ITK Antibody

What is ITK and why are ITK antibodies important in immunological research?

ITK, also known as Interleukin-2 inducible T-cell kinase, is a member of the Tec family of kinases that includes Tec, Btk, Bmx, and Txk. It functions as a tyrosine kinase expressed primarily in T-cells and contains both SH2 and SH3 domains . ITK plays an indispensable role in the activation of phospholipase C and calcium-dependent signaling cascades following antigen receptor triggering .

ITK antibodies are crucial research tools because they enable the investigation of T cell development, activation, and function. Mice congenitally lacking ITK show profound defects in T cell development and signaling, highlighting its importance . Additionally, defects in ITK are associated with lymphoproliferative syndrome EBV-associated autosomal type 1 (LPSA1), and it plays a role in various T cell-mediated inflammatory disorders including atopic dermatitis, psoriasis, and allergic asthma . Recent research has also identified ITK as a potential therapeutic target in melanoma and other solid tumors resistant to immune checkpoint blockade therapy .

What applications are most reliable for ITK antibody use in experimental settings?

ITK antibodies have been validated for multiple experimental applications, with varying degrees of reliability depending on the specific antibody clone and experimental conditions. Common applications include:

• Western blotting: Used to detect ITK protein expression levels and assess phosphorylation status .

• Immunohistochemistry (IHC): Particularly useful for formalin-fixed paraffin-embedded tissue sections to visualize ITK expression patterns in tissues .

• Flow cytometry: Enables quantitative analysis of ITK expression in different cell populations .

• Immunoprecipitation: Critical for studying ITK interactions with other signaling molecules like SLP-76 .

• Microscopy: Including immunofluorescence techniques to examine ITK localization within cells .

When using ITK antibodies for IHC, high pH antigen retrieval methods have been recommended, with optimal antibody concentrations at or below 1 μg/mL . For maximum reliability, it is essential to carefully titrate the antibody for each specific experimental setup and include appropriate controls.

How should ITK phosphorylation be assessed in activated T cells?

Assessment of ITK phosphorylation is crucial for studying T cell activation. The recommended methodological approach involves:

• Cell stimulation: Activate T cells through T-cell receptor engagement, typically using anti-CD3 antibodies or peptide stimulation .

• Cell lysis: Prepare precleared lysates under conditions that preserve phosphorylation status.

• Immunoprecipitation: Use anti-ITK antibodies to pull down ITK protein from the lysates .

• SDS-PAGE and immunoblotting: Resolve the immunoprecipitated proteins and sequentially blot with phosphospecific anti-ITK Tyr 511 antibody followed by total anti-ITK antibody .

• Quantification: Analyze immunoblots by densitometry (e.g., using NIH Image J software) to calculate the percent ITK phosphorylation by dividing the pixel intensity of phosphorylated ITK by the pixel intensity of total ITK .

This approach allows for accurate determination of ITK activation status following various stimulation conditions or in the presence of inhibitors.

What are the critical differences between monoclonal and polyclonal ITK antibodies?

Both monoclonal and polyclonal ITK antibodies are available for research use, each with distinct characteristics:

Monoclonal ITK Antibodies:

• Recognize a single epitope on the ITK protein

• Example: The 2F12 clone recognizes human, mouse, and rat ITK

• Provide consistent lot-to-lot reproducibility

• Typically higher specificity but might be more sensitive to epitope masking

• Useful for applications requiring high specificity, such as distinguishing ITK from other Tec family kinases

• Often preferred for quantitative applications

Polyclonal ITK Antibodies:

• Recognize multiple epitopes on the ITK protein

• Example: Rabbit polyclonal antibodies like ab137359

• May provide stronger signals due to binding to multiple epitopes

• More tolerant to protein denaturation in some applications

• Can show batch-to-batch variation

• Particularly useful for applications where protein conformation may be altered

The choice between monoclonal and polyclonal antibodies should be based on the specific experimental requirements, with validation for the particular application being essential regardless of antibody type.

How can ITK antibodies be utilized to study T cell exhaustion in cancer immunotherapy?

T cell exhaustion is a critical barrier to effective cancer immunotherapy, and ITK antibodies provide valuable tools for investigating this phenomenon:

• Detection of exhaustion markers alongside ITK: Researchers can perform multi-parameter flow cytometry using ITK antibodies in combination with antibodies against exhaustion markers (PD-1, Lag3, Tigit, and Tim3) to correlate ITK expression or phosphorylation with exhaustion states .

• Analysis of ITK activity in exhausted T cells: Exhausted T cells exhibit constitutive ITK and PLC-γ1 phosphorylation, which can be detected using phospho-specific ITK antibodies in immunoblotting assays . This allows for assessment of baseline ITK activity in different T cell states.

• Evaluation of ITK inhibitor effects: When studying ITK inhibitors like BMS-509744, ITK antibodies can be used to confirm target engagement and monitor downstream effects on exhaustion marker expression and cytokine production .

• Intracellular cytokine staining: Following ITK inhibitor treatment, antibodies against IL-2 and TNF-α can be combined with ITK staining to assess functional restoration of exhausted T cells .

• Transcription factor analysis: ITK inhibition has been shown to downregulate the exhaustion-associated transcription factor TOX. ITK antibodies can be used alongside TOX and TCF1 antibodies to assess the molecular mechanisms of exhaustion reversal .

Recent research has demonstrated that intermittent ITK inhibition can significantly improve immune checkpoint blockade therapy in previously resistant solid tumors by directly reinvigorating exhausted cytotoxic T cells . This highlights the importance of ITK as a therapeutic target and the utility of ITK antibodies in studying these mechanisms.

What techniques can detect ITK-SLP-76 interactions in primary T cells?

The interaction between ITK and the adaptor protein SLP-76 is crucial for T cell activation. Several techniques using ITK antibodies can be employed to study this interaction:

• Co-immunoprecipitation (Co-IP): The most direct approach involves immunoprecipitating ITK from T cell lysates using anti-ITK antibodies, followed by immunoblotting for SLP-76 . This can be performed under various stimulation conditions to assess how TCR engagement affects the interaction.

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. Using primary antibodies against ITK and SLP-76 from different species, followed by species-specific secondary antibodies conjugated to complementary oligonucleotides, researchers can detect close proximity (<40 nm) through fluorescent signal generation.

• FRET (Fluorescence Resonance Energy Transfer): By labeling ITK and SLP-76 antibodies with compatible fluorophores, researchers can detect energy transfer between molecules in close proximity, indicating direct interaction.

• Microscopy-based co-localization: Immunofluorescence with antibodies against ITK and SLP-76 can demonstrate co-localization at the immunological synapse following T cell activation.

In vivo data have confirmed the significance of ITK-SLP-76 interaction for cytokine production . When analyzing these interactions, it's critical to consider the timing of stimulation, as these interactions are dynamic and can change rapidly during T cell activation.

What experimental approaches can distinguish ITK from other Tec family kinases in complex samples?

Distinguishing ITK from other Tec family members (Tec, Btk, Bmx, and Txk) is crucial for specificity in research. Several approaches can be employed:

• Antibody validation with knockout controls: Using samples from ITK knockout models or cells treated with ITK-specific siRNA/shRNA is essential to confirm antibody specificity . This approach helps ensure the antibody does not cross-react with other Tec family members.

• Two-dimensional gel electrophoresis: This technique can separate proteins based on both isoelectric point and molecular weight, allowing for better discrimination between similar kinases. Western blotting with ITK antibodies after 2D separation can provide increased specificity .

• Tissue-specific expression analysis: ITK is predominantly expressed in T cells, while other Tec family members show different expression patterns. Using ITK antibodies on tissues with known expression profiles can help distinguish specific staining.

• Phospho-specific detection: Using antibodies specific for ITK phosphorylation sites that differ from those in other Tec family members (such as Y511) can provide kinase-specific detection .

• Mass spectrometry validation: Following immunoprecipitation with ITK antibodies, mass spectrometry can confirm the identity of the captured protein and distinguish it from other Tec family members.

Researchers should select antibodies that have been specifically validated for discriminating between Tec family members and consider the use of multiple antibodies targeting different epitopes for confirmation of results.

How can ITK antibodies be employed to evaluate ITK inhibitor efficacy in preclinical models?

ITK inhibitors such as BI 10N and BMS-509744 are being investigated as potential therapeutics for various conditions, including cancer immunotherapy. ITK antibodies are essential tools for evaluating inhibitor efficacy:

• Target engagement assessment: Phospho-specific ITK antibodies can be used to quantify the reduction in ITK phosphorylation following inhibitor treatment, confirming on-target activity .

• Functional readouts: After inhibitor treatment, ITK antibodies can be used alongside functional markers to assess downstream effects:

  • Flow cytometry to measure changes in inhibitory receptor expression (PD-1, Lag3, Tigit, Tim3)

  • Intracellular cytokine staining to evaluate IL-2 and TNF-α production

  • Immunoblotting to assess changes in PLCγ1 phosphorylation

• Ex vivo analysis from treated animals: ITK antibodies can be used to analyze samples from inhibitor-treated animals to confirm in vivo target engagement and effects on T cell populations. This is particularly important when using intermittent dosing schedules, which have shown improved efficacy in some models .

• Combination therapy assessment: In studies combining ITK inhibitors with immune checkpoint blockade, ITK antibodies can help distinguish the mechanisms of each therapeutic component by analyzing ITK activity and downstream signaling .

Recent research has demonstrated that intermittent ITK inhibition significantly improved immune checkpoint blockade therapy in previously resistant solid tumors including melanoma, mesothelioma, and pancreatic cancer . The inhibition directly reinvigorated exhausted cytotoxic T cells by enhancing cytokine production, decreasing inhibitory receptor expression, and downregulating the transcription factor TOX .

What quality control measures are essential when validating new lots of ITK antibodies?

Rigorous quality control is critical when working with ITK antibodies to ensure experimental reproducibility:

• Purity assessment: Confirm antibody purity is greater than 90% as determined by SDS-PAGE, and aggregation is less than 10% as determined by HPLC .

• Cross-reactivity testing: Test new antibody lots against samples from multiple species if cross-reactivity is expected (e.g., human, mouse, and rat ITK for the 2F12 clone) .

• Functional validation in multiple applications: Validate each new lot in all intended applications (western blotting, flow cytometry, IHC, etc.) using positive control samples with known ITK expression .

• Titration optimization: Perform careful titration experiments to determine the optimal concentration for each application. For IHC, concentrations at or below 1 μg/mL have been recommended for some antibodies .

• Specificity controls: Include appropriate negative controls such as:

  • ITK-knockout or knockdown samples

  • Isotype control antibodies

  • Blocking peptide competition assays

  • Secondary-only controls

• Performance comparison with previous lots: Always run side-by-side comparisons with previously validated lots to ensure consistent performance in terms of signal intensity, background levels, and specificity.

• Reproducibility testing: Perform replicate experiments to ensure consistent results across multiple runs and different operators.

These quality control measures help ensure that experimental results remain reliable and comparable across studies, which is particularly important for longitudinal studies or when comparing results between laboratories.

What are the optimal fixation and antigen retrieval methods for ITK immunohistochemistry?

Successful ITK immunohistochemistry requires careful attention to fixation and antigen retrieval methods:

The Bond fully-automated slide staining system has been successfully used for single and dual immunofluorescence staining of ITK in cell line arrays and tissue microarrays .

How should ITK antibodies be used in multiparameter flow cytometry panels?

Incorporating ITK antibodies into multiparameter flow cytometry panels requires careful consideration of several factors:

• Surface vs. intracellular staining sequence: Since ITK is an intracellular protein, surface markers should be stained first, followed by fixation, permeabilization, and then ITK staining.

• Fixation and permeabilization: For optimal ITK detection, use:

  • Formaldehyde-based fixatives (2-4%) for 10-15 minutes at room temperature

  • Permeabilization with either saponin-based (for reversible permeabilization) or methanol-based (for more stringent permeabilization) reagents

• Fluorophore selection: Choose a bright fluorophore for ITK antibodies (PE, APC, or BV421) when expression levels might be low or variable. Consider the following when designing panels:

  • Avoid fluorophore combinations with significant spectral overlap

  • Place ITK in a channel with minimal compensation requirements if it's a key analyte

  • Consider using directly conjugated ITK antibodies to reduce background and simplify protocols

• Controls for phospho-ITK staining:

  • Positive controls: Stimulated T cells (anti-CD3/CD28 or PMA/ionomycin)

  • Negative controls: Unstimulated cells and isotype controls

  • Biological controls: ITK inhibitor-treated cells

• Panel design for T cell exhaustion studies: Combine ITK antibodies with:

  • T cell markers: CD3, CD4, CD8

  • Exhaustion markers: PD-1, LAG3, TIM3, TIGIT

  • Functional markers: Intracellular cytokines (IL-2, TNF-α)

  • Transcription factors: TOX, TCF1

• Compensation: Proper compensation is critical, especially in panels with multiple fluorochromes. Single-stained controls should be prepared with the same cells and under the same conditions as the experimental samples.

Anti-ITK flow cytometry antibody products are available from multiple suppliers and in various formats (conjugated and unconjugated), allowing flexibility in panel design .

What troubleshooting approaches are recommended for inconsistent ITK western blot results?

Western blotting for ITK can present several challenges. Here are methodological approaches to troubleshoot common issues:

• Weak or no signal:

  • Increase antibody concentration: Try using higher concentrations of primary antibody (within manufacturer's recommended range)

  • Extend incubation time: Overnight incubation at 4°C may improve signal

  • Enrich for ITK: Consider immunoprecipitation before western blotting to concentrate ITK protein

  • Check expression levels: Ensure the cell type being analyzed expresses sufficient ITK (T cells are recommended as positive controls)

  • Optimize lysis conditions: Use RIPA or NP-40 based lysis buffers with protease and phosphatase inhibitors

• High background:

  • Increase blocking: Use 5% BSA or milk in TBST for 1-2 hours at room temperature

  • Adjust antibody dilution: More dilute antibody solutions may reduce background

  • Increase washing steps: Add extra washing steps with TBST between antibody incubations

  • Filter secondary antibody: Pre-adsorb or filter secondary antibodies to reduce non-specific binding

• Multiple bands or unexpected band size:

  • Verify specificity: Use ITK knockout or knockdown samples as negative controls

  • Check for degradation: Ensure protease inhibitors are fresh and adequate

  • Assess post-translational modifications: ITK undergoes phosphorylation and other modifications that can alter migration

  • Consider isoforms: Verify if multiple bands represent known isoforms or splice variants

• Inconsistent phospho-ITK detection:

  • Maintain phosphorylation status: Use phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in all buffers

  • Control activation state: Compare with positive controls (stimulated T cells) and negative controls (ITK inhibitor-treated cells)

  • Optimize sample handling: Process samples quickly and keep them cold throughout

• Loading control considerations:

  • Use appropriate controls: GAPDH has been validated as an effective loading control for ITK western blots

  • Consider cell-type specific controls: For T cell-specific analysis, CD3 or Lck may be appropriate controls

For assessing ITK phosphorylation specifically, immunoprecipitation with anti-ITK antibody followed by immunoblotting with phospho-specific antibodies (e.g., anti-ITK Tyr 511) has proven effective .

How can ITK antibodies contribute to melanoma and solid tumor research?

ITK antibodies have emerging applications in cancer research, particularly for melanoma and other solid tumors:

• Expression profiling in tumors: ITK antibodies can be used to assess ITK expression in tumor samples through IHC or western blotting. Studies have shown that ITK promoter CpG sites are hypomethylated in melanomas compared to nevi, suggesting potential altered expression .

• Tumor microenvironment analysis: Multiparameter flow cytometry or multiplex IHC using ITK antibodies alongside immune cell markers can characterize T cell populations within the tumor microenvironment.

• Response prediction biomarkers: ITK expression or phosphorylation status might serve as potential biomarkers for predicting response to immunotherapy. This can be assessed using ITK antibodies in pre-treatment biopsies .

• ITK inhibitor development: ITK antibodies are essential tools for:

  • Target engagement studies with novel ITK inhibitors like BI 10N

  • Pharmacodynamic assessment of ITK inhibition in preclinical models

  • Correlation of ITK inhibition with functional T cell outcomes

• Combination therapy approaches: Recent research has demonstrated that intermittent ITK inhibition can significantly improve immune checkpoint blockade therapy in previously resistant solid tumors . ITK antibodies can help characterize:

  • Changes in tumor-infiltrating lymphocytes following combination therapy

  • Alterations in T cell exhaustion phenotypes

  • Effects on cytokine production and inhibitory receptor expression

Research has shown that ITK inhibition directly reinvigorates exhausted cytotoxic T cells by enhancing cytokine production, decreasing inhibitory receptor expression, and downregulating the transcription factor TOX . These findings highlight ITK as a promising therapeutic target for overcoming resistance to immune checkpoint blockade in solid tumors.

What role do ITK antibodies play in studying T cell-mediated inflammatory disorders?

ITK antibodies are valuable tools for investigating T cell-mediated inflammatory conditions, including atopic dermatitis, psoriasis, and allergic asthma :

• Tissue expression analysis: ITK antibodies enable assessment of ITK expression in affected tissues through:

  • Immunohistochemistry of skin biopsies from patients with atopic dermatitis or psoriasis

  • Analysis of bronchial biopsies from asthma patients

  • Comparative expression studies between affected and unaffected tissues

• T cell subset characterization: Flow cytometry with ITK antibodies can help identify:

  • Differential ITK expression or activation in Th1, Th2, and Th17 cells

  • Correlation between ITK activity and disease-specific T cell responses

  • Changes in ITK phosphorylation following allergen challenge

• Mechanistic studies: Co-immunoprecipitation with ITK antibodies can reveal:

  • Disease-specific alterations in ITK-associated signaling complexes

  • Changes in ITK interaction partners in different inflammatory conditions

  • Potential therapeutic targets within the ITK signaling pathway

• Therapeutic monitoring: In clinical trials of ITK inhibitors for inflammatory conditions, ITK antibodies are essential for:

  • Confirming target engagement in patient samples

  • Monitoring changes in ITK activity during treatment

  • Correlating ITK inhibition with clinical response

• Genetic variant characterization: For patients with ITK mutations or polymorphisms, ITK antibodies can help determine:

  • Expression levels of variant ITK proteins

  • Functional consequences on T cell activation and cytokine production

  • Potential mechanisms linking genetic variants to disease susceptibility

These applications highlight the importance of ITK antibodies not only in basic research but also in translational studies aimed at developing new therapies for T cell-mediated inflammatory disorders.

ITK Antibody Performance Comparison Across Applications

Effect of ITK Inhibition on T Cell Exhaustion Markers

ITK Antibody Validation Methods