recombinant mouse IL-7 require careful consideration of several critical factors:

• Protein formulation selection:

  • BSA-containing formulations are recommended for cell or tissue culture applications and as ELISA standards

  • Carrier-free preparations should be used for applications where BSA might interfere

• Cell responsiveness assessment:

  • Different lymphocyte subsets show varying sensitivity to IL-7

  • Pre-assessment of IL-7 receptor (IL-7R) expression on target cells is advisable

  • Low SOCS3 expression correlates with enhanced IL-7 responsiveness

• Dose optimization:

  • Establish dose-response curves for each experimental system

  • Standard range for in vitro applications: 0.1-10 ng/mL

  • Higher doses may be required for in vivo applications

• Timing considerations:

  • Maximum effects on proliferation typically observed after 3-4 days of culture

  • In vivo effects may require multiple administrations (e.g., three doses over 5 days)

• Combinatorial approaches:

  • IL-7 synergizes with TCR agonists for γδ 27− cells

  • Consider combination with other cytokines or stimuli depending on the target cell population

How can researchers distinguish between direct and indirect effects of IL-7 in complex experimental systems?

Distinguishing direct from indirect effects of IL-7 requires methodical experimental approaches:

• Cell isolation and purification:

  • Use highly purified cell populations to identify direct responders

  • Example: Studies with purified CD44hi γδ 27−, CD44hi γδ 27+, or CD44lo γδ 27+ thymocytes demonstrated that IL-7 directly activates γδ 27− thymocytes rather than promoting conversion of other populations

• Receptor expression analysis:

  • Quantify IL-7R expression on different cell populations

  • Cells lacking IL-7R are unlikely to respond directly to IL-7

• Signaling pathway inhibition:

  • Use specific inhibitors of IL-7 signaling pathways

  • Example: STAT3 inhibition reduced the preferential enrichment of γδ 27− cells by >50% and severely attenuated the generation of IL-17–producing cells

• Genetic approaches:

  • Use cell-specific knockout models for IL-7R

  • Employ lineage tracing systems like Il17aCreR26ReYFP mice to track cell fate

• Timing analysis:

  • Direct effects typically occur rapidly (minutes to hours for signaling, 1-4 days for proliferation)

  • Indirect effects may require longer time periods

These approaches help researchers delineate the precise mechanisms through which IL-7 exerts its biological effects in complex systems.

How do findings from mouse IL-7 research translate to human systems?

While mouse and human IL-7 share only 58-60% amino acid sequence identity, they demonstrate significant cross-species activity , making mouse models valuable for translational research. Key translational aspects include:

• Functional conservation:

  • Both human and mouse IL-7 play essential roles in lymphocyte development and homeostasis

  • Both selectively promote IL-17-producing γδ cells

  • Similar signaling pathways activated in both species

• Clinical development:

  • Long-acting IL-7 (NT-I7) being tested in clinical trials for patients with high-grade gliomas (NCT03687957)

  • Preclinical findings in mouse models support clinical applications for combating treatment-induced lymphopenia

• Differences requiring consideration:

  • Dose adjustments may be necessary between species

  • Human lymphocyte subsets may display different sensitivity thresholds

  • Mouse studies should be validated in human cell systems before clinical translation

A thorough understanding of both the similarities and differences between mouse and human IL-7 biology is essential for effective translational research.

What are the emerging therapeutic applications of recombinant IL-7 based on mouse model findings?

Research with mouse models has identified several promising therapeutic applications for recombinant IL-7:

• Cancer immunotherapy:

  • Reversal of lymphopenia in cancer patients receiving radiotherapy and chemotherapy

  • Enhancement of anti-tumor immune responses by increasing cytotoxic T cells and decreasing Tregs

  • Combination with other immunotherapies to overcome tumor-induced immunosuppression

• Infectious disease:

  • Restoration of T-cell numbers and function during chronic viral infections

  • Enhancement of vaccine efficacy through improved T-cell responses

• Autoimmune regulation:

  • Selective modulation of specific T-cell subsets (e.g., promoting IL-17-producing cells)

  • Potential applications in autoimmune conditions requiring immune reconstitution

• Hematopoietic stem cell transplantation:

  • Acceleration of immune reconstitution post-transplantation

  • Reduction of opportunistic infection risk during recovery

These applications are supported by findings from mouse models, such as the significant survival improvement observed in glioblastoma models treated with NT-I7 in combination with radiotherapy .

What control experiments are essential when working with recombinant mouse IL-7?

These controls ensure that observed effects are specific to IL-7 activity and help distinguish direct from indirect mechanisms of action.

How can researchers quantitatively assess the biological activity of recombinant mouse IL-7 preparations?

Quantitative assessment of recombinant mouse IL-7 biological activity can be performed using several complementary approaches:

• Proliferation assays:

  • Standard: PHA-activated human peripheral blood lymphocytes (ED50: 0.15-0.3 ng/mL)

  • Alternative: Purified mouse thymocytes or lymph node T cells

  • Readout: 3H-thymidine incorporation, CFSE dilution, or Ki67 staining

• STAT phosphorylation:

  • Flow cytometry-based detection of p-STAT5 or p-STAT3 in responsive cells

  • Western blot analysis of phosphorylated signaling proteins

  • Expected timing: 15-30 minutes post-stimulation

• Survival assessment:

  • Measurement of anti-apoptotic protein induction (Bcl-2)

  • Annexin V/PI staining to quantify viable cells

  • Cell counting over time course (typically 4-7 days)

• Functional readouts:

  • IL-17 production by γδ 27− cells following activation

  • CD69 upregulation (activation marker)

  • CD44 expression changes

• In vivo activity:

  • Lymphocyte counts in blood, lymph nodes, spleen after administration

  • Phenotypic changes in T-cell subsets (CD44/CD69 upregulation)

  • Functional capacity (cytokine production upon ex vivo stimulation)

These assays provide comprehensive assessment of biological activity across multiple parameters, ensuring the quality and consistency of recombinant IL-7 preparations.

What are the key unanswered questions regarding mouse IL-7 biology and function?

Despite extensive research, several important questions about mouse IL-7 remain unanswered:

• Molecular mechanism of selectivity:

  • How does IL-7 selectively expand certain lymphocyte subsets (e.g., γδ 27− cells) while having minimal effects on others?

  • What are the transcriptional networks that determine IL-7 responsiveness beyond SOCS3 expression?

• Tissue microenvironment effects:

  • How does local production of IL-7 in specific tissue microenvironments influence resident and infiltrating immune cells?

  • What is the interplay between IL-7 and other tissue-derived factors?

• Memory formation:

  • What is the precise role of IL-7 in generating and maintaining memory T cells of different subsets?

  • How does IL-7 interact with antigen persistence in shaping memory responses?

• Developmental programming:

  • What epigenetic changes are induced by IL-7 signaling during lymphocyte development?

  • How do these changes influence long-term cell fate decisions?

• Non-lymphoid effects:

  • Does IL-7 have significant direct effects on non-lymphoid cells that express IL-7R?

  • What are the consequences of IL-7 therapy on non-immune tissues?

Addressing these questions will require innovative experimental approaches and may reveal new applications for IL-7-based therapeutics.

How might advanced technologies enhance research on recombinant mouse IL-7?

Emerging technologies offer exciting possibilities for advancing IL-7 research:

• Single-cell analysis:

  • Single-cell RNA sequencing to identify heterogeneity in IL-7 responsive populations

  • Single-cell proteomics to map signaling network activation at individual cell level

  • Spatial transcriptomics to understand IL-7 effects in tissue context

• Advanced protein engineering:

  • Structure-guided modification of IL-7 to enhance stability or specificity

  • Development of cell subset-specific IL-7 variants through directed evolution

  • Creating bifunctional molecules combining IL-7 with other cytokines or targeting moieties

• In vivo imaging:

  • Real-time tracking of IL-7-responsive cells using reporter mice

  • Intravital microscopy to visualize cellular interactions following IL-7 treatment

  • PET imaging with labeled IL-7 to map tissue distribution and receptor occupancy

• Systems biology approaches:

  • Computational modeling of IL-7 signaling networks

  • Integration of multi-omics data to understand global effects of IL-7

  • Prediction of optimal combination therapies based on network analysis

• CRISPR-based screening:

  • Genome-wide screens to identify novel regulators of IL-7 responsiveness

  • Precise genetic manipulation to test mechanistic hypotheses

  • In vivo CRISPR screens to identify resistance mechanisms

These technological advances promise to deepen our understanding of IL-7 biology and accelerate the development of IL-7-based therapeutic strategies.

What are the most robust and reproducible experimental systems for studying recombinant mouse IL-7?

Based on the available evidence, several experimental systems have demonstrated particular robustness for investigating IL-7 biology:

• For in vitro studies:

  • Purified CD44hi γδ 27− cells from mouse thymus or lymph nodes

  • PHA-activated human peripheral blood lymphocytes for cross-species activity testing

  • Freshly isolated thymocytes from wildtype or reporter mice (e.g., Il17aCreR26ReYFP)

• For in vivo applications:

  • Orthotopic glioma models (GL261 or CT2A) for studying IL-7 effects on tumor immunity

  • Lymphopenia models induced by sublethal irradiation

  • Adoptive transfer systems with CFSE-labeled cells to track proliferation and migration

• For mechanistic investigations:

  • STAT3 inhibition studies to assess signaling requirements

  • Cell-specific genetic deletion models for IL-7 receptor components

  • Competitive bone marrow chimeras to assess cell-intrinsic effects

These systems provide reliable platforms for studying the diverse biological activities of IL-7 and allow for rigorous testing of hypotheses regarding its mechanisms of action.

What methodological approaches would advance understanding of recombinant mouse IL-7's therapeutic potential?

To further explore and develop the therapeutic potential of recombinant mouse IL-7, researchers should consider the following methodological approaches:

• Combination therapy optimization:

  • Systematic testing of IL-7 with immune checkpoint inhibitors

  • Evaluation of sequential vs. concurrent administration with conventional therapies

  • Identification of synergistic drug combinations through high-throughput screening

• Delivery system development:

  • Long-acting formulations beyond NT-I7

  • Targeted delivery to specific tissues or cell populations

  • Controlled release systems for sustained local concentration

• Predictive biomarker identification:

  • Correlation of baseline immune parameters with IL-7 responsiveness

  • Development of ex vivo assays to predict in vivo efficacy

  • Identification of genetic markers associated with optimal response

• Mechanism-based combination strategies:

  • Combining IL-7 with agents targeting complementary pathways

  • Sequential modulation of immune microenvironment before IL-7 treatment

  • Rational combinations based on systems biology approaches

• Translational model development:

  • Humanized mouse models for more accurate prediction of human responses

  • Patient-derived xenograft models for personalized therapy assessment

  • Ex vivo human tissue systems for rapid screening