IL2RA Human, sf9

What is IL2RA and what is its role in immune regulation?

IL2RA, also known as CD25, is a subunit of the high-affinity interleukin-2 (IL-2) receptor complex. It functions in conjunction with IL-2Rβ and the common gamma chain (γc) to form the complete receptor that mediates IL-2 signaling. The IL2RA is primarily involved in the regulation of immune tolerance by controlling regulatory T cells (Tregs) activity, which in turn suppresses the activation and expansion of autoreactive T-cells . This receptor is crucial for maintaining the balance between immunity and tolerance, with differential effects on various immune cell populations including T cells, B cells, natural killer cells, and dendritic cells .

What is the structure of IL2RA Human produced in sf9 expression systems?

IL2RA produced in Sf9 Baculovirus cells is a single, glycosylated polypeptide chain containing 461 amino acids (specifically residues 22-240 of the native sequence) with a molecular mass of approximately 52.1kDa. When analyzed by SDS-PAGE, it typically appears as a band between 50-70kDa due to its glycosylation pattern. Commercial preparations often include a C-terminal hIgG-His tag (239 amino acids) to facilitate purification through proprietary chromatographic techniques . This expression system is chosen for its ability to produce properly folded mammalian proteins with post-translational modifications.

How does IL2RA interact with other IL-2 receptor components to form a functional receptor?

IL2RA forms part of the tripartite IL-2 receptor complex. The high-affinity IL-2 receptor consists of three subunits: IL2RA (α chain), IL2RB (β chain), and IL2RG (γc chain). When IL-2 binds to this receptor complex, it leads to heterodimerization of the IL-2Rβ and γc subunits, which initiates downstream signaling . Homodimeric α chains (IL2RA) alone result in low-affinity binding to IL-2, while homodimeric β chains produce medium-affinity receptors . Full signaling capacity requires all three components, with IL2RA significantly enhancing the binding affinity of IL-2 to the receptor complex .

What are the optimal conditions for storing and handling IL2RA Human, sf9 in laboratory settings?

For optimal preservation of IL2RA Human, sf9 protein activity, the recombinant protein should be stored at -80°C for long-term storage or at -20°C for short-term storage. Repeated freeze-thaw cycles should be avoided as they can lead to protein degradation and loss of functionality. When handling the protein, maintain it on ice and use sterile, protein low-binding tubes. For experiments, reconstitute the lyophilized protein in sterile, filtered buffer (PBS with 0.1% BSA is often suitable) at an appropriate concentration. For working solutions, dilutions should be prepared fresh before each experiment to ensure consistent activity.

What methods can be used to verify the functionality of IL2RA Human, sf9 preparations?

To verify the functionality of IL2RA Human, sf9, several complementary approaches can be employed:

• Binding assays: Surface plasmon resonance (SPR) can measure binding of IL2RA to IL-2 or antibodies directed against conformational epitopes .

• Cell-based assays: Flow cytometry using fluorescently labeled IL-2 can assess binding to cells transfected with IL2RA.

• Signaling assays: Monitor STAT5 phosphorylation in appropriate cell lines like activated CD8+ T cells when exposed to IL-2 in the presence of IL2RA .

• Co-immunoprecipitation experiments: Verify proper interaction with IL2RB and IL2RG components.

• Single-molecule tracking: Advanced microscopy techniques can be used to assess receptor dimerization upon ligand binding, similar to the co-diffusion studies performed with IL-2 muteins .

How can researchers accurately quantify IL2RA expression levels in experimental samples?

Quantification of IL2RA expression can be performed using several complementary techniques:

• Flow cytometry: For cell surface expression, using anti-IL2RA antibodies conjugated to fluorophores allows quantification on a per-cell basis. Calibration beads with known antibody binding capacity can convert fluorescence intensity to absolute receptor numbers.

• Western blotting: For total protein levels, using validated anti-IL2RA antibodies with appropriate loading controls. Densitometric analysis can provide semi-quantitative results.

• qRT-PCR: For mRNA expression levels, using primers specific to IL2RA transcripts normalized to housekeeping genes.

• ELISA: For soluble IL2RA (sIL2RA) in culture supernatants or biological fluids.

• Mass spectrometry: For absolute quantification, using isotope-labeled standards of IL2RA peptides.

Each method has specific advantages and limitations that should be considered based on experimental objectives.

How do IL2RA variants affect receptor dimerization and downstream signaling pathways?

IL2RA variants can significantly alter receptor dimerization efficiency and subsequent signaling outcomes. Research using engineered IL-2 muteins (modified IL-2 molecules) has demonstrated that alterations in the interface between receptor components can create a spectrum of signaling responses . For example, mutations that affect the interaction between IL-2Rβ and γc chains (like those in the H9-RET and H9-RETR variants) result in impaired heterodimerization, which can be quantified using co-tracking techniques such as single-molecule tracking .

The functional consequences of altered dimerization include:

• Differential phosphorylation of STAT5 - a key transcription factor in IL-2 signaling

• Varied patterns of gene induction, with some variants activating only subsets of the normal IL-2 response genes

• Creation of partial agonists or antagonists of IL-2 signaling

• Cell type-specific responses, depending on the expression levels of receptor components

This mechanistic understanding allows researchers to design IL-2 variants with precisely calibrated signaling properties for experimental or therapeutic applications.

What are the distinct signaling outcomes of IL2RA pathway activation in different T cell subsets?

IL2RA signaling produces markedly different outcomes depending on the T cell subset, a phenomenon that reflects both the differential expression of receptor components and the unique intracellular signaling environments:

In regulatory T cells (Tregs):

• High constitutive expression of IL2RA (CD25) enables response to low IL-2 concentrations

• Signaling promotes proliferation, survival, and maintenance of suppressive function

• Critical for immune tolerance and prevention of autoimmunity

In effector CD8+ T cells:

• IL2RA expression is induced upon activation

• Signaling promotes proliferation, differentiation, and effector function

• The level of IL-2 signaling determines the balance between memory and effector cell fate

In CD4+ T helper subsets:

• Promotes Th1, Th9, and Treg differentiation

• Inhibits Th17 cell differentiation

• IL-2 antagonists (like H9-RET and H9-RETR) reverse these effects, inhibiting Th1, Th9, and Treg differentiation while augmenting Th17 development

These differential effects highlight how the same receptor can mediate context-dependent outcomes based on cell type and activation state.

How is IL2RA implicated in autoimmune diseases and what are the mechanisms involved?

IL2RA plays a central role in multiple autoimmune diseases through several mechanisms:

In Multiple Sclerosis (MS):

• The IL-2–IL-2R pathway determines the balance between immunity and tolerance

• Genetic variants in IL2RA affect susceptibility to MS

• Soluble IL2RA (sIL2RA) levels are elevated in MS patients and may serve as a biomarker

• The therapeutic monoclonal antibody daclizumab targets IL2RA and unexpectedly involves NK-cells in its mechanism of action

In Rheumatoid Arthritis (RA):

• The IL2RA-rs2104286 genetic variant is associated with disease persistence

• This variant is linked to both joint destruction and RA-persistence

• Soluble IL2RA levels correlate with disease activity and persistence

• This underscores the relevance of IL2RA for RA pathogenesis and chronicity

In Type 1 Diabetes:

• IL2RA variants influence disease susceptibility

• These variants affect the function of regulatory T cells, which normally prevent autoimmunity

Common mechanisms across autoimmune diseases include altered IL-2 sensitivity of regulatory T cells, imbalanced effector T cell responses, and modulation of immune tolerance thresholds.

How can engineered IL2RA-based molecules be used as research tools to study immune regulation?

Engineered IL2RA-based molecules offer powerful tools to dissect immune regulatory mechanisms:

• Receptor "clamps": Engineered IL-2 variants like H9-RETR act as IL-2-receptor-signaling "clamps" that allow fine-tuning of signaling amplitude . These molecules can:

  • Partially or completely block IL-2 signaling

  • Inhibit both IL-2 and IL-15 signaling due to shared receptor components

  • Create graded responses to study dose-dependent effects

• Cell-specific targeting: By modulating the binding properties of IL2RA ligands, researchers can selectively target specific cell populations:

  • Treg-biased agonists that preferentially activate regulatory T cells

  • Effector-biased agonists that preferentially stimulate cytotoxic T cells

• Signaling dissection: These molecules allow researchers to:

  • Separate different branches of IL-2 signaling

  • Identify gene expression patterns linked to specific signaling thresholds

  • Study compensatory mechanisms when IL-2 signaling is partially blocked

• Disease models: IL2RA-targeted reagents can modulate disease in experimental models:

  • Prolonged survival in murine models of graft versus host disease

  • Inhibition of spontaneous proliferation of adult T-cell leukemia cells

These tools provide precise control over IL-2 signaling, enabling detailed dissection of immune regulatory mechanisms.

What are the common pitfalls in experiments using IL2RA Human, sf9 and how can they be avoided?

Researchers working with IL2RA Human, sf9 frequently encounter several challenges that can impact experimental outcomes:

• Protein Aggregation Issues:

  • Problem: IL2RA can form aggregates during storage or experimental handling

  • Solution: Add 0.1% BSA as a carrier protein; use gentle mixing rather than vortexing; centrifuge solutions briefly before use to remove any aggregates

• Inconsistent Glycosylation Patterns:

  • Problem: Sf9-expressed IL2RA may have variable glycosylation affecting function and molecular weight appearance (50-70kDa range on SDS-PAGE)

  • Solution: Characterize each batch by lectin blotting; consider deglycosylation experiments to assess functional impact; include appropriate controls

• Interference from Endogenous IL-2:

  • Problem: Endogenous IL-2 production by cells in culture may confound results

  • Solution: Use IL-2 blocking antibodies as controls; consider IL-2 knockout or knockdown systems for clean background

• Tag Interference:

  • Problem: The C-terminal hIgG-His tag (239 amino acids) may affect function

  • Solution: Include tag-only controls; compare with untagged versions where possible; consider enzymatic tag removal

• Cell Type-Specific Responses:

  • Problem: IL2RA signaling varies dramatically between different cell types and activation states

  • Solution: Carefully characterize receptor expression levels on target cells; include multiple cell types as controls; consider the activation state of cells

How can researchers accurately interpret contradictory results when studying IL2RA signaling?

When faced with contradictory results in IL2RA signaling studies, a systematic approach can help resolve discrepancies:

• Receptor Expression Level Differences:

  • IL2RA signaling outcomes depend heavily on the expression levels of all three receptor components

  • Quantify IL2RA, IL2RB, and IL2RG expression in your experimental system

  • Compare with published data - note that CD8+ T cells express more IL-2Rβ and IL-2Rγ than CD4+ T cells, affecting response thresholds

• Activation State Considerations:

  • Results may differ between resting and activated cells

  • H9-RET induces different functional outcomes in distinct cell subsets and activation states

  • Document and control for the activation status of cells

• Temporal Dynamics Analysis:

  • Create time-course experiments to capture the full signaling profile

  • Early vs. late signaling events may show different patterns

  • Use phospho-flow cytometry to track signaling kinetics at single-cell resolution

• Dose-Response Relationships:

  • IL-2 signaling is highly dose-dependent

  • Create full dose-response curves rather than single-dose experiments

  • Document the concentration and specific activity of IL-2 used

• Multi-parameter Analysis Approaches:

  • Measure multiple signaling outputs (pSTAT5, pS6, gene expression)

  • A partial agonist may affect some pathways but not others

  • For example, H9-RET lowered BCL6 expression while H9-RETR did not, despite similar effects on other parameters

What are emerging applications of IL2RA Human, sf9 in advanced immunotherapy research?

Emerging applications of IL2RA in immunotherapy research show significant promise across several frontiers:

• Engineered IL-2 Therapeutics Development:

  • Fine-tuned IL-2 partial agonists like H9-RET serve as "receptor-signaling clamps" that calibrate immune responses precisely

  • These engineered molecules allow titration of signaling amplitude to achieve desired biological outcomes

  • Potential applications in autoimmune diseases where complete blockade may be detrimental

• Cell-Type Selective Targeting:

  • Manipulating the binding properties of IL-2 to IL2RA creates therapeutics that selectively activate specific immune cell populations

  • Treg-selective IL-2 variants for autoimmune disease treatment

  • Effector T cell-selective variants for cancer immunotherapy

• Combination Immunotherapy Approaches:

  • IL2RA-targeted agents combined with checkpoint inhibitors show synergistic potential

  • Dual targeting of IL-2 and IL-15 pathways through IL2RA/IL2RB-focused approaches

  • Rational design of combination protocols based on mechanistic understanding

• Biomarker Development:

  • Soluble IL2RA as a diagnostic and prognostic marker in multiple sclerosis

  • IL2RA genetic variants as predictors of treatment response

  • Dynamic monitoring of IL2RA expression patterns to guide therapy selection

How might single-cell analysis technologies advance our understanding of IL2RA signaling heterogeneity?

Single-cell technologies offer unprecedented insights into IL2RA signaling heterogeneity that were previously masked in bulk population analyses:

• Receptor Expression Mapping:

  • Single-cell proteomics can quantify absolute copy numbers of IL2RA, IL2RB, and IL2RG on individual cells

  • This reveals how receptor stoichiometry influences signaling outcomes

  • Identification of previously unrecognized cell subpopulations with unique receptor expression patterns

• Signaling Network Reconstruction:

  • Single-cell phospho-proteomics can track multiple signaling nodes simultaneously

  • Reveals how IL2RA signaling branches into distinct pathways in different cells

  • Identification of critical nodes that determine cell fate decisions

• Transcriptional Response Profiling:

  • Single-cell RNA-seq captures the full spectrum of transcriptional responses to IL-2

  • Allows linking of specific gene expression programs to receptor levels and signaling intensity

  • Studies have shown that IL-2 and its variants induce or repress specific STAT5 target genes (IL2RA, LTA, CISH, IL7R, BCL6) with different potencies

• Spatial Context Analysis:

  • Imaging mass cytometry and spatial transcriptomics reveal how IL2RA signaling is influenced by cellular microenvironment

  • Particularly relevant in tissues where IL-2 availability may be limited and spatially regulated

  • Critical for understanding in vivo relevance of in vitro findings

• Trajectory Analysis:

  • Time-resolved single-cell data enables reconstruction of cellular response trajectories

  • Reveals how initial IL2RA signaling events propagate to determine cell fate

  • Important for understanding memory vs. effector T cell differentiation decisions

What statistical approaches are most appropriate for analyzing IL2RA expression data across different experimental conditions?

When analyzing IL2RA expression data, selecting appropriate statistical methods is crucial for valid biological interpretation:

For Flow Cytometry Data:

• Use median fluorescence intensity (MFI) rather than mean when distributions are non-normal

• Apply appropriate transformations (often logicle or arcsinh for flow data)

• For comparing multiple conditions:

  • ANOVA with post-hoc tests for normally distributed data

  • Kruskal-Wallis with Dunn's post-test for non-parametric data

• For bimodal expressions (positive/negative populations):

  • Report both percentage positive and MFI of positive population

  • Consider mixture modeling approaches for complex distributions

For Gene Expression Data:

• Normalization considerations:

  • Select appropriate housekeeping genes validated for stability in your experimental system

  • Consider geometric averaging of multiple reference genes

• For comparing fold changes across conditions:

  • Log-transform data prior to statistical analysis

  • Apply false discovery rate correction for multiple comparisons

• For time-course experiments:

  • Consider repeated measures ANOVA or mixed effects models

  • Functional data analysis for detailed temporal patterns

For Protein Quantification:

• Use calibration curves with recombinant standards for absolute quantification

• Include technical and biological replicates to estimate variability sources

• Consider measurement uncertainty in downstream analyses

Advanced Approaches:

• Multivariate analysis techniques:

  • Principal component analysis to identify major sources of variation

  • Partial least squares regression for correlating IL2RA with functional outcomes

• Bayesian approaches:

  • Particularly valuable when integrating prior knowledge with new data

  • Useful for small sample sizes common in complex IL2RA experiments

How can researchers effectively compare findings across different IL2RA experimental systems and model organisms?

Cross-system and cross-species comparisons of IL2RA research require careful consideration of multiple factors:

Standardization Approaches:

• Develop common reporting standards for:

  • Receptor quantification methods

  • Signaling readouts

  • Functional assays

• Use calibrated units where possible:

  • Absolute receptor numbers rather than relative expression

  • Standardized activity units for cytokines

Cross-Species Considerations:

• Account for species-specific differences in:

  • IL2RA structure (85% homology between human and mouse)

  • Expression patterns across cell types

  • Signaling thresholds and kinetics

• Perform parallel experiments in multiple species when possible

• Focus on conserved signaling nodes and outcomes

System Equivalence Assessment:

• For cell lines vs. primary cells:

  • Quantify receptor component ratios in both systems

  • Compare dose-response relationships

  • Validate key findings in primary cells

• For in vitro vs. in vivo models:

  • Consider microenvironmental factors absent in vitro

  • Validate with ex vivo analysis of in vivo treated samples

Data Integration Frameworks:

• Network-based approaches:

  • Map findings onto canonical IL-2 signaling networks

  • Identify conserved vs. system-specific network modules

• Multi-omics integration:

  • Correlate transcriptomic, proteomic, and functional data

  • Identify robust cross-platform biomarkers

Translational Considerations:

• Focus on mechanistic equivalence rather than exact parameter matching

• Consider pathway activation status rather than absolute levels

• Develop and validate translational assays that work across systems

How should experiments be designed to study the differential effects of IL2RA signaling on various T cell subsets?

Designing experiments to capture the diverse effects of IL2RA signaling across T cell subsets requires careful consideration of multiple factors:

Cell Isolation and Characterization:

• Use high-purity isolation techniques:

  • Fluorescence-activated cell sorting (FACS) for highest purity

  • Sequential magnetic separation for higher yields

• Comprehensive phenotyping of starting populations:

  • Surface markers: CD4/CD8, naïve/memory markers, activation markers

  • Critical to quantify baseline IL2RA (CD25), IL2RB (CD122), and IL2RG (CD132) expression

  • Consider intracellular markers like FOXP3 for Treg identification

Experimental Matrix Design:

• Cell type variables:

  • Include multiple T cell subsets in parallel (CD4+ Tconv, CD8+, Tregs)

  • Consider both resting and pre-activated states for each subset

• Stimulation variables:

  • Dose titration of IL-2 (at least 5-6 concentrations spanning 4 logs)

  • Include IL-2 variants with different receptor binding properties

  • Consider temporal aspects (acute vs. sustained signaling)

Multilevel Readout Strategy:

• Signaling readouts:

  • Phospho-flow cytometry for pSTAT5, pS6, and other signaling nodes

  • Western blotting for total protein changes

  • Consider multiplexed approaches to capture pathway cross-talk

• Transcriptional responses:

  • Key IL-2 responsive genes: IL2RA, CISH, BCL6, IL7R

  • Genome-wide approaches for unbiased assessment

• Functional outcomes:

  • Proliferation (CFSE dilution, Ki-67 expression)

  • Cytokine production profiles

  • Differentiation markers

  • Survival/apoptosis indicators

Control Considerations:

• Blocking controls:

  • Anti-IL-2 antibodies to block endogenous IL-2

  • JAK/STAT inhibitors as pathway controls

• Competing ligands:

  • IL-15 shares IL2RB and IL2RG components

  • Include IL-15 controls to assess pathway specificity

What advanced techniques can be used to monitor IL2RA receptor dynamics and trafficking in live cells?

Advanced imaging and molecular techniques provide powerful approaches to study IL2RA dynamics:

Single-Molecule Imaging Approaches:

• Total Internal Reflection Fluorescence (TIRF) microscopy:

  • Allows visualization of cell surface receptor movements with high signal-to-noise ratio

  • Can track individual IL2RA molecules to study diffusion properties

• Single-particle tracking (SPT):

  • Label IL2RA with quantum dots or small fluorophores

  • Track receptor dimerization by measuring co-diffusion with IL2RB and IL2RG

  • Can reveal membrane microdomains that regulate receptor clustering

Resonance Energy Transfer Techniques:

• Förster Resonance Energy Transfer (FRET):

  • Label IL2RA and IL2RB/IL2RG with compatible fluorophore pairs

  • Measures receptor proximity at nanometer scale

  • Can detect conformational changes upon ligand binding

• Bioluminescence Resonance Energy Transfer (BRET):

  • Alternative to FRET with lower background

  • Useful for detecting receptor interactions in living animals

Receptor Trafficking Visualization:

• pH-sensitive fluorophores:

  • Fluorophores like pHluorin that change brightness based on pH

  • Can distinguish surface IL2RA from internalized receptor in acidic endosomes

• Photoactivatable fluorescent proteins:

  • Activate fluorescence in specific cellular regions

  • Track receptor fate from specific membrane domains

Quantitative Endocytosis Assays:

• Antibody-based internalization assays:

  • Surface biotinylation followed by internalization

  • Flow cytometry with dual-color antibodies (one for total, one for surface)

• Recycling vs. degradation tracking:

  • Pulse-chase approaches with different color labels

  • Can determine receptor half-life and recycling rates

Advanced Molecular Approaches:

• CRISPR-based endogenous tagging:

  • Insert fluorescent tags into endogenous IL2RA locus

  • Maintains native expression levels and regulation

• Optogenetic control of receptor activation:

  • Light-inducible receptor dimerization systems

  • Allows precise spatial and temporal control of signaling