TOX2 Antibody

What is TOX2 and what cellular functions does it regulate?

TOX2 is a transcription factor belonging to the TOX (thymocyte selection-associated HMG box) family that shares a highly conserved high mobility group DNA-binding domain with other TOX family members . Unlike its family member TOX, which primarily regulates T cell exhaustion, TOX2 exhibits diverse functions across immune cell populations:

• In central memory T cells (TCM): TOX2 positively regulates differentiation and maintenance

• In T follicular helper cells (TFH): TOX2 is critical for long-term survival and functional maintenance

• In Natural Killer cells: TOX2 is preferentially expressed and required for differentiation from CD34+ hematopoietic stem cells

TOX2 binds to the promoters of numerous TCM genes and coordinates with TET2 to regulate chromatin accessibility, suggesting its fundamental role in transcriptional programming of memory T cell populations .

How do TOX2 expression patterns differ across tissue types?

TOX2 demonstrates highly tissue-specific expression patterns, which is significant for researchers considering experimental models and therapeutic applications.

This expression profile contrasts with TET2, which is widely expressed throughout the body, making TOX2 potentially a superior target for immune cell-specific manipulation .

What are the optimal conditions for using TOX2 antibodies in Western blot analysis?

When conducting Western blot analysis with TOX2 antibodies, researchers should consider several methodological factors:

• Dilution range: Optimal results are typically achieved using dilutions between 1:5000-1:50000, though this should be titrated for each experimental system

• Molecular weight considerations: While the calculated molecular weight of TOX2 is 52 kDa (488 amino acids), the observed molecular weight is approximately 70 kDa , potentially indicating post-translational modifications

• Sample preparation: TOX2 antibodies have been validated with multiple cell types including A549, HepG2, HeLa, HEK-293, and MCF-7 cells, as well as mouse and rat liver tissue

• Storage conditions: Store antibodies at -20°C in PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) for optimal stability

Researchers should be aware that variations in molecular weight might occur due to post-translational modifications, protein isoforms, or degradation products.

How can TOX2 antibodies be effectively employed in flow cytometry?

For intracellular flow cytometry applications:

• Recommended concentration: Use 0.25 μg per 10^6 cells in a 100 μl suspension

• Validated cell types: A549 and MCF-7 cells have been confirmed as positive controls

• T cell applications: When analyzing T cell populations, researchers should consider dual staining with surface markers like CD45RO and CCR7 to distinguish memory T cell subsets (TCM: CD45RO+CCR7+; TEM: CD45RO+CCR7-)

• Co-staining considerations: For exhaustion studies, consider co-staining with PD-1 to identify T cell exhaustion-precursor cells (TPEX: PD-1+CCR7+)

Fixation and permeabilization protocols should be optimized specifically for nuclear transcription factors to ensure adequate antibody access to the nuclear compartment.

How does TOX2 function differ from TOX in T cell regulation?

Despite their structural similarities, TOX2 and TOX exhibit distinct and sometimes opposing functions in T cells:

This functional dichotomy highlights the importance of specifically targeting and studying TOX2 independent of other TOX family members in T cell research applications.

What experimental approaches can assess TOX2's impact on T cell memory formation?

Researchers investigating TOX2's role in T cell memory can employ several methodological approaches:

• Serial stimulation experiments: Conduct "stress test" assays using engineered target cells (e.g., irradiated K562 cells expressing CD19) to mimic features of antigen-induced exhaustion during long-term coculture, allowing assessment of TOX2's impact on proliferative capacity

• Flow cytometry memory subset analysis: Use markers such as:

  • CD45RO and CCR7 (TCM: CD45RO+CCR7+; TEM: CD45RO+CCR7-)

  • CD45RO and CD27 (TCM: CD45RO+CD27+)

• Gene expression profiling: Compare differentially expressed genes between TOX2 knockdown and control groups using gene sets associated with various T cell states

• Chromatin accessibility assays: Assess TOX2's impact on chromatin structure at memory-associated gene loci

• Combinatorial manipulation: Compare TOX2 and TET2 manipulation to disentangle their coordinated effects on T cell memory programming

How can TOX2 manipulation potentially improve CAR T cell therapy?

Emerging evidence suggests several promising approaches for leveraging TOX2 in CAR T cell therapy development:

• Targeted upregulation: TOX2 overexpression significantly increases the proportion of CD45RO+CCR7+ TCM cells, which are associated with improved persistence and anti-tumor efficacy

• Balancing expression levels: Careful titration of TOX2 expression may be necessary, as excessive overexpression can increase PD-1+ cells, potentially promoting T cell exhaustion-precursor (TPEX) differentiation

• Selective targeting: Due to its T cell-specific expression compared to more broadly expressed genes like TET2, TOX2 represents a potentially superior target for manipulation in CAR T cells

• CD4+ T cell applications: TOX2 may have particular utility in promoting long-term maintenance of CD4+ T cells, which exhibit longer persistence in CAR T cell therapy

Researchers should note that while TOX2 knockdown reduced proliferation, mere overexpression did not increase proliferation above baseline, suggesting the need for coordinated manipulation of multiple factors .

What is known about TOX2's role in T follicular helper cells and antibody responses?

TOX2 plays a critical role in T follicular helper (TFH) cell biology with implications for vaccine development and antibody-mediated immunity:

• Memory formation: TOX2 is essential for establishing TFH cell memory, which is crucial for long-term immunity after vaccination

• Cellular stability: Overexpression of TOX2 insulates TFH cells from spontaneous transformation upon TCR stimulation, indicating its role in maintaining TFH cell identity and persistence

• Functional impact: Mice with deficient TOX2 show low levels of TFH cells in the blood and poor antibody responses to infection, demonstrating its importance in antibody-mediated immunity

• Distinct mechanism: Unlike its exhaustion-promoting role in some T cell subsets, TOX2 has a contrasting function in TFH cells, promoting their survival and functional maintenance

These findings suggest that targeting TOX2 could be used therapeutically to modulate TFH cells for enhanced vaccine responses and improved antibody production in immunodeficiencies .

What technical considerations are important when studying TOX2 in NK cells?

When investigating TOX2 in Natural Killer (NK) cells, researchers should consider:

• Developmental context: TOX2 is preferentially expressed in human NK cells among various leukocyte populations and is required for in vitro and in vivo human NK cell differentiation from UCB-derived CD34+ hematopoietic stem cells

• Antibody selection: Choose antibodies validated specifically for NK cell applications, considering cross-reactivity with other immune cell types

• Experimental models: Consider species differences when designing experiments, as TOX2 antibodies may show different reactivity patterns across human, mouse, and rat samples

• Functional assays: Design experiments that assess both NK cell differentiation and cytotoxic function to fully characterize TOX2's impact

Researchers should validate antibody specificity in NK cells alongside appropriate controls, particularly when examining developmental stages where expression levels may fluctuate.

How can contradictory findings about TOX2 function be reconciled across different experimental systems?

The apparently contradictory roles of TOX2 across different immune cell populations present significant interpretive challenges:

• Cell type specificity: TOX2 promotes beneficial central memory differentiation in CAR T cells but may contribute to exhaustion in other contexts, suggesting highly context-dependent functions

• Expression level effects: Threshold effects may exist where moderate TOX2 expression promotes memory while excessive expression drives exhaustion precursor populations (TPEX)

• Temporal dynamics: The timing of TOX2 expression during immune cell differentiation likely influences its functional impact

• Methodological approach: Different knockout or overexpression systems may produce varying phenotypes due to compensatory mechanisms or off-target effects

• Protein interactions: TOX2 functions in coordination with other factors like TET2 , suggesting its role depends on the broader molecular environment

Researchers should design experiments with carefully matched conditions across cell types, include appropriate time-course analyses, and consider combinatorial manipulation of TOX2 with its known interaction partners.

What are the current limitations in detecting post-translational modifications of TOX2?

Understanding TOX2's post-translational state presents several technical challenges:

• Molecular weight discrepancy: The observed molecular weight of TOX2 (70 kDa) differs significantly from its calculated weight (52 kDa) , suggesting extensive post-translational modifications

• Antibody epitope access: Modifications may mask antibody binding sites, potentially leading to false negatives in certain detection methods

• Tissue-specific modifications: Different cell types may produce distinctly modified TOX2 proteins, complicating cross-experimental comparisons

• Functional impact assessment: Correlating specific modifications with functional outcomes requires sophisticated approaches combining mass spectrometry, mutational analysis, and functional assays

Researchers should consider combining multiple antibodies targeting different epitopes and employing mass spectrometry-based approaches to fully characterize TOX2's post-translational landscape.

What emerging areas show promise for TOX2-targeted therapeutic development?

TOX2 research opens several promising avenues for therapeutic development:

• CAR T cell engineering: Manipulating TOX2 expression could enhance persistence and function of adoptive cell therapies by promoting central memory phenotypes

• Vaccine adjuvants: Given TOX2's role in TFH cells and antibody responses, targeting this pathway could enhance vaccine efficacy by promoting durable antibody responses

• NK cell-based immunotherapies: Leveraging TOX2's role in NK cell development could improve NK cell-based cancer therapies

• Combination approaches: Coordinated manipulation of TOX2 with other factors (e.g., TET2) might produce synergistic effects on immune cell function

• Cell type-selective targeting: The tissue-restricted expression of TOX2 makes it a promising target for selective immune manipulation without broad off-target effects

Future research should focus on developing technologies for temporal and cell type-specific control of TOX2 expression to maximize therapeutic benefits while minimizing potential adverse effects.