NR2F6 Antibody, FITC conjugated

What is NR2F6 and why is it significant in immunological research?

NR2F6 is a nuclear orphan receptor that functions as a transcriptional repressor in lymphocytes. It has gained significant attention because it potently antagonizes the ability of T helper cells to induce expression of key cytokine genes such as IL-2 and IL-17 . NR2F6 directly interferes with the DNA binding of nuclear factor of activated T cells (NF-AT) and activator protein 1 (AP-1), subsequently affecting transcriptional activity of cytokine promoters . Its significance lies in its role as an intracellular immune checkpoint that suppresses adaptive anti-cancer immune responses, making it a promising target for next-generation immuno-oncological approaches .

How does NR2F6 function at the molecular level in T cells?

At the molecular level, NR2F6 acts as a PKC substrate and essential regulator of CD4+ T cell activation responses. When activated, it binds to promoter/enhancer response elements that contain imperfect 5'-AGGTCA-3' direct or inverted repeats, which are recognized by other nuclear hormone receptors as well . NR2F6 inhibits NFATC transcription factor DNA binding and subsequently its transcriptional activity . This inhibition results in suppression of key inflammatory cytokines, particularly IL-17 in Th17-differentiated CD4+ T cells . Studies have shown that acute silencing of Nr2f6 in both mouse and human T cells induces hyper-responsiveness, establishing its non-redundant T-cell-inhibitory function .

What are the physiological consequences of NR2F6 deficiency in model organisms?

Nr2f6-deficient mice exhibit several notable phenotypes that demonstrate the physiological importance of this receptor:

• Hyperreactive lymphocytes and development of late-onset immunopathology

• Hypersusceptibility to Th17-dependent experimental autoimmune encephalomyelitis

• High susceptibility to dextran sodium sulfate (DSS)-induced colitis, characterized by enhanced weight loss, increased colonic tissue destruction, and immune cell infiltration

• Altered intestinal permeability and reduced Muc2 expression, leading to spontaneous late-onset colitis

• Enhanced T and B lymphocyte numbers and decreased sensitivity to apoptosis induced by antigen-receptor ligation

• Changes in skeletal muscle oxidative metabolism and contractile function

These diverse phenotypes highlight NR2F6's multifunctional role across different tissue systems.

What are the primary applications for NR2F6 antibody, FITC conjugated in research settings?

FITC-conjugated NR2F6 antibodies serve several critical functions in research:

• Flow cytometric analysis of NR2F6 expression in immune cell populations, particularly T cells

• Immunofluorescence microscopy for visualization of NR2F6 localization within cells and tissues

• Monitoring changes in NR2F6 expression levels in response to stimuli or during disease progression

• Tracking intracellular translocation of NR2F6 between cytoplasm and nucleus

• Analysis of NR2F6 expression in tumor-infiltrating lymphocytes (TILs) from patient samples

The fluorescent conjugation enables direct detection without secondary antibodies, streamlining workflows and enabling multiparameter analyses alongside other markers.

What experimental protocols yield optimal results when using FITC-conjugated NR2F6 antibodies?

For optimal results with FITC-conjugated NR2F6 antibodies, researchers should consider the following protocol guidelines:

• Flow cytometry protocol:

  • Perform fixation and permeabilization steps using commercial kits designed for nuclear antigen detection

  • Typical antibody concentration: 2-5 μg per 1×10^6 cells

  • Include proper compensation controls for spectral overlap with other fluorophores

  • Use 488nm laser excitation with 530/30 bandpass filter for detection

• Immunofluorescence microscopy:

  • Fix cells with 4% paraformaldehyde followed by permeabilization with 0.1-0.3% Triton X-100

  • Block with 5% normal serum from the same species as the secondary antibody

  • Incubate with FITC-conjugated NR2F6 antibody (typically 1:100-1:200 dilution)

  • Include nuclear counterstain (e.g., DAPI)

  • Mount with anti-fade reagent to prevent photobleaching

• Tissue section staining:

  • Perform antigen retrieval (citrate buffer pH 6.0 or EDTA buffer pH 9.0)

  • Block endogenous fluorescence with 0.1% sodium borohydride

  • Use longer incubation times (overnight at 4°C) for better penetration

Validation with appropriate positive and negative controls is essential for all applications.

How can researchers distinguish between cytoplasmic and nuclear localization of NR2F6?

Since NR2F6 functions as a nuclear receptor that can translocate between cytoplasm and nucleus, distinguishing its localization is critical:

• Confocal microscopy approach:

  • Use FITC-conjugated NR2F6 antibody alongside nuclear stains like DAPI or Hoechst

  • Perform z-stack imaging to ensure proper localization in three dimensions

  • Analyze colocalization coefficients (Pearson's or Mander's) to quantify nuclear vs. cytoplasmic distribution

• Subcellular fractionation approach:

  • Separate nuclear and cytoplasmic fractions using commercial kits

  • Analyze fractions by Western blot using the unconjugated form of the same NR2F6 antibody clone

  • Use proper loading controls for each fraction (e.g., HDAC1 for nuclear, GAPDH for cytoplasmic)

• Flow cytometry approach:

  • Use differential permeabilization techniques (e.g., digitonin for cytoplasmic-only vs. Triton X-100 for total)

  • Compare median fluorescence intensity (MFI) between permeabilization conditions

Note that proper controls and validation are essential as NR2F6 localization may vary depending on cell activation status and tissue context.

What are common technical challenges when using FITC-conjugated NR2F6 antibodies and how can they be addressed?

Several technical challenges may arise when working with FITC-conjugated NR2F6 antibodies:

• Low signal intensity:

  • Cause: Low expression levels of NR2F6 or epitope masking

  • Solution: Optimize fixation/permeabilization conditions; consider signal amplification (e.g., tyramide signal amplification)

• High background fluorescence:

  • Cause: Non-specific binding or autofluorescence

  • Solution: Increase blocking time/concentration; use tissues from Nr2f6-deficient mice as negative controls; include autofluorescence quenching steps

• Photobleaching:

  • Cause: FITC's susceptibility to photobleaching during microscopy

  • Solution: Minimize exposure time; use anti-fade mounting media; consider alternative more photostable fluorophores if available

• Cross-reactivity:

  • Cause: Antibody binding to related nuclear receptors

  • Solution: Validate specificity using Nr2f6-deficient samples or knockdown approaches; consider competitive binding assays with recombinant NR2F6 protein

• Nuclear penetration issues:

  • Cause: Inadequate permeabilization for nuclear antigens

  • Solution: Extend permeabilization time; try alternative permeabilization reagents (e.g., methanol)

Each of these challenges requires systematic optimization for the specific experimental context.

How should researchers validate the specificity of NR2F6 antibodies?

Rigorous validation of NR2F6 antibody specificity is critical for reliable research outcomes:

• Genetic validation approaches:

  • Test antibody in tissues/cells from Nr2f6-knockout mice

  • Use siRNA or CRISPR-based knockdown of NR2F6 in relevant cell lines

• Protein-based validation:

  • Perform blocking experiments with recombinant human NR2F6 protein

  • Conduct Western blot analysis to confirm single band of expected molecular weight

• Comparative validation:

  • Test multiple antibody clones against the same samples

  • Compare staining patterns with published immunohistochemical data

• Functional validation:

  • Correlate NR2F6 detection with known biological functions

  • Verify increased expression in contexts where NR2F6 is known to be upregulated (e.g., in tumor-infiltrating lymphocytes from NSCLC patients)

A combination of these approaches provides the strongest evidence for antibody specificity.

How can NR2F6 antibodies be incorporated into multi-parametric flow cytometry panels for tumor immunology studies?

For comprehensive immunophenotyping in tumor immunology, FITC-conjugated NR2F6 antibodies can be integrated into multi-parametric panels:

Sample panel design for TIL analysis:

This panel allows researchers to:

• Quantify NR2F6 expression levels across T cell subsets

• Correlate NR2F6 with other established checkpoint molecules (PD-1, CTLA-4)

• Assess the relationship between NR2F6 expression and proliferative capacity

• Compare expression patterns between tumor-infiltrating and peripheral blood T cells

Based on clinical samples, NR2F6 expression has been found to be upregulated in more than 50% (164 of 303) of NSCLC patients' TILs and significantly correlates with PD-1 and CTLA-4 expression .

What approaches can be used to study NR2F6 function in combination with checkpoint blockade therapies?

To investigate NR2F6 function in the context of checkpoint blockade therapies, researchers can employ several sophisticated approaches:

• In vivo experimental design:

  • Use tumor models in wild-type and Nr2f6-deficient mice

  • Administer anti-PD-L1/PD-1 antibodies and monitor response

  • Analyze survival curves and tumor growth kinetics

  • Compare combination effects to single interventions

• Ex vivo T cell functional assays:

  • Isolate T cells from patients or experimental models

  • Use siRNA to acutely silence Nr2f6

  • Measure functional responses (proliferation, cytokine production) with and without PD-1/PD-L1 blocking antibodies

  • Analyze synergistic or additive effects using appropriate statistical models

• Transcriptomic analyses:

  • Perform RNA-seq on intratumoral T cells from different treatment conditions

  • Identify target genes deregulated upon genetic ablation of Nr2f6 alone or together with PD-L1 blockade

  • Conduct pathway analysis to identify mechanistic overlaps and distinctions

Research has demonstrated that genetic ablation of Nr2f6, particularly in combination with established cancer immune checkpoint blockade, efficiently delays tumor progression and improves survival in experimental mouse models .

What is the relationship between NR2F6 and other immune checkpoint molecules?

NR2F6 exhibits important relationships with established immune checkpoint molecules:

• Expression correlation:

  • NR2F6 protein expression in tumor-infiltrating lymphocytes from NSCLC patients significantly correlates with both PD-1 and CTLA-4 expression

  • This suggests potential coordinated regulation or functional interaction

• Mechanistic distinctions:

  • Unlike PD-1 and CTLA-4, which are cell surface receptors, NR2F6 functions as an intracellular nuclear receptor

  • NR2F6 directly interferes with transcription factor binding (NF-AT:AP-1) rather than signaling cascade inhibition

  • This creates potential for complementary rather than redundant inhibitory functions

• Therapeutic implications:

  • The non-redundant inhibitory function suggests potential for combination therapies

  • Current data indicates that targeting NR2F6 together with PD-L1 blockade reveals multiple advantageous transcriptional alterations in T cells

Understanding these relationships is essential for developing rational combination immunotherapy approaches that might overcome resistance to current checkpoint inhibitors.

How is NR2F6 expression being studied in human cancer specimens?

Researchers are employing several methodologies to investigate NR2F6 expression in human cancer specimens:

• Tissue microarray (TMA) analysis:

  • Immunohistochemical staining of TMAs containing numerous patient samples

  • Development of standardized scoring systems (0-4 scale) for NR2F6 expression in TILs

  • Correlation with clinicopathological features and patient outcomes

• Single-cell approaches:

  • Isolation of TILs from fresh tumor specimens

  • Flow cytometric analysis of NR2F6 expression in defined immune cell subsets

  • Correlation with functional markers and other checkpoint molecules

• Molecular analyses:

  • Comparison of NR2F6 mRNA expression between tumor-infiltrating T cells and peripheral blood T cells

  • Studies have shown significantly higher NR2F6 mRNA expression in CD3+ T cells from tumor tissue of NSCLC patients compared to PBMCs

Current data indicates that NR2F6 protein expression is upregulated in 54% of NSCLC cases examined (164 of 303 patients) , suggesting its potential relevance as a biomarker.

What are the current hypotheses about targeting NR2F6 for therapeutic purposes?

Several hypotheses are being explored regarding NR2F6 as a therapeutic target:

The non-redundant T-cell-inhibitory function of NR2F6 established through acute silencing in both mouse and human T cells provides a strong foundation for these therapeutic hypotheses .

What methodological advances are needed to better understand NR2F6 function across different tissue contexts?

Several methodological advances would enhance our understanding of NR2F6 biology:

• Tissue-specific conditional knockout models:

  • Current models show that disease susceptibility is not dependent on NR2F6 expression in the immune compartment but on its protective role in the intestinal epithelium

  • Development of inducible, tissue-specific models would help dissect context-dependent functions

• Improved structural biology approaches:

  • Crystal structures of NR2F6 bound to DNA and cofactors

  • Structure-guided development of small molecule modulators

• Advanced imaging techniques:

  • Intravital microscopy to track NR2F6 dynamics in living tissues

  • Single-molecule approaches to understand NR2F6 interactions with chromatin

• Systems biology integration:

  • Multi-omics approaches (transcriptomics, proteomics, metabolomics) in Nr2f6-deficient models

  • Network analysis to understand the broader impact of NR2F6 on cellular function

• Humanized mouse models:

  • Human immune system mice to better translate findings toward clinical applications

  • Patient-derived xenograft models with manipulation of NR2F6 in human TILs

These methodological advances would address current knowledge gaps, particularly regarding the context-dependent functions of NR2F6 across different tissues and disease states.

NR2F6 Expression Profiles Across Human Tissues and Disease States

Correlation of NR2F6 with Other Immune Checkpoints in Human NSCLC

This data demonstrates that NR2F6 expression in TILs correlates with established checkpoint molecules but does not independently predict survival in the examined NSCLC cohort .