TSLPR Antibody

What is TSLPR and how does it function in immune signaling?

TSLPR (Thymic Stromal Lymphopoietin Receptor) is an approximately 50 kDa transmembrane protein with significant structural similarity to the common gamma-chain. TSLPR forms a heteromeric complex with IL-7Rα (CD127) to create the high-affinity receptor for thymic stromal lymphopoietin (TSLP) . This receptor complex is primarily expressed on monocytes and CD11c+ dendritic cells in humans .

When TSLP binds to this receptor complex, it activates signaling pathways that induce the expression of Th2 cell-attracting chemokines like CCL17 and CCL22. Additionally, TSLPR-mediated activation of dendritic cells indirectly enhances the secretion of Th2 cytokines (IL-4, IL-5, and IL-13), which play crucial roles in allergic inflammation and CD4+ T cell homeostasis . The TSLP-TSLPR axis represents a master regulatory pathway in allergic inflammation at barrier surfaces including the lung, skin, and gut .

What are the key differences between TSLP and TSLPR antibodies in research applications?

Anti-TSLP antibodies target the cytokine itself (thymic stromal lymphopoietin), while anti-TSLPR antibodies target its receptor. Anti-TSLP antibodies are primarily used to neutralize TSLP activity by preventing its binding to the receptor complex, making them valuable for both research and therapeutic applications . In contrast, anti-TSLPR antibodies are predominantly used for detection of receptor expression and blocking receptor-ligand interactions in experimental settings .

Anti-TSLP antibodies like TAVO101 and tezepelumab have demonstrated potent neutralization of TSLP activities in various in vitro, ex vivo, and in vivo models, with applications extending to potential treatments for TSLP-mediated diseases . Anti-TSLPR antibodies such as eBio1A6 are more commonly used for research applications including flow cytometry and immunoblotting to study receptor expression patterns and interactions .

How are human TSLPR expression patterns characterized across different cell types?

Human TSLPR expression is primarily characterized using flow cytometry, immunohistochemistry, and western blotting techniques with validated anti-TSLPR antibodies. Flow cytometry is particularly valuable for quantifying TSLPR expression on intact cells, with antibodies such as eBio1A6 (clone 1A6) being effective at concentrations of ≤0.5 μg per test when analyzing TSLPR-transfected cell lines .

In humans, TSLPR is predominantly expressed on monocytes and CD11c+ dendritic cells, where it plays a crucial role in allergic and inflammatory responses . For western blot characterization, anti-TSLPR antibodies can detect the receptor at approximately 45-60 kDa under reducing conditions, as demonstrated with NS0 mouse myeloma cell lines transfected with human TSLPR . For comprehensive expression profiling, researchers typically employ a combination of protein and mRNA detection methods, including RT-PCR analysis to complement antibody-based detection systems.

What validation steps should be performed before using a new TSLPR antibody?

Before incorporating a new TSLPR antibody into experimental protocols, researchers should conduct comprehensive validation through multiple approaches:

• Specificity testing: Compare antibody binding in TSLPR-expressing versus non-expressing cells (e.g., TSLPR-transfected versus mock-transfected cells) . Western blot analysis should show specific bands at the expected molecular weight of 45-60 kDa for human TSLPR .

• Cross-reactivity assessment: Test the antibody against TSLPR from different species if cross-species applications are planned. For example, antibodies like TAVO101 may not bind to cynomolgus monkey or mouse TSLP, limiting their use in certain animal models .

• Functional validation: For neutralizing antibodies, confirm their ability to block TSLP-TSLPR interactions through competition assays or functional reporter systems like STAT5 activation assays .

• Application-specific optimization: Determine optimal antibody concentrations for specific applications (e.g., ≤0.5 μg per test for flow cytometry) . Titration experiments across a range of concentrations are essential for identifying optimal signal-to-noise ratios.

• Positive and negative controls: Include properly validated positive controls (TSLPR-expressing cells) and negative controls (blocking peptides or isotype controls) in all experiments.

How should researchers design experiments to study TSLP-TSLPR signaling mechanisms?

To effectively study TSLP-TSLPR signaling mechanisms, researchers should implement multi-faceted experimental approaches:

• Reporter gene assays: Utilize STAT5 luciferase reporter systems in cells co-transfected with IL-7Rα and TSLPR expression constructs. This system can quantitatively measure TSLP-induced signaling and evaluate the neutralizing capacity of anti-TSLP antibodies .

• Cell proliferation assays: Employ BaF3 cells (mouse pro-B cells) transfected with human IL-7Rα and TSLPR to assess TSLP-driven proliferation. Measure cell growth using resazurin-based fluorescence detection with excitation at 544 nm and emission at 590 nm .

• Chemokine production assays: Investigate TSLP-induced release of CCL17 from primary human CD1c+ dendritic cells as a functional readout of receptor activation .

• Signal transduction analysis: Monitor TSLP-mediated phosphorylation of downstream signaling molecules (particularly STAT5) through western blotting or flow cytometry with phospho-specific antibodies.

• Receptor complex formation studies: Use co-immunoprecipitation with anti-TSLPR antibodies to isolate and analyze the composition of the receptor complex (TSLPR and IL-7Rα) under various stimulation conditions.

What is the optimal protocol for flow cytometric detection of TSLPR using monoclonal antibodies?

For optimal flow cytometric detection of TSLPR, researchers should follow this methodological approach:

• Cell preparation:

  • Harvest cells (primary cells or cell lines expressing TSLPR)

  • Wash cells 2-3 times in flow cytometry buffer (PBS with 2% FBS and 0.1% sodium azide)

  • Adjust cell concentration to 1-5 × 10^6 cells/mL

• Antibody staining:

  • Aliquot 100 μL of cell suspension (1-5 × 10^5 cells) per test tube

  • Add ≤0.5 μg of anti-TSLPR antibody (such as eBio1A6) per test

  • Include appropriate isotype control antibody in separate tubes

  • Incubate for 30 minutes at 4°C in the dark

• Washing and secondary detection (if using unconjugated primary antibody):

  • Wash cells twice with 2 mL flow cytometry buffer

  • Add fluorochrome-conjugated secondary antibody

  • Incubate for 30 minutes at 4°C in the dark

  • Wash twice with flow cytometry buffer

• Analysis:

  • Resuspend cells in 200-500 μL of flow cytometry buffer

  • Analyze on flow cytometer with appropriate laser and filter configuration

  • Gate on viable cells using appropriate exclusion dyes

  • Compare TSLPR antibody staining to isotype control to determine positive populations

• Controls and validation:

  • Include TSLPR-transfected cells (e.g., BaF3 cells expressing human TSLPR) as positive controls

  • Use blocking experiments with recombinant TSLP to confirm specificity

How can researchers quantitatively assess TSLP-TSLPR neutralization in functional assays?

Quantitative assessment of TSLP-TSLPR neutralization requires rigorous functional assays with clear readouts and proper controls:

• STAT5 reporter gene assay methodology:

  • Transfect HEK293T cells with constructs expressing IL7Rα, TSLPR, and pGL4.52 STAT5 luciferase reporter

  • Plate 10,000 cells/well in 96-well white flat-bottom plates

  • Treat with serial dilutions of TSLP (3-fold, starting at 100 ng/mL)

  • For neutralization assays, add antibody dilutions (3-fold, starting at 10 μg/mL) with a fixed TSLP concentration (3 ng/mL)

  • After 24 hours, measure luciferase activity using ONE-Glo EX Luciferase Assay kit

  • Calculate IC50 values using non-linear regression analysis

• Cell proliferation neutralization assay:

  • Use BaF3 cells co-transfected with human IL-7Rα and TSLPR

  • Wash cells three times with RPMI medium

  • Seed 5,000 cells per well in 96-well plates

  • Add 0.5 ng/mL hTSLP with serial dilutions of neutralizing antibodies (3-fold, starting at 1 μg/mL)

  • Incubate for 2 days at 37°C, 5% CO2

  • Add 10 μL/well of 0.1 mg/mL resazurin solution and incubate for 16-20 hours

  • Measure fluorescence (excitation 544 nm, emission 590 nm)

  • Calculate IC50 values using non-linear regression analysis

The following table summarizes key parameters for quantitative neutralization assays:

What troubleshooting strategies should be employed when TSLPR antibodies show inconsistent results?

When encountering inconsistent results with TSLPR antibodies, researchers should systematically troubleshoot using these methodological approaches:

• Antibody quality assessment:

  • Verify antibody purity (should be >90% by SDS-PAGE)

  • Check for aggregation (should be <10% as determined by HPLC)

  • Confirm proper storage conditions and avoid freeze-thaw cycles

• Technical optimizations:

  • Titrate antibody concentration across a broader range

  • Modify incubation conditions (time, temperature, buffer composition)

  • For western blotting, test different sample preparation methods (reducing vs. non-reducing conditions)

  • For flow cytometry, optimize fixation and permeabilization protocols if detecting intracellular epitopes

• Cell system validation:

  • Confirm TSLPR expression levels in your cell system using RT-PCR

  • For transfected cells, verify expression by multiple methods (flow cytometry, western blot, RT-PCR)

  • Monitor cell culture conditions that might affect receptor expression

• Antibody epitope considerations:

  • Determine if post-translational modifications (like glycosylation) affect antibody binding

  • Consider using antibodies recognizing different epitopes

  • For neutralizing antibodies, confirm the epitope location relative to receptor binding sites

• Species cross-reactivity issues:

  • Verify antibody specificity for the species being studied

  • Note that antibodies like TAVO101 may not cross-react with cynomolgus monkey or mouse TSLP

How can researchers distinguish between the different isoforms of TSLPR in experimental systems?

Distinguishing between TSLPR isoforms requires specialized approaches targeting isoform-specific features:

• Western blot analysis with isoform resolution:

  • Use gradient gels (4-15% or 4-20%) to achieve better separation of closely related isoforms

  • Employ reducing conditions to maximize size differences

  • Look for TSLPR-specific bands in the 45-60 kDa range

  • Compare band patterns between different cell types known to express specific isoforms

• RT-PCR with isoform-specific primers:

  • Design primers that span isoform-specific exon junctions

  • Implement quantitative RT-PCR to measure relative expression levels of different isoforms

  • Validate PCR products by sequencing to confirm isoform identity

• Isoform-specific antibody selection:

  • Choose antibodies raised against epitopes unique to specific isoforms

  • Validate antibody specificity using cells transfected with individual isoform constructs

  • Compare staining patterns across tissue types with known differential isoform expression

• Functional discrimination:

  • Assess differential signaling outcomes of various isoforms

  • Compare STAT5 activation profiles and downstream gene expression patterns

  • Examine differences in receptor internalization and trafficking

• Mass spectrometry analysis:

  • Immunoprecipitate TSLPR using validated antibodies

  • Perform tryptic digestion and analyze peptide fragments by mass spectrometry

  • Identify isoform-specific peptide sequences to confirm isoform identity

How are TSLPR antibodies utilized in animal models of allergic and inflammatory diseases?

TSLPR antibodies serve as valuable tools in animal disease models, with several methodological approaches:

• Model selection and development:

  • Use humanized mice (hTSLP/hTSLPR double knock-in mice) to evaluate human-specific TSLP antibodies

  • Employ established models such as TSLP/OVA-induced asthma and imiquimod-induced psoriasis models

  • Consider timing of antibody administration (prophylactic vs. therapeutic)

• Dosing and administration protocols:

  • Administer antibodies via appropriate routes (intraperitoneal, subcutaneous, or intravenous)

  • Establish appropriate dosing schedules based on antibody pharmacokinetics

  • Monitor serum antibody levels throughout the study duration

• Efficacy assessment parameters:

  • Measure inflammatory markers in serum and tissue

  • Quantify immune cell infiltration through histopathology and flow cytometry

  • Evaluate physiological parameters specific to the disease model (e.g., airway hyperresponsiveness in asthma models)

  • Assess tissue-specific changes through histological scoring systems

• Mechanistic investigations:

  • Analyze changes in cytokine and chemokine profiles

  • Characterize dendritic cell activation states

  • Monitor Th2 cell responses and associated cytokines (IL-4, IL-5, IL-13)

  • Evaluate alterations in tissue remodeling and repair mechanisms

What are the key considerations when developing and optimizing anti-TSLPR antibodies for translational research?

Developing anti-TSLPR antibodies for translational research requires careful optimization across multiple parameters:

• Antibody engineering strategies:

  • Humanize mouse-derived antibodies to reduce immunogenicity in human studies

  • Optimize CDR regions while preserving binding affinity

  • Consider Fc engineering to modify antibody properties:

    • Reduce Fcγ receptor binding for improved safety profile

    • Enhance FcRn binding for extended half-life

• Binding characteristics optimization:

  • Achieve high-affinity binding (nanomolar to sub-nanomolar range)

  • Verify binding to relevant epitopes that effectively block receptor-ligand interactions

  • Test binding under physiologically relevant conditions

• Functional validation requirements:

  • Confirm potent neutralization in cell-based assays

  • Demonstrate efficacy in ex vivo human cell systems

  • Validate in appropriate animal models with humanized targets

• Pharmacokinetic considerations:

  • Assess circulating half-life through in vivo studies

  • Evaluate tissue distribution patterns

  • Compare pharmacokinetic profiles to existing therapeutic antibodies like tezepelumab

• Manufacturing and stability parameters:

  • Ensure consistent glycosylation patterns

  • Optimize formulation for long-term stability

  • Develop reliable production methods with high yields

How do TSLPR-targeting therapeutic antibodies differ from TSLP-targeting approaches in clinical development?

The strategic differences between TSLPR-targeting and TSLP-targeting therapeutic approaches include:

• Target accessibility and distribution:

  • TSLP is a secreted cytokine present in circulation and tissues, making it readily accessible to antibodies

  • TSLPR is a membrane-bound receptor with expression limited to specific cell types, potentially offering more selective targeting

• Mechanism of action differences:

  • Anti-TSLP antibodies (like tezepelumab and TAVO101) neutralize the cytokine before receptor binding, blocking signaling at the ligand level

  • Anti-TSLPR antibodies block receptor function, potentially affecting both ligand-dependent and independent functions

• Clinical development status:

  • TSLP-targeting approaches are more advanced, with tezepelumab approved for severe asthma treatment

  • TSLP-targeting antibodies like TAVO101 show promising features compared to tezepelumab:

    • Recognize different epitopes

    • Demonstrate similar potency in neutralizing TSLP activities

    • Engineered for longer circulating half-life

• Safety and selectivity profiles:

  • TSLP-targeting approaches affect all TSLP-responsive cells

  • TSLPR-targeting might offer more selective inhibition based on receptor expression patterns

  • Fc engineering to reduce Fcγ receptor binding can improve safety profiles of both approaches

• Clinical application considerations:

  • Prolonged treatment is typically required due to unpredictable allergen exposure

  • Extended half-life antibodies may improve patient compliance and convenience

  • Combination approaches targeting both ligand and receptor might provide synergistic effects in resistant cases

What are the optimal conditions for using TSLPR antibodies in Western blotting applications?

For optimal Western blotting with TSLPR antibodies, researchers should follow these methodological guidelines:

• Sample preparation:

  • Lyse cells in appropriate buffer (RIPA buffer with protease inhibitors)

  • For membrane proteins like TSLPR, include 1% NP-40 or Triton X-100 in lysis buffer

  • Determine protein concentration using BCA or Bradford assay

  • Prepare samples under reducing conditions for best detection of TSLPR

• Gel electrophoresis parameters:

  • Load 20-30 μg total protein per lane

  • Use 10-12% SDS-PAGE gels for optimal resolution of TSLPR (45-60 kDa)

  • Include positive controls (TSLPR-transfected cells) and negative controls (mock-transfected cells)

• Transfer and blocking conditions:

  • Transfer to PVDF membrane (preferred over nitrocellulose for TSLPR)

  • Block with 5% non-fat dry milk or 3% BSA in TBST for 1 hour at room temperature

• Antibody incubation protocol:

  • Dilute primary anti-TSLPR antibody to 0.5-1.0 μg/mL in blocking buffer

  • Incubate overnight at 4°C with gentle rocking

  • Wash 3-4 times with TBST (5 minutes each)

  • Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Wash 4-5 times with TBST (5 minutes each)

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) detection reagents

  • Expose membrane to film or use digital imaging system

  • Expect TSLPR-specific bands at approximately 45-60 kDa

  • Multiple bands may represent different glycosylation states

How can researchers optimize ELISA protocols for measuring TSLP-TSLPR interactions?

Optimizing ELISA protocols for TSLP-TSLPR interaction studies requires careful attention to technical details:

• Direct binding ELISA setup:

  • Coat high-binding ELISA plates with recombinant human TSLP (2 μg/mL in PBS) overnight at 4°C

  • Block with 3% BSA in PBS-T for 1-2 hours at room temperature

  • Add serial dilutions of anti-TSLP antibodies (starting at 1-10 μg/mL)

  • Calculate EC50 values for binding (TAVO101: 29.06 ng/mL; tezepelumab: 63.12 ng/mL)

• Competitive ELISA methodology:

  • Coat plates with recombinant TSLPR-Fc fusion protein (1-2 μg/mL)

  • Block with 3% BSA in PBS-T

  • Pre-incubate biotinylated TSLP with serial dilutions of neutralizing antibodies

  • Add the mixture to the plate and detect with streptavidin-HRP

  • Calculate IC50 values for inhibition of TSLP-TSLPR binding

• Sandwich ELISA for TSLP detection:

  • Coat plates with capture anti-TSLP antibody (2 μg/mL)

  • Add samples containing TSLP

  • Detect with biotinylated detection anti-TSLP antibody targeting a different epitope

  • Use streptavidin-HRP and appropriate substrate for colorimetric detection

  • Include standard curve with recombinant TSLP (0.1-1000 pg/mL)

• Critical assay parameters:

  • Optimize antibody concentrations through checkerboard titration

  • Determine optimal incubation times and temperatures

  • Select appropriate detection system based on sensitivity requirements

  • Include controls for non-specific binding

What are the best approaches for multiplex analysis of TSLPR signaling pathways?

For comprehensive multiplex analysis of TSLPR signaling pathways, researchers should implement these methodological strategies:

• Phospho-flow cytometry protocol:

  • Stimulate cells with TSLP (10-50 ng/mL) for various timepoints (5-60 minutes)

  • Fix cells with 1.6% paraformaldehyde for 10 minutes at room temperature

  • Permeabilize with ice-cold methanol for 30 minutes

  • Stain with fluorochrome-conjugated antibodies against:

    • Phospho-STAT5 (primary TSLPR signaling pathway)

    • Phospho-JAK1/JAK2

    • Phospho-ERK1/2

    • Phospho-AKT

  • Analyze by flow cytometry with appropriate compensation controls

• Multiplex bead-based immunoassays:

  • Use Luminex or similar bead-based technology

  • Measure multiple phosphoproteins simultaneously from cell lysates

  • Quantify downstream cytokines and chemokines (IL-4, IL-5, IL-13, CCL17, CCL22)

  • Compare signaling profiles between different cell types and conditions

• RNA-seq/transcriptomic analysis:

  • Stimulate cells with TSLP (10 ng/mL) for 6-24 hours

  • Extract total RNA and prepare libraries for RNA-seq

  • Identify differentially expressed genes in response to TSLP

  • Perform pathway enrichment analysis to map activated signaling networks

  • Compare effects of TSLPR antibody blockade on gene expression profiles

• Protein array technology:

  • Use reverse-phase protein arrays (RPPA) to analyze multiple signaling nodes

  • Compare phosphorylation patterns before and after TSLP stimulation

  • Assess effects of TSLPR antibody blockade on signaling networks

  • Identify novel components of the TSLPR signaling pathway

• Multiparameter imaging approaches:

  • Implement multiplexed immunofluorescence to visualize signaling components

  • Use spectral imaging to overcome fluorophore limitations

  • Analyze co-localization of TSLPR with signaling components

  • Quantify nuclear translocation of transcription factors like STAT5