CXE6 Antibody

What is CXCR6 and what cell types express this receptor?

CXCR6 (also known as Bonzo and STRL33) is a G protein-coupled chemokine receptor that plays significant roles in immune cell trafficking and function. The receptor is predominantly expressed on specific immune cell populations including activated T cells, helper T type 1 (Th1) cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), and various cell types within the tumor microenvironment (TME) .

Expression patterns differ between resting and activated states. For example, research shows that IL-2 stimulation significantly increases CXCR6 expression on human peripheral blood mononuclear cells (PBMCs), particularly on CD3+ T cells, as demonstrated through flow cytometry analysis . This differential expression makes CXCR6 a valuable marker for studying activated immune populations in various disease contexts.

What experimental applications are CXCR6 antibodies commonly used for?

CXCR6 antibodies serve multiple experimental applications in immunology and oncology research:

Each application requires specific optimization protocols and considerations for accurate results and interpretation .

How can I distinguish between human and mouse CXCR6 antibodies in my experimental design?

Species-specific CXCR6 antibodies are critical for accurate research outcomes. Human and mouse CXCR6 antibodies are developed through different immunization strategies and recognize distinct epitopes:

Human CXCR6 antibodies (such as clones 56811 and 1010910) are typically generated using human CXCR6-transfected cell lines as immunogens and recognize the full-length protein (Met1-Leu342, Accession # O00574) .

In contrast, mouse-specific antibodies like Cx6Mab-1 (rat IgG1, kappa) have been developed through N-terminal peptide immunization strategies in rats. This antibody specifically recognizes mouse CXCR6 with high affinity (KD = 1.7 × 10-9 M for overexpressed CXCR6 and approximately 3.4-3.8 × 10-7 M for endogenous expression) .

When designing cross-species experiments, researchers must carefully select antibodies validated for their target species and avoid assuming cross-reactivity without explicit validation.

How can I validate CXCR6 antibody specificity for flow cytometry applications?

Rigorous validation of CXCR6 antibody specificity is essential for reliable flow cytometry results. A comprehensive validation approach includes:

• Overexpression systems: Compare staining between CXCR6-transfected cells and control transfectants. For example, HEK293 cells transfected with human CXCR6 versus irrelevant transfectants provide a clear positive/negative comparison .

• Biological expression modulation: Test antibody detection in models where CXCR6 expression is physiologically induced. IL-2 stimulation of human PBMCs provides an excellent model, as it significantly upregulates CXCR6 expression on T cells after 6-9 days of culture .

• Isotype controls: Always include appropriate isotype control antibodies to set quadrant markers and distinguish specific from non-specific binding .

• Kinetic binding analysis: For advanced validation, determine dissociation constants (KD) through kinetic analysis. The Cx6Mab-1 antibody demonstrated KD values of 1.7 × 10-9 M for overexpressed mouse CXCR6 and 3.4-3.8 × 10-7 M for endogenously expressed protein .

• Secondary detection system optimization: When using indirect staining, optimize secondary antibody selection to minimize cross-reactivity (e.g., Allophycocyanin-conjugated Anti-Mouse IgG Secondary Antibody) .

What methodologies exist for inducing and measuring CXCR6+ T cells in experimental systems?

Several approaches can induce and expand CXCR6+ T cell populations for functional studies:

In vitro induction methods:

• Cytokine stimulation: Treatment of human PBMCs with IL-2 (20 ng/mL) for 6-9 days significantly increases CXCR6+CD3+ populations, as demonstrated by flow cytometry analysis .

• Tumor cell co-culture: Mixing naive T cells with tumor cells (e.g., MC38 at 1:100 ratio) supplemented with IL-2 (10 ng/mL) and CD3/CD28 stimulation (5 μg/mL each) induces CXCR6 expression .

• Transwell tumor tissue exposure: A sophisticated approach uses shredded tumor tissues in upper transwell chambers with naive T cells in lower chambers, supplemented with IL-2 and CD3/CD28 antibodies. This allows for soluble factor exchange while preventing contamination with pre-existing CXCR6+ tumor-infiltrating lymphocytes .

• Th1 polarization: CXCR6 expression can be induced in human CD3+ PBMCs through Th1 polarization using plate-bound anti-CD3/CD28 antibodies, anti-IL-4 antibodies (20 μg/mL), and IL-12 (10 ng/mL) for 5 days .

Measurement approaches:

• Flow cytometry with anti-CXCR6 antibodies (either directly conjugated or with secondary detection)

• Dual staining with lineage markers (CD3, CD4) to identify specific CXCR6+ subpopulations

• Western blotting for population-level expression analysis

What is the role of CXCR6 in tumor immunity and how can antibodies help investigate this function?

CXCR6 plays a critical role in antitumor immunity, particularly through CD8+ T cell function:

• Tumor infiltration: CXCR6+CD8+ T cells demonstrate enhanced tumor-infiltrating capacity compared to CXCR6- cells, making CXCR6 a potential marker for effective tumor-reactive T cells .

• Tumor antigen specificity: Induced CXCR6+CD8+ T cells possess tumor antigen specificity, suggesting their importance in targeted antitumor responses .

• Immunotherapy enhancement: CXCR6+CD8+ T cells can enhance the efficacy of checkpoint inhibitor therapy, specifically anti-PD-1 blockade, in retarding tumor progression .

• Differential expression in tumor tissue: CXCR6 expression levels differ significantly between non-neoplastic lung tissues and lung cancer specimens (both adenocarcinoma and squamous cell carcinoma), suggesting altered CXCR6 signaling in the tumor microenvironment .

CXCR6 antibodies facilitate investigation of these mechanisms through:

• Flow cytometric identification and isolation of CXCR6+ tumor-infiltrating lymphocytes

• In vivo depletion studies using anti-CXCR6 antibodies (e.g., clone 19A5) to assess the functional significance of CXCR6+ cells in tumor control

• Immunohistochemical quantification of CXCR6 expression in tumor tissues versus normal tissues

• Analysis of CXCR6-CXCL16 axis through complementary assessment of the ligand CXCL16 in tumor tissues via Western blotting

What are the optimal protocols for detecting CXCR6 via flow cytometry in different sample types?

Successful detection of CXCR6 via flow cytometry requires optimized protocols based on sample type:

For PBMCs and primary immune cells:

• Harvest cells and wash in flow cytometry buffer (PBS with 1% BSA and 0.1% sodium azide)

• Block Fc receptors (10 minutes, 4°C) to reduce non-specific binding

• Stain with anti-CXCR6 antibody (e.g., clone 56811 or 1010910 for human samples) at pre-titrated concentrations

• For indirect detection: Add appropriate secondary antibody (e.g., Allophycocyanin-conjugated Anti-Mouse IgG)

• Include lineage markers (e.g., CD3, CD4) for identifying specific CXCR6+ populations

• Set quadrant markers based on isotype control staining patterns

For transfected cell lines (validation controls):

• Culture CXCR6-transfected cells alongside control transfectants

• Harvest cells at 70-80% confluence for optimal surface expression

• Proceed with standard surface staining protocol

• Include markers for transfection efficiency (e.g., eGFP) when applicable

For tumor-infiltrating lymphocytes:

• Generate single-cell suspensions from tumor tissues using gentle enzymatic digestion

• Implement density gradient separation to enrich lymphocyte populations

• Include viability dye to exclude dead cells

• Stain with anti-CXCR6 antibodies alongside T cell markers

• Consider using fluorescence-minus-one (FMO) controls for accurate gating

What controls should be included when working with CXCR6 antibodies?

A robust experimental design for CXCR6 antibody applications should include multiple controls:

When analyzing experimental data, researchers should always set quadrant markers based on control antibody staining patterns to ensure accurate identification of CXCR6-positive populations .

How can I optimize Western blotting protocols for CXCR6 detection?

Western blotting for CXCR6 requires careful optimization due to its nature as a seven-transmembrane G protein-coupled receptor:

• Sample preparation:

  • For cell lines: Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

  • For tissues: Homogenize in RIPA buffer, centrifuge to remove debris

  • Quantify protein using BCA protein assay kit

• Protein separation:

  • Use appropriate percentage SDS-PAGE gels (10-12%) for optimal resolution

  • Load adequate protein amount (30-50 μg for endogenous expression)

• Transfer conditions:

  • For transmembrane proteins like CXCR6, use PVDF membranes with pore size 0.45 μm

  • Consider extended transfer times or semi-dry transfer systems

• Blocking and antibody incubation:

  • Block membranes thoroughly (5% non-fat milk or BSA in TBST)

  • Incubate with primary antibody overnight at 4°C (e.g., Cx6Mab-1 for mouse CXCR6)

  • Use HRP-conjugated secondary antibodies appropriate for primary antibody species

• Detection:

  • Employ chemiluminescent substrate systems (e.g., LumiGLO) for visualization

  • Include β-actin as loading control

The established anti-mouse CXCR6 antibody Cx6Mab-1 has been validated for Western blotting applications with endogenous CXCR6 in P388 and J774-1 cell lines, demonstrating its utility for protein expression analysis .

What should researchers consider when developing adoptive cell therapy protocols with CXCR6+ T cells?

When developing adoptive cell therapy (ACT) protocols utilizing CXCR6+ T cells, researchers should consider several critical factors:

• Induction and expansion strategies:

  • Optimize culture conditions for generating large numbers of CXCR6+ T cells

  • Consider transwell systems to induce CXCR6 expression via tumor-derived factors without contamination from tumor-resident CXCR6+ cells

  • Monitor expansion kinetics to determine optimal harvest timing (days 2, 4, and 7 post-induction show different percentages of CXCR6+CD8+ T cells)

• Functional validation:

  • Confirm tumor antigen specificity of induced CXCR6+ cells

  • Assess cytokine production, cytotoxicity, and proliferative capacity

  • Evaluate persistence potential through phenotypic characterization

• Combination approaches:

  • Investigate synergistic effects with checkpoint inhibition (e.g., anti-PD-1 therapy)

  • Consider the relationship between CXCR6 expression and the CXCL16 gradient in tumors

• Administration protocol:

  • Determine optimal cell dose, route, and schedule for adoptive transfer

  • Consider lymphodepletion strategies to enhance engraftment

• Monitoring strategies:

  • Track CXCR6+ T cell trafficking using flow cytometry of tissue samples

  • Correlate tumor volume measurements with therapeutic efficacy (V=1/2(a×b²))

How does CXCR6 expression in tumors correlate with patient outcomes and therapeutic responses?

CXCR6 expression patterns in tumors provide valuable insights into prognostic implications and therapeutic responsiveness:

• Expression in lung cancer subtypes:
  Immunohistochemical analysis reveals significant differences in CXCR6 expression between non-neoplastic lung tissues, adenocarcinoma, and squamous cell carcinoma. Quantification using image analysis software (Aperio ImageScope) demonstrates highly significant variation between these groups (p ≤ 0.0001) .

• Relationship with immunotherapy response:
  CXCR6+CD8+ T cells enhance the efficacy of anti-PD-1 blockade in retarding tumor progression, suggesting that CXCR6 expression levels might predict responsiveness to checkpoint inhibitor therapy .

• CXCR6-CXCL16 axis in tumor microenvironment:
  The interaction between CXCR6 on lymphocytes and its ligand CXCL16 in tumor tissues regulates immune cell trafficking and retention in the tumor microenvironment. Assessment of both receptor and ligand provides more comprehensive understanding of this signaling pathway .

• Potential as therapeutic target:
  CXCR6 has been proposed as a therapeutic target against tumors through regulation of the tumor microenvironment, highlighting its importance in cancer immunobiology .

Researchers investigating CXCR6 in clinical contexts should consider correlative analyses between CXCR6 expression patterns, infiltrating lymphocyte populations, and clinical outcomes to better understand its prognostic and predictive value.

What methodological approaches can quantify CXCR6 expression in patient tumor samples?

Multiple methodological approaches allow precise quantification of CXCR6 expression in patient tumor samples:

• Immunohistochemistry (IHC) with digital quantification:

  • Stain tissue sections with validated anti-CXCR6 antibodies

  • Capture images using digital pathology systems (e.g., TissueFAXS cell analysis system)

  • Quantify immuno-intensities using image analysis software like Aperio ImageScope

  • Compare expression between tumor and adjacent normal tissues with statistical analysis

• Flow cytometry of dissociated tumor tissues:

  • Generate single-cell suspensions from fresh tumor samples

  • Stain with fluorophore-conjugated anti-CXCR6 antibodies alongside lineage markers

  • Analyze CXCR6+ cell frequencies among different immune cell subpopulations

  • Compare with matched peripheral blood samples when available

• Western blotting for bulk tissue analysis:

  • Prepare protein lysates from tumor tissues using RIPA buffer with protease inhibitors

  • Quantify protein concentration using BCA assay

  • Detect CXCR6 expression using validated antibodies

  • Normalize to housekeeping proteins (e.g., β-actin) for accurate quantification

• Immunofluorescence multiplex imaging:

  • Perform multiplexed staining including CXCR6 and relevant cell markers

  • Analyze spatial relationships between CXCR6+ cells and other cell types in the tumor microenvironment

  • Quantify using specialized software for spatial tissue analysis

Each methodology offers distinct advantages, and selection should be based on specific research questions, available sample types, and required resolution of analysis.