IRF8 Antibody

What is IRF8 and why is it important in immunological research?

IRF8 (Interferon Regulatory Factor 8), also known as ICSBP1 (Interferon Consensus Sequence Binding Protein 1), is a 48-50 kDa transcription factor belonging to the IRF family. IRF8 specifically binds to the upstream regulatory region of type I IFN and IFN-inducible MHC class I genes, functioning as both a transcriptional activator and repressor depending on its binding partners .

IRF8 is crucial in immunological research because it:

• Plays a critical role in myeloid cell differentiation and lineage commitment

• Is essential for dendritic cell (DC) development, particularly plasmacytoid DCs and CD8a+ DCs

• Regulates B cell development and self-tolerance mechanisms

• Functions in both innate and adaptive immunity

• Has implications in autoimmune diseases and cancer immunology

Recent studies have demonstrated that IRF8 deficiency leads to expansion of marginal zone B cells and B1 B cells, increased production of anti-dsDNA antibodies, and breaching of B cell anergy, indicating its importance in preventing autoimmunity .

What are the main applications of IRF8 antibodies in research?

IRF8 antibodies are utilized across multiple research applications:

These applications enable researchers to investigate IRF8 expression patterns, protein interactions, transcriptional regulation, and its role in various biological processes .

How should I validate the specificity of an IRF8 antibody?

Thorough validation is critical due to reported cross-reactivity issues with some IRF8 antibodies:

• Peptide blocking controls: Incubate the antibody with a 5-10 fold excess of the immunizing peptide before staining. This should significantly reduce specific binding as demonstrated in previous studies .

• Positive and negative controls: Use tissues/cells known to express high levels of IRF8 (e.g., spleen tissue, lymphoma tissue, monocytes) and those with minimal expression .

• Knockout/knockdown validation: Compare staining in IRF8 wild-type versus knockout/knockdown samples. Several studies have used IRF8-/- mice for this purpose .

• Nuclear versus cytoplasmic localization: Perform subcellular fractionation or imaging flow cytometry to verify nuclear localization, which is the primary location for functional IRF8 .

• Multiple antibody comparison: Use antibodies targeting different epitopes of IRF8 and compare results .

Research has shown that polyclonal antibodies may have cross-reactivity with cytoplasmic components, making validation particularly important for accurate interpretation .

How can I differentiate between nuclear and cytoplasmic IRF8 localization?

Distinguishing nuclear from cytoplasmic IRF8 is methodologically important as research has shown that total cellular IRF8 presence doesn't always correlate with its nuclear localization, particularly in disease states .

Recommended approaches:

• Imaging flow cytometry (IFC): This technique combines flow cytometry with microscopy, allowing quantification of nuclear versus cytoplasmic localization:

  • Use nuclear dyes (e.g., DAPI) alongside IRF8 antibody staining

  • Calculate the "similarity score" between nuclear and IRF8 images

  • Analyze specific nuclear fluorescence intensity

• Confocal microscopy: For fixed cells or tissues:

  • Co-stain with nuclear markers

  • Use z-stack imaging to confirm intranuclear localization

  • Employ digital image analysis for quantification

• Subcellular fractionation: For biochemical analysis:

  • Separate nuclear and cytoplasmic fractions

  • Perform western blotting on isolated fractions

  • Include fraction-specific controls (e.g., Lamin B for nuclear, GAPDH for cytoplasmic)

Research findings indicate that during certain pathologies, IRF8's nuclear-cytoplasmic distribution may be altered, which could affect its function despite unchanged total expression levels .

What protocol is recommended for detecting IRF8 by flow cytometry?

Flow cytometric detection of IRF8 requires careful optimization:

• Sample preparation:

  • For peripheral blood: Use fresh samples when possible; cryopreservation protocols should include DNase (70 IU/mL) during thawing

  • For tissues: Generate single-cell suspensions using appropriate digestion protocols

• Fixation and permeabilization:

  • Use Foxp3/Transcription Factor Staining Buffer Set or equivalent

  • Fix cells with 4% methanol-free formaldehyde for 10 minutes at room temperature

  • Permeabilize with 0.1% Triton-X-100 in staining buffer

• Antibody staining:

  • Surface markers: Stain before fixation

  • Block with normal human IgG (or species-appropriate) for 10 minutes

  • IRF8 antibody: Typically use 0.25-0.5 μg per test in 100 μL volume

  • Incubate for 20-30 minutes at room temperature

• Controls:

  • Include isotype control at matched concentration

  • Consider peptide-blocked antibody as negative control

  • Include known positive cell populations (e.g., monocytes)

• Analysis:

  • Gate on specific cell populations

  • Analyze using geometric mean fluorescence intensity

  • Consider analyzing mean fluorescence intensity ratio (compared to isotype)

Research shows this approach can effectively detect IRF8 in various cell types including monocytes, dendritic cells, and B cells .

How can IRF8 antibodies be used to study dendritic cell development?

IRF8 antibodies are valuable tools for investigating dendritic cell (DC) development, as IRF8 plays essential roles in DC lineage determination:

• Flow cytometric analysis of progenitor populations:

  • Combine IRF8 antibodies with markers of DC progenitors

  • Track IRF8 expression levels during differentiation stages

  • Compare expression between different DC subsets (pDCs vs. conventional DCs)

• Functional studies:

  • Use IRF8 antibodies to correlate expression with functional maturation

  • Analyze IRF8 levels after TLR stimulation

  • Investigate IRF8's role in cytokine production

• Developmental research approach:

  • Analyze DC populations in IRF8-deficient models

  • Restore IRF8 expression via retroviral transduction

  • Track DC development with IRF8 antibodies

Research has demonstrated that IRF8-/- mice are deficient in both plasmacytoid DCs and CD8a+ DCs, while CD8a- DCs are present but functionally impaired upon TLR stimulation. Importantly, IRF8 deficiency doesn't affect DC precursor frequency or viability, and retroviral IRF8 transduction can restore pDC development from DC progenitors in IRF8-/- mice .

What's the role of IRF8 in cancer immunology and how can antibodies help investigate this?

IRF8 has emerged as an important factor in cancer immunology, with research applications of IRF8 antibodies including:

• Tumor infiltrating lymphocyte (TIL) analysis:

  • Use multiplex immunohistochemistry to correlate IRF8 expression with CD8+ T cell infiltration

  • Analyze IRF8 levels in tumor versus adjacent normal tissue

  • Correlate expression with clinical outcomes

• Predictive biomarker research:

  • Study has shown IRF8 expression can predict complete pathological response to monoclonal antibody therapy or select chemotherapy combinations (e.g., FAC: fluorouracil, adriamycin, cytoxan) in ER-negative breast cancer

  • Use IRF8 antibodies to stratify patient samples

• Therapeutic resistance mechanisms:

  • IRF8 regulates CD20 expression and controls the depleting capacity of anti-CD20 antibodies

  • Use IRF8 antibodies to investigate resistance to CD20-targeting immunotherapies

• Experimental approach:

  • Perform CRISPR/Cas9 screening to identify IRF8-related pathways

  • Validate findings with IRF8 antibodies in knockout models

  • Correlate IRF8 and target gene expression (e.g., CD20)

Research has demonstrated that high IRF8 expression correlates with CD8+ T cell infiltration, and analysis of immune cell infiltration indicates a strong correlation between activated/effector CD8+ T cells and tumoral IRF8 expression, suggesting IRF8 may influence tumor immunogenicity .

How are IRF8 antibodies used to investigate B cell tolerance mechanisms?

B cell tolerance is crucial for preventing autoimmunity, and IRF8 antibodies help elucidate this process:

• B cell anergy studies:

  • Use flow cytometry with IRF8 antibodies to analyze expression in anergic versus responsive B cells

  • Correlate IRF8 levels with B cell receptor signaling components

  • Track IRF8 expression during B cell maturation stages

• Autoimmunity research approach:

  • Measure anti-dsDNA antibodies in IRF8-deficient models

  • Use hen egg lysozyme (HEL) double transgenic models to study B cell anergy

  • Compare IRF8 expression between normal and autoimmune-prone B cells

• Experimental methodology:

  • Generate inducible IRF8 transgenic models using tetracycline-responsive elements

  • Induce IRF8 expression at specific developmental stages

  • Analyze effects on B cell tolerance using IRF8 antibodies

Research has shown that IRF8-deficient mice produce higher titers of anti-dsDNA IgM and IgG antibodies. In HEL transgenic models, IRF8-deficient mice produced significantly higher levels of anti-HEL antibodies. Furthermore, anergic B cells in IRF8-proficient backgrounds were blocked at the transitional stage, while anergic B cells in IRF8-deficient backgrounds matured further and regained responsiveness to antigen stimulation .

How do I interpret discrepancies between nuclear and cytoplasmic IRF8 staining?

Discrepancies between nuclear and cytoplasmic IRF8 staining require careful interpretation:

• Biological significance:

  • Nuclear IRF8 is typically associated with active transcriptional function

  • Cytoplasmic IRF8 may represent protein awaiting nuclear transport or retention due to regulatory mechanisms

  • Research has shown that in certain pathological conditions, nuclear-cytoplasmic distribution may be altered while total cellular levels remain unchanged

• Technical considerations:

  • Antibody specificity: Some IRF8 antibodies (particularly polyclonals) show cross-reactivity with cytoplasmic components

  • Peptide blocking typically has greater effect on nuclear IRF8 staining compared to cytoplasmic staining

  • Fixation methods can affect nuclear antigen accessibility

• Analytical approach:

  • Compare similarity scores between nuclear staining (DAPI) and IRF8 staining

  • Analyze specific nuclear IRF8 fluorescence intensity

  • Use peptide-blocked controls to determine specific versus non-specific binding

• Interpretation framework:

  • In healthy donor cells, total cellular IRF8 correlates well with nuclear localization

  • In certain disease states (e.g., myeloid-derived suppressor cells from renal carcinoma patients), this correlation may be lost

  • Consider nuclear IRF8 as potentially more relevant to transcriptional activity than total cellular measurements

Research indicates that analyzing nuclear localization of IRF8 by imaging flow cytometry may provide a more relevant correlate to its activity than whole cell assessments .

What explains variations in observed IRF8 molecular weight in western blots?

When analyzing western blot data for IRF8, researchers may observe variations in molecular weight:

• Expected molecular weight:

  • The calculated molecular weight of human IRF8 is 48 kDa

  • Observed molecular weight is typically reported as 48-50 kDa

• Sources of variation:

  • Post-translational modifications (phosphorylation, ubiquitination)

  • Protein isoforms (alternative splicing)

  • Species differences (human vs. mouse vs. rat)

  • Sample preparation methods (denaturing conditions, buffer composition)

• Methodological considerations:

  • Reduction conditions: Perform experiments under reducing conditions

  • Buffer selection: Use appropriate buffer systems (e.g., Immunoblot Buffer Group 8)

  • Positive controls: Include lysates from tissues known to express IRF8 (e.g., spleen tissue)

• Validation approach:

  • Run multiple antibodies targeting different epitopes

  • Include recombinant IRF8 protein as a standard

  • Use lysates from IRF8 knockout/knockdown samples as negative controls

If concerned about antibody specificity, peptide competition assays can help confirm that the observed band represents IRF8, though this may not eliminate all cytoplasmic cross-reactivity as observed in some studies .

What are the common pitfalls in IRF8 detection and how can they be addressed?

Several technical challenges can affect IRF8 detection:

• Non-specific binding:

  • Issue: Some polyclonal IRF8 antibodies show cross-reactivity with cytoplasmic components

  • Solution: Use monoclonal antibodies when possible; include peptide-blocked controls; perform subcellular fractionation

• Low signal-to-noise ratio:

  • Issue: High background obscuring specific IRF8 signal

  • Solution: Optimize blocking conditions; titrate antibody concentration; use more specific detection systems; include appropriate controls

• Inconsistent fixation/permeabilization:

  • Issue: Variable access to nuclear IRF8

  • Solution: Standardize protocols; use methanol-free formaldehyde (4%) for fixation; optimize permeabilization with 0.1% Triton-X-100

• Sample-dependent variability:

  • Issue: Different tissue types may require different protocols

  • Solution: Optimize conditions for each tissue type; human lymphoma tissue works well for IHC with suggested antigen retrieval using TE buffer pH 9.0 or alternative citrate buffer pH 6.0

• Storage and handling issues:

  • Issue: Antibody degradation affecting performance

  • Solution: Store at -20°C; antibodies are stable for one year after shipment; aliquoting is unnecessary for -20°C storage; some preparations contain 0.1% BSA for stability

Research indicates that as newer monoclonal alternatives become available, some of these issues may be mitigated, though careful validation remains essential for all applications .

How can ChIP-seq with IRF8 antibodies provide insights into transcriptional networks?

ChIP-seq with IRF8 antibodies enables genome-wide analysis of IRF8 binding sites and transcriptional networks:

• Experimental design considerations:

  • Use 20 μl of antibody and 10 μg of chromatin (approximately 4 × 10^6 cells) per IP

  • Consider SimpleChIP Enzymatic Chromatin IP Kits for optimal results

  • Include appropriate positive controls (known IRF8 target genes) and negative controls

• Target identification approach:

  • Identify direct IRF8 targets by integrating ChIP-seq with transcriptomic data

  • Look for enrichment of the IFN-stimulated response element (ISRE) motif

  • Analyze co-occurrence with binding sites of known IRF8 partners (e.g., BATF-JUNB)

• Functional network analysis:

  • Identify AICE sequences (5'-TGAnTCA/GAAA-3'), which are immune-specific regulatory elements

  • Study cooperative binding of IRF8 with partners like BATF

  • Correlate binding patterns with cell type-specific functions

Research has shown that IRF8 forms complexes with the BATF-JUNB heterodimer in immune cells, leading to recognition of AICE sequences followed by cooperative binding and activation of genes involved in dendritic cell differentiation, particularly CD8+ dendritic cells .

How does IRF8 expression change during cellular differentiation and activation?

IRF8 expression dynamics during differentiation and activation can be studied using antibodies:

• Hematopoietic differentiation:

  • Track IRF8 expression during myeloid differentiation

  • Monitor levels during B cell development stages

  • Analyze expression in DC precursors versus mature DCs

• Cell activation dynamics:

  • Measure IRF8 changes after:

    • IFN-γ stimulation (originally described as inducing IRF8)

    • IFN-α stimulation

    • IL-12 stimulation in NK and T cells

  • Correlate with functional outcomes

• Methodological approach:

  • Time-course experiments with flow cytometry or western blotting

  • Single-cell analysis to capture population heterogeneity

  • Correlation with other differentiation markers

Research indicates that IRF8 expression is variably regulated in hematopoietic cells including monocytes, macrophages, dendritic cells, and B cells. IRF8 has a conserved N-terminal DNA-binding domain and a divergent C-terminal regulatory domain that mediates interactions with other IRF family members, transcription factors, or cofactors .

What is the significance of IRF8 in autoimmune disease research?

IRF8 antibodies provide valuable insights into autoimmune disease mechanisms:

• B cell tolerance breakdown:

  • IRF8 deficiency leads to production of anti-dsDNA antibodies

  • Breaching of B cell anergy occurs in IRF8-deficient models

  • IRF8 expression is elevated in anergic B cells

• Experimental approaches:

  • Compare IRF8 expression in immune cells from autoimmune patients versus healthy controls

  • Analyze correlation between IRF8 levels and disease severity

  • Study IRF8 regulation of genes implicated in autoimmunity

• Autoimmune models:

  • Use IRF8 conditional knockout mice to study tissue-specific effects

  • Analyze IRF8 expression in models like lupus-prone mice

  • Investigate therapeutic approaches targeting IRF8-dependent pathways

Research has demonstrated that IRF8-deficient mice produce anti-dsDNA antibodies by 3 months of age, while conditional B cell-specific IRF8 knockout mice develop these antibodies by 5 months of age. This suggests that IRF8 plays a critical role in preventing B cell-mediated autoimmunity through maintenance of B cell tolerance .