FCRL4 Antibody

What is FCRL4 and why is it significant for immunological research?

FCRL4 is an immunoregulatory receptor belonging to the Fc receptor-like (FCRL) family. In humans, the canonical protein has 515 amino acid residues with a molecular mass of 57.2 kDa and is localized in the cell membrane. It functions as an inhibitor of B-cell receptor signaling through recruitment of SHP1 and SHP2 tyrosine phosphatases to tyrosine-based inhibitory motif (ITIM) consensus sequences. FCRL4 is significant because it serves as a low-affinity receptor for systemic IgA (but not mucosal secretory IgA) and acts as a molecular switch between adaptive and innate immune responses by dampening BCR signaling while enhancing TLR signaling .

What are the common applications for FCRL4 antibodies in research?

FCRL4 antibodies are predominantly used for flow cytometry, Western blotting, and immunohistochemistry. They allow researchers to identify and isolate FCRL4-expressing memory B cells, particularly from lymphoid tissues such as tonsils, lymph nodes, and the appendix where FCRL4 is notably expressed. These antibodies can also be utilized for studying the inhibitory functions of FCRL4 on B-cell receptor signaling and for investigating its role in normal immune responses versus pathological conditions like HIV infection, malaria, and rheumatoid arthritis .

What is the expression pattern of FCRL4 in healthy individuals?

In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) that are predominantly localized in sub-epithelial regions of lymphoid tissues, particularly in the mucosa-associated lymphoid tissue. FCRL4+ memory B cells represent a distinct population with a unique gene expression profile that includes high expression of CD20, CD11c, CD40, and several chemokine receptors (CCR1, CCR5, and CCR6). Additionally, FCRL4+ memory B cells in peripheral blood express the phenotype CD21loCD11chi, though at lower levels compared to those found in inflamed tissues or lymphoid organs .

What tissue samples are optimal for FCRL4 antibody detection?

Human tonsil tissue represents an optimal sample for FCRL4 antibody detection due to the significant presence of FCRL4+ memory B cells in this lymphoid organ. Other suitable tissues include lymph nodes and the appendix. In disease states, synovial fluid from patients with rheumatoid arthritis and peripheral blood mononuclear cells (PBMCs) from individuals with certain chronic infections may also contain detectable populations of FCRL4+ B cells, though expression levels in peripheral blood are typically lower than in tissue-resident populations .

How can researchers differentiate between FCRL4+ and FCRL4- memory B cell populations?

The gold standard for differentiating FCRL4+ from FCRL4- memory B cells is through flow cytometric analysis using fluorescently labeled anti-FCRL4 antibodies (clone 1A3 provides stronger signal compared to clone 4-2A6). For a comprehensive phenotypic characterization, researchers should use a panel including CD19, CD38, IgD, and IgM markers. FCRL4+ memory B cells can be identified as CD19+CD38-IgD-IgM- cells that stain positively for FCRL4. Additionally, these cells typically co-express higher levels of CD11c, CD20, CD40, and CCR6 compared to their FCRL4- counterparts, providing further discriminatory markers .

What are the key differences in antibody repertoires between FCRL4+ and FCRL4- memory B cells?

FCRL4+ memory B cells display several distinct characteristics in their antibody repertoires compared to FCRL4- memory B cells:

• Lower levels of somatic mutations in both heavy and light chain sequences

• Similar frequencies of variable gene family usage

• Slightly longer CDR-H3 regions (complementarity-determining region 3 of heavy chains)

• Similar CDR-H3 hydrophobicity

• Comparable J gene usage patterns

Importantly, antibodies derived from FCRL4+ memory B cells show increased reactivity to commensal microbiota antigens, although this is not due to polyreactive binding characteristics .

How can FCRL4 antibodies be used to study the role of FCRL4+ memory B cells in disease pathology?

FCRL4 antibodies provide crucial tools for investigating the expansion and altered functionality of FCRL4+ memory B cells in various disease contexts:

What experimental approaches can reveal the functional significance of FCRL4 in B cell biology?

Several experimental approaches can elucidate FCRL4's functional significance:

• Signaling studies: Using anti-IgM/IgG to stimulate BCR signaling in FCRL4+ versus FCRL4- B cells and measuring calcium flux, phosphorylation of downstream signaling molecules, and gene expression changes.

• Receptor-ligand interaction assays: Analyzing IgA binding to FCRL4 using purified IgA and recombinant FCRL4, or by treating cells with human colostrum IgA and measuring surface capture.

• Genetic manipulation: CRISPR/Cas9-mediated knockout or overexpression of FCRL4 in B cell lines to assess effects on receptor signaling, antibody production, and response to stimuli.

• Ex vivo stimulation: Treating FCRL4+ B cells with TLR7/9 agonists and TGF-β to assess changes in FCRL4 expression and subsequent functional alterations.

• Co-culture experiments: Establishing co-cultures with T cells or dendritic cells to investigate how FCRL4+ B cells interact with other immune cells .

What sample preparation techniques optimize FCRL4 detection in immunohistochemistry?

For optimal FCRL4 detection in immunohistochemistry, the following protocol is recommended:

• Fix tissue samples in 10% neutral buffered formalin and embed in paraffin.

• Section tissues at 5 μm thickness.

• Perform heat-induced epitope retrieval using an antigen retrieval reagent with basic pH.

• Block endogenous peroxidase activity with 3% hydrogen peroxide.

• Apply primary anti-FCRL4 antibody at 1 μg/mL and incubate for 1 hour at room temperature.

• Use an appropriate secondary antibody system, such as Anti-Goat IgG HRP Polymer.

• Develop with DAB (brown) and counterstain with hematoxylin (blue).

This approach has been validated for detection of FCRL4 in human tonsil sections, where specific staining localizes to cell membranes in lymphocytes .

What are the critical considerations when selecting anti-FCRL4 antibodies for specific applications?

When selecting anti-FCRL4 antibodies, researchers should consider:

• Epitope specificity: Antibodies targeting different epitopes (e.g., between Gln16-Asp385) may have different detection capabilities and functional effects.

• Application compatibility: Ensure the antibody has been validated for your specific application (flow cytometry, IHC, WB, ICC).

• Species reactivity: Most commercial antibodies target human FCRL4, but check cross-reactivity if working with animal models (documented orthologs exist in mouse, bovine, and chimpanzee).

• Clonality: Polyclonal antibodies may provide better detection sensitivity but potentially lower specificity compared to monoclonals.

• Conjugation: For flow cytometry, consider directly conjugated antibodies to minimize background and simplify staining protocols.

• Clone selection: For flow cytometry, clone 1A3 provides stronger signal compared to clone 4-2A6 .

How should researchers optimize flow cytometry protocols for FCRL4 detection?

For optimal flow cytometry detection of FCRL4:

• Prepare single-cell suspensions from tissues (tonsil, lymph node) or isolate PBMCs from blood using density gradient centrifugation.

• Use a comprehensive staining panel: CD19 (B cell marker), CD38 (activation marker), IgD and IgM (to exclude naive B cells), and additional markers like CD11c and CD21 to further characterize FCRL4+ populations.

• Include appropriate isotype controls to set gates correctly.

• Consider using clone 1A3 anti-FCRL4 antibody for stronger signal intensity.

• When analyzing peripheral blood samples, be aware that FCRL4 expression is typically lower than in tissue-resident B cells.

• For sorting FCRL4+ cells for downstream applications (e.g., single-cell RT-PCR), collect cells directly into appropriate buffers containing RNase inhibitors to preserve RNA integrity .

What controls are essential when studying IgA binding to FCRL4?

When investigating IgA binding to FCRL4, these controls are essential:

• Negative control: FCRL4- B cells should show minimal IgA binding compared to FCRL4+ cells.

• Blocking control: Pre-incubation with anti-FCRL4 antibodies should diminish IgA binding if the interaction is specific.

• Removal control: Treatment with 0.1M glycine buffer (pH3) should remove previously bound IgA from cell surfaces.

• Specificity control: Testing binding of other immunoglobulin isotypes (IgG, IgM) to confirm specificity of the FCRL4-IgA interaction.

• Expression threshold control: Compare cells with different FCRL4 expression levels to determine the threshold required for detectable IgA binding.

These controls help confirm that observed IgA binding is specifically mediated by FCRL4 rather than by other receptors or non-specific interactions .

How is FCRL4 expression altered in pathological conditions, and what implications does this have for research?

FCRL4 expression undergoes significant alterations in various pathological conditions:

These alterations suggest that FCRL4 antibodies can serve as valuable tools for monitoring disease progression, studying mechanism of pathogenesis, and potentially as biomarkers for treatment response .

What is the relationship between FCRL4 expression and commensal microbiota reactivity?

FCRL4+ memory B cells display increased reactivity to commensal microbiota antigens compared to FCRL4- memory B cells. This characteristic is not due to polyreactive binding properties but represents genuine specificity for microbial antigens. This finding links the immunoregulatory function of FCRL4 with recognition of commensal bacteria, suggesting that FCRL4 may play a role in maintaining tolerance to the microbiome while allowing for appropriate responses to pathogens. Researchers studying host-microbiome interactions should consider incorporating FCRL4 antibodies into their experimental designs to assess whether alterations in commensal populations influence FCRL4+ B cell responses .

How can FCRL4 antibodies be used to study exhausted B cell phenotypes in cancer?

FCRL4 antibodies represent valuable tools for studying exhausted B cell phenotypes in cancer:

What are common challenges in detecting FCRL4 in peripheral blood samples and how can they be addressed?

Common challenges in detecting FCRL4 in peripheral blood include:

• Low expression levels: FCRL4 expression is typically lower in peripheral blood compared to tissue-resident B cells.

  • Solution: Use highly sensitive detection methods, brighter fluorophores for flow cytometry, and consider enriching for B cells before analysis.

• Variable expression across subjects: FCRL4 expression can vary significantly between individuals.

  • Solution: Include appropriate healthy controls matched for age and sex, and consider analyzing larger sample sizes.

• Distinguishing from other FCRL family members: Cross-reactivity with other FCRL family proteins can occur.

  • Solution: Use highly specific antibody clones and validate with positive and negative controls.

• Threshold for functional relevance: Low-level FCRL4 expression may be insufficient for IgA binding.

  • Solution: Perform functional assays (such as IgA binding) alongside expression analysis to determine functional relevance of detected FCRL4 .

How can researchers distinguish between true FCRL4+ memory B cells and other cell populations with potential cross-reactivity?

To accurately identify true FCRL4+ memory B cells and avoid misidentification:

• Use a comprehensive panel of markers:

  • Positive markers: CD19, CD20 (B cell lineage)

  • Negative markers: CD38, IgD, IgM (to exclude naive B cells and plasmablasts)

  • Confirmatory markers: CD11c, CCR6 (typically co-expressed with FCRL4)

• Include isotype controls to set appropriate gates and minimize false positives.

• Validate findings with orthogonal methods, such as immunohistochemistry or RT-PCR for FCRL4 mRNA expression.

• Consider functional validation through IgA binding assays, as FCRL4 serves as a low-affinity IgA receptor.

• When analyzing tissue samples, confirm anatomical localization (FCRL4+ cells typically localize to sub-epithelial regions in lymphoid tissues) .

What factors can affect FCRL4 detection in experimental settings?

Several factors can impact FCRL4 detection:

• Sample handling: Delay in processing tissue or blood samples can affect cell viability and surface marker expression.

  • Recommendation: Process samples promptly and maintain strict protocols for storage and handling.

• Fixation conditions: Excessive fixation can mask epitopes.

  • Recommendation: Optimize fixation time and conditions; for paraffin-embedded tissues, ensure proper antigen retrieval.

• Antibody clone selection: Different clones recognize different epitopes with varying efficiency.

  • Recommendation: For flow cytometry, clone 1A3 provides stronger signal than clone 4-2A6.

• Stimulation status: In vitro stimulation with TLR agonists or TGF-β can upregulate FCRL4 expression.

  • Recommendation: Consider the activation state of cells when interpreting results and maintain consistent culture conditions.

• Disease state: Expression levels vary significantly between healthy and disease states.

  • Recommendation: Include appropriate controls and consider the clinical context when interpreting results .