Anti-Human TCR Vα24-Jα18 (iNKT cell)- Purified in vivo PLATINUM™ Functional Grade

Clone 6B11 has specific limitations and applications in mouse studies due to its selective reactivity profile. While this monoclonal antibody was originally raised in CD1d knockout mice, it does not react with murine invariant natural killer T (iNKT) cells or other murine cells.

Transgenic Mouse Model Applications

The primary in vivo mouse application for clone 6B11 involves studies using V?24J?18 transgenic mice. These are genetically modified mice that express the human invariant TCR chains, making them reactive to the 6B11 antibody.

In immunohistochemistry studies with these transgenic mice, 6B11 has been successfully used to:

• Identify and localize iNKT cells in tissue sections, specifically in spleen frozen sections
• Demonstrate tissue distribution of transgenic iNKT cells, which were found to be predominantly located in the splenic T cell zones (peri-arteriolar lymphoid sheath)
• Confirm specificity through double staining experiments, where essentially all 6B11-positive splenic cells co-stained with mouse CD3

Technical Limitations in Standard Mouse Studies

For conventional wild-type mouse studies, clone 6B11 has no utility because it does not recognize endogenous murine iNKT cells. Wild-type mouse spleen frozen sections show no specific 6B11 staining and only react with mouse CD3 antibodies.

Cross-Species Applications

Interestingly, while 6B11 doesn't work with murine cells, it does react with iNKT cells from Rhesus macaques and other non-human primates, expanding its potential applications to primate research models.

The antibody's inability to work on paraffin-embedded sections further limits its histological applications, requiring the use of frozen tissue sections for immunohistochemistry studies.

The correct storage temperature for sterile packaged clone 6B11 monoclonal antibody is between 2°C and 8°C (typically at 4°C), undiluted, and protected from prolonged exposure to light. Do not freeze the antibody.

• This storage range is standard for monoclonal antibodies like clone 6B11 used in research, including those supplied by BD Biosciences and BioLegend.
• Do not store at room temperature for extended periods, as antibody activity can be compromised.
• Do not freeze the antibody, since repeated freeze-thaw cycles or storage below 0°C can denature the protein and reduce its effectiveness.

These guidelines apply to research-use 6B11, such as FITC, PE, or APC conjugates for flow cytometry. Always refer to the specific product’s data sheet for any special instructions, but 2°C to 8°C is the accepted standard for 6B11 storage.

Based on the research literature, 6B11 antibody is commonly used in combination with several other antibodies and proteins, particularly in flow cytometry applications and cancer immunotherapy studies.

Flow Cytometry Co-staining Partners

The most frequently reported combination involves CD3 antibodies for T cell identification. In flow cytometric analysis, 6B11 is routinely paired with CD3 antibodies conjugated to different fluorophores, such as CD3-FITC or CD3-APC, to distinguish between different T cell populations. This combination is essential because 6B11 recognizes the invariant CDR3 region of TCR V?24-J?18, which is specific to invariant natural killer T (iNKT) cells, while CD3 serves as a pan-T cell marker.

Isotype control antibodies are also standard companions to 6B11 in experimental protocols. Mouse IgG1 ? isotype controls are used at equivalent concentrations to validate the specificity of 6B11 staining and control for non-specific binding.

Cancer Immunotherapy Applications

In the context of ovarian cancer treatment, 6B11 is used alongside several key immunological components. The antibody works in conjunction with anti-CD3 antibodies and cytokines such as IL-2 in the development of 6B11-OCIK (ovarian cancer immunotherapy using cytokine-induced killer cells). This combination stimulates both specific cytotoxic T lymphocytes and non-specific cytokine-induced killer cells.

Dendritic cell (DC) phenotyping markers are frequently analyzed together with 6B11 in immunotherapy studies. These include CD86, CD80, CD1a, CD83, HLA-DR, CD54, and CD40, which are used to assess dendritic cell activation status when evaluating the effectiveness of 6B11-based treatments.

Secondary Detection Systems

For immunofluorescence and flow cytometry applications, 6B11 is often paired with F(ab')2 anti-mouse IgG conjugated to phycoerythrin (PE) or other fluorophores for indirect detection methods. This secondary antibody system allows for signal amplification and flexible fluorophore selection in multi-parameter flow cytometry panels.

The combinations described reflect 6B11's primary role in identifying and functionally characterizing iNKT cells, as well as its therapeutic applications in cancer immunotherapy protocols where comprehensive immune cell profiling is essential.

Key Findings from Clone 6B11 Scientific Literature

The monoclonal antibody clone 6B11 is notable for its specificity and utility in studying human invariant natural killer T (iNKT) cells. Below are the key findings from the scientific literature regarding 6B11:

Specificity and Application

• 6B11 is highly specific for the invariant V?24-J?18 T cell receptor (TCR) ?-chain characteristic of human iNKT cells.
• Detection efficiency: When used for flow cytometry or FACS, 6B11 paired with anti-V?24 or anti-V?11 antibodies is highly selective for isolating invariant, CD1d-restricted T cell clones.
• Comparison with tetramer staining: The frequency of iNKT cells detected with 6B11 is approximately 95% of those detected by CD1d tetramer staining, making it a reliable tool for identifying and quantifying iNKT cells in circulation.
• Enrichment: Single-round 6B11 purification from peripheral blood mononuclear cells (PBMC) yields up to 90% pure iNKT cells, a 360-fold enrichment ex vivo.
• Identification of variants: Rare clones with single amino acid substitutions (e.g., serine to threonine or asparagine) in the invariant CDR3 loop of the TCR can still be detected by 6B11, but further substitutions (e.g., serine to glycine) result in loss of 6B11 reactivity, underscoring its specificity for the canonical sequence.

Biological Insights Gained Using 6B11

• Phenotype of iNKT cells: 6B11 studies revealed that most peripheral blood iNKT cells display a memory phenotype (CD45R0?), with high expression of CD27, CD28, and CD161, but low expression of CD56 and CD16 (classical NK markers).
• Activation status: Despite their memory phenotype, circulating iNKT cells have low expression of activation markers (CD25, CD38, CD95, CD154, HLA-DR), with only slightly elevated CD69.
• Cytokine production: Pure iNKT cell lines stimulated with 6B11 secrete 2–3 log units more cytokine than lines generated with anti-V?24 alone, indicating 6B11’s superior ability to selectively expand and activate iNKT cells.

Practical Applications

• 6B11 enables selective expansion of iNKT cells without the absolute requirement for antigen-presenting cells (APCs), highlighting its potential for generating large numbers of functional iNKT cells for research and therapy.
• Clinical relevance: The antibody has been used to study iNKT cell frequency and phenotypes in healthy individuals and in diseases such as type 1 diabetes, aiding in understanding immune dysregulation in these contexts.

Summary Table

Clone 6B11 thus stands out as a highly specific and effective tool for the identification, isolation, characterization, and expansion of human iNKT cells, facilitating both basic research and clinical studies.