TRBV7-9 Antibody, Biotin conjugated

What is TRBV7-9 and what is its significance in T cell biology?

TRBV7-9 (according to IMGT nomenclature; also known as TCRVβ6.4 in the Arden nomenclature) is a specific T cell receptor beta variable gene expressed on a subset of T lymphocytes. It forms part of the T cell receptor (TCR) complex that recognizes antigens. TRBV7-9 expressing T cells can be selectively activated by certain superantigens, including engineered proteins like ABR-217620 (naptumomab estafenatox), which has been studied in cancer immunotherapy .

Methodologically, TRBV7-9 expression on T cells can be identified using flow cytometric methods based on staining with fluorochrome-conjugated multimeric complexes. This allows researchers to track and analyze this specific T cell subset in various immunological contexts .

How does biotin conjugation enhance TRBV7-9 antibody detection?

Biotin conjugation significantly improves TRBV7-9 antibody detection through several mechanisms:

• Signal amplification: The biotin-streptavidin interaction creates multimeric complexes that enhance detection sensitivity

• Flexibility in experimental design: Researchers can use various streptavidin-conjugated fluorophores with the same biotin-conjugated antibody

• Formation of multimeric complexes: Data shows optimal detection using a 6:1 molar ratio of biotin-conjugated ABR-217620 to streptavidin-PE

The multimeric nature of these complexes more closely mimics natural TCR-antigen interactions, providing more sensitive detection of TRBV7-9+ T cells compared to conventional monomeric antibody approaches .

What are the optimal conditions for flow cytometric detection of TRBV7-9?

Based on published protocols, optimal flow cytometric detection of TRBV7-9+ T cells requires:

After complex formation, cells should be resuspended in the fluorochrome-conjugated multimeric solution and co-stained with other appropriate markers according to standard procedures. Single and viable cells that bind the multimeric complex can be identified based on laser scatter profile and fluorescence intensity .

How is TRBV7-9 involved in disease-specific T cell responses?

TRBV7-9 expressing T cells appear to play significant roles in several immunological contexts:

• Cancer immunotherapy: ABR-217620 selectively activates TRBV7-9 expressing T lymphocytes through low-affinity interactions with the TCR, triggering cytotoxic activity against tumor cells expressing the 5T4 antigen .

• Celiac disease: Following gluten challenge, celiac patients show enrichment of specific CDR3 motifs within TRBV7-9-expressing T cells. The GN motif at positions 6-7 within the CDR3 region occurred in 40% (16/40) of unique TCRβ clones using TRBV7-9, compared to only 0.13% (12/9,584) in reference databases (p<0.0001) .

• T cell clonal expansion: Studies show that TRBV7-9+ T cells with specific CDR3 motifs undergo clonal expansion in response to antigens, demonstrating their functional importance in adaptive immunity .

These disease-specific associations make TRBV7-9 an important marker for studying targeted immune responses .

What experimental approaches can detect TRBV7-9 in low-frequency T cell populations?

Detecting rare TRBV7-9+ T cell populations requires specialized approaches:

• Multimeric staining: Form complexes of biotin-conjugated TRBV7-9 antibody with streptavidin-PE at 6:1 molar ratio to enhance sensitivity

• Cell enrichment: Employ magnetic bead-based enrichment prior to analysis to concentrate target populations

• High-event acquisition: Collect at least 500,000-1,000,000 total events during flow cytometry to capture sufficient numbers of rare cells

• Optimized panel design: Use bright fluorophores (PE, APC) for TRBV7-9 detection with minimal spectral overlap from other markers

• Cell sorting validation: Confirm population purity post-sorting by functional assays or molecular analysis

The detection sensitivity can be further enhanced by combining these approaches with genetic analysis of sorted populations, as demonstrated in studies of T cell repertoires .

How can TRBV7-9 detection be integrated with functional assays?

TRBV7-9 detection can be effectively integrated with several functional assays:

• Reporter systems: Researchers have developed TRBV7-9+ cell lines with NFκB-luciferase reporters to measure activation following stimulation. Activity is quantified in relative luminescence units (RLUs) to assess TCR engagement .

• Cytotoxicity assays: ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells, allowing for targeted cytotoxicity assessment .

• Co-culture systems: TRBV7-9+ cells can be co-cultured with target cells (e.g., Caki-2 cells) and different concentrations of stimulating agents to measure activation in a physiologically relevant context .

• Antigen presentation models: Latex beads coated with 5T4Fc and mouse monoclonal anti-human CD2 have been used to study TRBV7-9+ cell activation in controlled antigen presentation systems .

• Surface plasmon resonance analysis: This technique allows measurement of binding kinetics between TRBV7-9 and various ligands, providing quantitative data on receptor-ligand interactions .

Each of these approaches provides unique insights into TRBV7-9+ T cell functionality while maintaining the ability to track this specific T cell subset .

What are the critical factors for validating TRBV7-9 antibody specificity?

Comprehensive validation of TRBV7-9 antibody specificity requires:

• Engineered cell lines: Generate and test cell lines expressing TRBV7-9, as demonstrated in studies where "Individual clones growing in selection medium were expanded to mass cultures and assayed for TRBV7-9-expression by flow cytometry using the [ABR-217620-biotin/SA-PE]-complex"

• Correlation with sequencing data: Compare antibody staining patterns with TCR sequencing results to confirm targeting of TRBV7-9+ populations

• Functional validation: Confirm that TRBV7-9+ cells identified by the antibody respond specifically to known TRBV7-9-targeting superantigens like SEA/E-120

• Cross-reactivity assessment: Test binding on cells expressing other TRBV family members to ensure specificity

• Controls: Include isotype controls and TRBV7-9 negative samples in each experiment

Proper validation ensures that downstream analyses correctly identify the target population without false positives that could confound experimental results .

How do TCR CDR3 motifs in TRBV7-9 correlate with T cell function?

The correlation between TRBV7-9 CDR3 motifs and T cell functionality has been demonstrated through several key observations:

These specific motifs demonstrate:

• Antigen-driven selection: The significant enrichment of these motifs suggests strong selection pressure during antigen exposure

• Convergent evolution: Same motifs appearing in different patients indicates their functional importance in antigen recognition

• Clonal expansion: In one patient, the GN motif occurred in 14 of 19 unique TCRβ clones, with 5 of these converging on two identical TCRβs, indicating selective proliferation of functionally important clones

This correlation provides insight into how specific TRBV7-9 CDR3 sequences contribute to immune responses in different disease contexts .

What methodological approaches enable accurate TRBV7-9 TCR sequencing analysis?

Accurate TRBV7-9 TCR sequencing requires specialized methodological approaches:

• Cell isolation strategy: Use flow cytometry sorting of antibody-labeled TRBV7-9+ cells to ensure a pure starting population

• CDR3 motif analysis: Examine positions 6-7 within the CDR3 region for motifs like GN or GT that may indicate functional significance

• Reference comparison: Compare frequency of identified motifs against large reference databases (>9,000 sequences) to determine statistical significance of enrichment

• Clonal expansion assessment: Evaluate the degree of sequence repetition to determine clonality, which indicates antigen-driven selection

• Convergent evolution analysis: Look for identical amino acid sequences arising from different nucleotide arrangements, suggesting strong selection pressure

The search results describe analysis methods that successfully identified statistically significant (p<0.0001) enrichment of specific CDR3 motifs in TRBV7-9+ T cells, validating these approaches for TCR repertoire studies .

What are optimal conditions for activating and sorting TRBV7-9+ T cells?

The optimal conditions for activating and sorting TRBV7-9+ T cells include:

• Activation conditions:

  • Cell density: 10^5 TRBV7-9+ cells co-cultured with 5x10^4 target cells

  • Stimulant concentration: Titrated ABR-217620 or SEA/E-120

  • Incubation time: 4-6 hours for activation assays

  • Temperature: 37°C for activation, room temperature (21°C) for staining

• Sorting parameters:

  • Staining complex: Biotin-conjugated antibody with streptavidin-fluorophore at 6:1 ratio

  • Gating strategy: Based on scatter profile and fluorescence intensity

  • Viability marker: Include appropriate viability dye to exclude dead cells

  • Cell recovery buffer: Protein-containing buffer to maintain viability post-sort

• Validation metrics:

  • Post-sort purity assessment

  • Viability check after sorting procedure

  • Functional confirmation of sorted population

These optimized conditions ensure effective isolation of viable, functional TRBV7-9+ T cells for downstream applications .

How are TRBV7-9 antibodies used in cancer immunotherapy research?

TRBV7-9 antibodies play crucial roles in cancer immunotherapy research:

• Tumor-targeted superantigen development: ABR-217620 (naptumomab estafenatox) was designed to selectively activate TRBV7-9+ T cells against 5T4-expressing tumor cells. This approach uses T cell/tumor cell affinity to mimic natural productive T cell responses .

• Mechanism studies: TRBV7-9 antibodies help elucidate how engineered immunotherapeutics like ABR-217620 engage T cells with low affinity while binding tumor cells with high affinity (K_D approximately 1 nM) .

• TCR engineering approaches: Understanding TRBV7-9 binding and activation informs the development of engineered T cell receptors for adoptive cell therapy.

• Monitoring therapeutic responses: Tracking expansion of TRBV7-9+ T cell populations during immunotherapy treatment provides insights into treatment efficacy .

• Target validation: Surface plasmon resonance analysis with TRBV7-9 antibodies confirms binding interactions between therapeutic agents and their intended T cell targets .

These applications demonstrate the importance of TRBV7-9 antibodies in developing and understanding targeted cancer immunotherapies .

What role does TRBV7-9 play in autoimmune disease research?

TRBV7-9 has emerged as an important focus in autoimmune disease research:

• Celiac disease: TRBV7-9+ T cells with specific CDR3 motifs show significant expansion following gluten challenge in celiac patients. The GN motif at CDR3 positions 6-7 was highly enriched in these cells (p<0.0001) compared to reference populations .

• Clonal tracking: TRBV7-9 antibodies enable tracking of disease-relevant T cell clones over time and in response to therapeutic interventions.

• Convergent evolution: Studies have identified identical TRBV7-9 TCRβ sequences across different celiac patients, suggesting strong selection pressure toward recognizing specific disease-relevant antigens .

• Antigen-specific responses: TRBV7-9 detection helps identify and characterize antigen-specific T cell populations that drive autoimmune pathology.

• Repertoire analysis: TRBV7-9 antibodies facilitate isolation of disease-relevant T cells for comprehensive TCR repertoire analysis, revealing insights into disease mechanisms .

These applications highlight how TRBV7-9 antibodies contribute to understanding the immunological basis of autoimmune conditions like celiac disease .

How can TRBV7-9 antibodies be used in multiplex immune profiling?

TRBV7-9 antibodies can be effectively integrated into multiplex immune profiling approaches:

• Multiparameter flow cytometry: Combine biotin-conjugated TRBV7-9 antibodies with markers for activation (CD38), tissue homing (αEβ7), exhaustion (PD-1), and lineage markers (CD3, CD4, CD8) to comprehensively profile T cell subsets .

• Sequential staining protocols: Implement protocols where "Cells were labeled with MHC-I tetramers at 21°C for 10 minutes. Anti-PD-1 antibody was added to the suspension and incubated for a further 20 minutes. Cells were washed and labeled in separate steps with intervening washes" .

• Antigen-specific T cell identification: Pair TRBV7-9 detection with MHC tetramers to identify antigen-specific T cells within the TRBV7-9+ population .

• Functional marker integration: Combine TRBV7-9 detection with functional readouts such as cytokine production or activation markers to correlate TCR usage with functionality .

• Single-cell approaches: Use TRBV7-9 antibodies to identify and isolate cells for single-cell RNA sequencing or other high-dimensional analyses .

These multiplex approaches provide comprehensive immunophenotyping of TRBV7-9+ T cells in various research contexts .

What emerging technologies may enhance TRBV7-9 detection and analysis?

Several emerging technologies show promise for enhancing TRBV7-9 detection and analysis:

• High-parameter cytometry: Spectral flow cytometry and mass cytometry (CyTOF) could enable simultaneous detection of TRBV7-9 with dozens of other markers, providing unprecedented phenotypic detail.

• Single-cell multi-omics: Combining TRBV7-9 detection with single-cell transcriptomics, epigenomics, and proteomics could reveal the full functional profile of these T cells.

• Advanced reporter systems: Building on the NFκB-luciferase reporter systems described in the literature , more sophisticated reporters could track multiple signaling pathways simultaneously.

• In vivo imaging: Development of imaging agents targeting TRBV7-9 could enable tracking of these T cell populations in living organisms.

• AI-assisted repertoire analysis: Machine learning approaches could identify subtle patterns in TRBV7-9 CDR3 sequences that correlate with functional properties or disease associations.

These technologies would build upon current methodologies while providing deeper insights into TRBV7-9+ T cell biology and function .

How might understanding TRBV7-9 T cell biology contribute to precision medicine?

Understanding TRBV7-9 T cell biology has significant implications for precision medicine:

• Personalized immunotherapy: Knowledge of TRBV7-9 distributions and functions could help predict individual responses to immunotherapies like ABR-217620 that specifically target these T cells .

• Biomarker development: The presence of specific TRBV7-9 CDR3 motifs could serve as biomarkers for disease susceptibility, progression, or treatment response .

• Targeted therapeutic approaches: Drugs specifically designed to modulate TRBV7-9+ T cell function could provide more precise intervention in conditions where these cells play a pathogenic role.

• Patient stratification: TCR repertoire analysis focusing on TRBV7-9 could help stratify patients into subgroups likely to respond to specific treatments.

• Disease monitoring: Tracking changes in TRBV7-9+ T cell populations over time could provide early indicators of disease recurrence or treatment failure.

The convergent evolution of TRBV7-9 CDR3 motifs across different patients with the same condition suggests common immunological targets that could be exploited for therapeutic development .