NCR3LG1 Antibody

Basic Research Questions

• What is NCR3LG1 and what is its significance in immunology?

  NCR3LG1 (Natural Cytotoxicity Triggering Receptor 3 Ligand 1), also known as B7-H6 or B7H6, is a member of the B7 family of immune receptors that plays a crucial role in immune response modulation. It has been identified as a tumor-associated antigen, particularly in gastrointestinal tumors, and functions primarily by regulating natural killer (NK) cell-mediated cytotoxicity . B7-H6 is involved in immune homeostasis and has implications for cancer research, autoimmune disorders, and chronic inflammatory diseases . Research methodologies for studying NCR3LG1 typically involve expression analysis in different cell types, functional assays to assess NK cell activation, and investigation of its potential as a therapeutic target in cancer immunotherapy approaches.

• What applications are NCR3LG1 antibodies commonly used for in research settings?

  NCR3LG1 antibodies are versatile research tools with multiple validated applications:

  • Western Blotting: For protein detection showing bands at approximately 51 kDa (full-length protein) with potential additional bands at 38 kDa and 32 kDa representing different isoforms

  • ELISA: For quantitative detection of NCR3LG1 in various sample types including serum and cell culture supernatants

  • Immunohistochemistry (IHC): For tissue localization studies with recommended dilutions of 1:20-1:200

  • Flow Cytometry: For cell surface expression analysis, particularly useful for detecting B7-H6 on cell lines and primary tumors

  • Immunoprecipitation: For protein-protein interaction studies

  Researchers should optimize antibody dilutions based on specific applications and consider appropriate detection systems based on the host species of the primary antibody. For Western blot applications, dilutions of 1:1000-1:5000 are typically recommended .

• How can I evaluate the efficacy of B7-H6/NCR3LG1-targeted therapeutic approaches?

  Evaluating B7-H6-targeted therapeutic approaches requires a multi-parameter assessment strategy:

  For in vitro evaluation:

  • Cytotoxicity assays: Measure lactate dehydrogenase concentrations in cell culture supernatants to assess cell lysis

  • T-cell activation: Analyze expression of activation markers (CD25, CD69) and effector molecules (granzyme B, CD107a) by flow cytometry

  • T-cell proliferation: Label PBMCs with CFDA-SE and stain with anti-CD3 antibodies to track division rates

  • Cytokine secretion: Quantify cytokine production using multiplex assays (e.g., V-PLEX Assays)

  For in vivo evaluation:

  • Mouse xenograft models: Utilize PBMC-humanized NSG or T cell-humanized NOG mice bearing B7-H6-positive tumors (e.g., HCT-15, NCI-H716)

  • Combination strategies: Test with checkpoint inhibitors (e.g., anti-PD-1) as data suggests enhanced activity with this combination

  • Tumor measurements: Monitor tumor regression, T-cell infiltration, and inflammatory markers in the tumor microenvironment

  Both monotherapy and combination approaches should be evaluated to determine optimal therapeutic strategies.

Advanced Research Questions

• How can I validate the specificity of NCR3LG1 antibodies in my experimental system?

  Validating antibody specificity requires a multi-faceted approach:

  • Positive and negative controls: Use cell lines with known expression patterns. BA/F-3-B7-H6 transfectants can serve as positive controls for B7-H6 detection

  • Genetic validation: Employ siRNA knockdown or CRISPR-Cas9 knockout of NCR3LG1 to confirm reduced antibody binding, as demonstrated in studies using genetic editing of NCR3LG1 on CAR T cells

  • Peptide blocking: Pre-incubate the antibody with purified recombinant NCR3LG1 protein before application to your sample

  • Cross-platform validation: Confirm expression using multiple detection methods

  • Western blot validation: Verify band patterns match expected molecular weights (51 kDa for full-length protein, with potential additional bands at 38 and 32 kDa)

  Perform dose-dependent studies with varying antigen concentrations to establish detection limits and linear range, as demonstrated in Western blot analyses using 0.1-0.6 μg of reducing antigen .

• What are the optimal conditions for using NCR3LG1 antibodies in multiparametric immune cell functional assays?

  For optimal results in multiparametric immune assays:

  • Sample preparation: Isolate PBMCs or specific immune cell populations using density gradient separation or magnetic sorting

  • Co-culture systems: For cytotoxicity assays, co-culture target cells with effector cells at various ratios (typically 5:1 to 20:1)

  • Staining panels for flow cytometry:

    • T-cell activation: Anti-CD25-APC, anti-CD69-PE-Cy7

    • Degranulation: Anti-CD107a-BV421

    • Effector molecules: Anti-GrzB-FITC (intracellular staining)

    • Proliferation: CFDA-SE labeling with anti-CD3 counterstaining

  • Blocking experiments: Use anti-B7-H6 antibodies at 1-10 μg/mL to assess functional relevance

  • Controls: Include isotype controls and single-stained compensation controls

  • Timing: Perform activation assays for 4-6 hours, proliferation assays for 48-72 hours

  Analyze results using multi-parameter flow cytometry software capable of dimensionality reduction for complex datasets.

• How do I optimize the use of NCR3LG1 antibodies for investigating B7-H6 expression in tumor microenvironment?

  Optimizing NCR3LG1 antibody use for tumor microenvironment studies requires:

  • Tissue processing: Use optimal fixation (10% neutral buffered formalin) and antigen retrieval methods

  • Antibody selection: Choose antibodies validated for IHC/IF applications, such as those that can detect B7-H6 on cytospins (frozen sections) of B7-H6 expressing cells

  • Multiplex staining: Combine B7-H6 detection with markers for:

    • Tumor cells (e.g., cytokeratins, tumor-specific markers)

    • Immune infiltrates (e.g., CD3, CD8, CD56)

    • Checkpoint molecules (e.g., PD-L1)

  • Signal amplification: Consider tyramide signal amplification for low-abundance antigens

  • Quantification: Use digital pathology and computational analysis to:

    • Quantify B7-H6 expression levels

    • Assess spatial relationships between B7-H6+ cells and immune infiltrates

    • Correlate with clinical outcomes

  Include appropriate controls including B7-H6 positive and negative tissues, and antibody absorption controls to validate staining specificity.

• What considerations are important when developing B7-H6-targeted bispecific antibodies for cancer immunotherapy?

  Developing B7-H6-targeted bispecific antibodies requires attention to several critical factors:

  • Format selection: IgG-like T-cell engagers (ITEs) have shown promising preclinical results for B7-H6 targeting

  • Epitope selection: Target the extracellular domain of B7-H6 for optimal accessibility

  • Affinity balancing: Optimize binding affinity for both B7-H6 and effector cell receptors (e.g., CD3)

  • Functional validation assays:

    • B7-H6-dependent tumor cell lysis

    • T-cell infiltration into tumor tissue

    • Inflammatory marker induction in tumor microenvironment

    • Tumor regression in xenograft models

  • Potential enhancements: Combination with checkpoint inhibitors (anti-PD-1) has demonstrated enhanced activity and creation of an inflamed tumor environment

  • Safety considerations: Monitor for cytokine release syndrome and off-target effects

  Comprehensive in vivo testing should include both monotherapy and combination approaches in appropriate mouse models with humanized immune components.

• How can I design experiments to investigate the mechanisms of B7-H6 shedding from tumor cells?

  To investigate B7-H6 shedding mechanisms:

  • Detection systems: Use the validated ELISA kits for quantifying soluble B7-H6 in culture supernatants

  • Baseline characterization: Compare B7-H6 surface expression (by flow cytometry) with soluble B7-H6 levels across different tumor cell lines

  • Shedding modulators: Test the effects of:

    • Metalloprotease inhibitors (e.g., TIMP-1, TIMP-2, GM6001)

    • Specific ADAM inhibitors

    • Inflammatory cytokines (TNF-α, IL-1β)

    • Hypoxic conditions

  • Kinetic analysis: Perform time-course experiments to determine shedding dynamics

  • Functional consequences: Assess how soluble B7-H6 affects:

    • NK cell receptor (NKp30) downregulation

    • NK cell cytotoxicity against B7-H6+ targets

    • Immune evasion in co-culture systems

  • Molecular identification: Use mass spectrometry to characterize cleaved B7-H6 fragments and identify precise cleavage sites

  These approaches will help determine whether shedding represents a tumor immune evasion mechanism and identify potential therapeutic intervention points.

• What are the best approaches for generating and characterizing high-affinity recombinant antibodies against NCR3LG1?

  For generating high-quality recombinant anti-NCR3LG1 antibodies:

  • Antibody format selection: Consider various formats including scFv fragments which have demonstrated efficacy in multiple applications (ELISA, FC, IHC)

  • Expression systems: Utilize animal-free production systems for consistent quality and sustainability

  • Quality control parameters:

    • Increased sensitivity

    • Confirmed specificity

    • High repeatability

    • Excellent batch-to-batch consistency

    • Sustainable supply

  • Validation requirements:

    • ELISA testing against purified NCR3LG1 protein

    • Western blot analysis with concentration gradients of reducing antigen (0.1-0.6 μg)

    • Flow cytometry validation on positive cell lines

    • Functional testing including blocking capacity where relevant

  • Application optimization: Different antibody clones may have distinct optimal applications (e.g., some primarily for ELISA, others for blocking or immunoprecipitation)

  Document all characterization data comprehensively to support reproducible research applications.

• How can I investigate the role of B7-H6 in T cell regulation and potential T cell immunosurveillance mechanisms?

  Recent findings suggest B7-H6 subjects activated T cells to NK cell-mediated surveillance . To investigate this phenomenon:

  • Expression analysis: Characterize physiological expression of B7-H6 on T cells following activation with various stimuli

  • Regulatory mechanisms: Determine factors controlling B7-H6 upregulation on T cells

  • Functional consequences: Examine:

    • NK cell recognition of B7-H6+ T cells

    • Cytotoxicity assays with activated T cells as targets

    • Impact on T cell responses in inflammatory settings

  • Intervention approaches:

    • Antibody blockade of B7-H6/NKp30 interaction

    • Genetic editing of NCR3LG1 on T cells or CAR T cells

  • In vivo relevance: Assess how B7-H6/NKp30 interactions affect:

    • T cell homeostasis

    • Immune responses to infection or vaccination

    • Autoimmunity models

    • CAR T cell persistence and function

  This research direction may reveal important immunoregulatory mechanisms and inform strategies to enhance T cell-based immunotherapies by preventing unwanted NK-mediated elimination.