FCRL3 Antibody

What is FCRL3 and what cell types express it?

FCRL3 (Fc Receptor-Like 3) is a transmembrane glycoprotein that belongs to the Fc receptor-like family of proteins. It is primarily expressed on specific immune cell populations including natural killer (NK) cells and subsets of T and B lymphocytes. Flow cytometric analysis reveals that FCRL3 is expressed at varying levels on different lymphocyte populations, with notably higher expression on regulatory T cells (Tregs) compared to CD8+ and TCRγδ+ T cells . Within the B cell compartment, FCRL3 expression can be detected on various subsets including follicular B cells, though expression levels vary significantly based on activation state and disease context .

The protein contains both immunoreceptor tyrosine-based inhibition motifs (ITIMs) and activation motifs, suggesting a potential dual role in immune regulation. This complex signaling capability makes FCRL3 particularly interesting in the context of autoimmune disease research, where dysregulated immune signaling contributes to pathogenesis .

How should researchers detect FCRL3 expression by flow cytometry?

For optimal detection of FCRL3 by flow cytometry, researchers should follow this methodological approach:

• Sample preparation: Isolate peripheral blood mononuclear cells (PBMCs) through density gradient centrifugation and resuspend cells in PBS containing 2% FBS .

• Antibody selection: Use a validated anti-FCRL3 (CD307c) monoclonal antibody such as clone H5, which recognizes an epitope within the extracellular domain of FCRL3 . For multicolor flow cytometry, PE-conjugated antibodies provide excellent signal separation.

• Staining protocol:

  • Incubate cells with surface marker antibodies for 30 minutes on ice

  • Wash three times with PBS containing 2% FBS

  • For FCRL3 detection with biotinylated antibodies, perform secondary staining with streptavidin-Qdot655

  • Wash three more times and fix in 1% paraformaldehyde

• Controls and gating strategy:

  • Use appropriate isotype controls to establish background staining levels

  • Include a fluorescence-minus-one (FMO) control for accurate gate setting

  • For FCRL3+ cell identification, set gates using negative control staining as there is a continuum of expression rather than distinct positive/negative populations

  • Use 10 μL of reagent per 100 μL of whole blood or per 10^6 cells in suspension

• Panel design considerations: Include dead cell exclusion dye (e.g., Aqua Live/Dead amine reactive stain) and markers to identify specific lymphocyte subsets such as anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD127, and anti-TCRγδ antibodies .

For intracellular FoxP3 staining to identify Tregs, use specialized fixation and permeabilization buffers following the manufacturer's protocol .

What is the relationship between FCRL3 genetic variants and protein expression?

The FCRL3 gene contains several functionally relevant polymorphisms, with the -169 T→C variant (rs7528684, also known as FCRL3_3) being particularly significant for protein expression. This relationship between genotype and expression follows specific patterns:

• Allele-specific expression patterns: Individuals carrying the FCRL3 -169C allele (either C/C or C/T genotype) express significantly higher levels of FCRL3 protein on their T regulatory cells, CD8+ T cells, and TCRγδ+ T cells compared to individuals with the T/T genotype . This difference in expression provides a direct molecular link between genetic variation and immune phenotype.

• Mechanism of enhanced expression: The -169 T→C substitution is located in the promoter region of the FCRL3 gene and results in enhanced NFκB binding capacity, which leads to increased FCRL3 promoter activity . This molecular mechanism explains how the polymorphism directly affects protein expression levels.

• Cell type-specific expression: While the -169C allele enhances FCRL3 expression across multiple lymphocyte subsets, the magnitude of effect varies. Studies have shown that among T cell subsets, FCRL3 expression is significantly higher on Tregs compared to CD8+ and TCRγδ+ T cells, regardless of genotype . This suggests additional cell type-specific regulatory mechanisms beyond the -169 polymorphism.

• B cell expression: Similar to T cells, B lymphocytes from individuals with the -169C variant demonstrate higher surface FCRL3 expression compared to those with the T/T genotype . This consistent pattern across different immune cell populations highlights the fundamental importance of this promoter variant in controlling FCRL3 expression.

Understanding this genotype-phenotype relationship is essential for interpreting FCRL3 expression data in research studies, particularly when investigating autoimmune conditions with heterogeneous genetic backgrounds.

How does FCRL3 expression correlate with autoimmune disease activity?

FCRL3 expression demonstrates significant correlations with autoimmune disease activity, particularly in rheumatoid arthritis (RA), with complex relationships that vary based on both genetic background and specific T cell subpopulations:

• Correlation with disease activity measures: In RA patients carrying the FCRL3 -169C allele, higher FCRL3 expression on T regulatory cells (Tregs) correlates significantly with multiple objective disease activity measures including Disease Activity Score (DAS), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels . This correlation was not observed in RA patients with the T/T genotype, suggesting a genotype-specific effect.

• Independent prediction of disease activity: Multivariate regression analysis has demonstrated that FCRL3 expression on Tregs serves as an independent predictor of RA disease activity (as measured by DAS and ESR), even when controlling for FCRL3 -169 genotype . This indicates that protein expression levels provide prognostic information beyond genotyping alone.

• Cell type-specific correlations: FCRL3 expression on CD8+ T cells and TCRγδ+ T cells also correlates with disease activity measures in patients with the FCRL3 -169C allele, though these correlations are generally weaker than those observed with Treg expression . This suggests that Treg dysfunction may be particularly important in the pathogenesis of autoimmune disease in genetically susceptible individuals.

• ACPA status and FCRL3 association: Studies have identified a significant association between FCRL3 variants and ACPA (anti-citrullinated protein antibodies) positivity in RA, suggesting that FCRL3 may contribute to pathogenesis through modulation of autoantibody production . This finding highlights FCRL3's potential role in B cell-mediated autoimmunity in addition to its effects on T cell function.

These correlations provide important evidence linking FCRL3 expression to disease mechanisms and suggest that FCRL3 may be a promising biomarker for disease activity and progression in autoimmune conditions.

What are the functional consequences of elevated FCRL3 expression on immune cells?

Elevated FCRL3 expression on immune cells produces several significant functional consequences that contribute to autoimmune pathogenesis:

These functional consequences highlight the mechanistic importance of FCRL3 in autoimmune disease and suggest potential therapeutic targets for intervention.

How does FCRL3 genetic variation contribute to erosive disease in rheumatoid arthritis?

The relationship between FCRL3 genetic variation and erosive disease in rheumatoid arthritis (RA) involves multiple interconnected mechanisms:

• Association with disease progression: Patients with erosive RA disease express higher levels of FcRL3 on their regulatory T cells compared to patients with non-erosive disease . Furthermore, the FCRL3 -169C allele is overrepresented in patients with erosive RA disease , suggesting that this genetic variant predisposes to more aggressive disease phenotypes.

• Mechanism of joint destruction: The link between FCRL3 and erosive disease likely involves impaired immune regulation. Higher FCRL3 expression on Tregs is associated with diminished suppressive capacity, potentially allowing unchecked inflammatory responses that drive joint destruction . Additionally, FCRL3 may influence osteoclast activation through its effects on inflammatory cytokine production.

• Autoantibody-mediated pathways: FCRL3 genetic variants show strong associations with autoantibody-positive RA . Both RF (rheumatoid factor) and ACPA (anti-citrullinated protein antibodies) positivity correlate with FCRL3 polymorphisms , and these autoantibodies are known risk factors for erosive disease. This suggests that FCRL3 may contribute to erosive disease through modulation of autoantibody production or function.

• Independent prediction of radiographic progression: Polymorphisms in FCRL3 have been reported to independently predict radiographic progression in RA , suggesting that genetic testing for FCRL3 variants may have clinical utility in identifying patients at high risk for erosive disease who might benefit from more aggressive therapeutic approaches.

• Population-specific effects: The association between FCRL3 variants and RA susceptibility appears stronger in Asian populations compared to other ethnic groups , highlighting the importance of considering genetic ancestry when evaluating FCRL3 as a risk factor for erosive disease.

These findings position FCRL3 as a key player in the pathogenesis of erosive joint disease in RA and suggest that targeting FCRL3 or its downstream pathways might represent a promising therapeutic strategy for preventing or limiting erosive progression.

How should researchers design experiments to study FCRL3 function in human samples?

Designing robust experiments to study FCRL3 function in human samples requires careful consideration of several methodological aspects:

• Sample stratification and controls:

  • Stratify subjects by FCRL3 genotype (-169 T→C, rs7528684) to account for genotype-specific effects on expression and function

  • Include age-matched and gender-matched healthy controls alongside disease cases

  • For autoimmune disease research, further stratify patients by autoantibody status (e.g., ACPA-positive vs. ACPA-negative in RA)

  • Consider disease duration and treatment status as potential confounding variables

• Cell isolation techniques:

  • For T cell studies, isolate specific T cell subsets (Tregs, CD8+, TCRγδ+) using fluorescence-activated cell sorting (FACS) or magnetic separation

  • Further separate Tregs into FCRL3-high and FCRL3-low populations for comparative functional studies

  • For B cell studies, separate naive, memory, and plasmablast populations to account for differential FCRL3 expression across B cell development stages

• Functional assays:

  • Assess Treg suppressive function using co-culture assays with CFSE-labeled responder T cells

  • Evaluate BCR signaling in B cells with or without FCRL3 engagement using phosphoflow cytometry

  • Assess cytokine production through intracellular staining and flow cytometry or multiplex bead arrays

  • Investigate interactions between TLR and FCRL3 signaling using combinatorial stimulation approaches

• Molecular analysis approaches:

  • Perform chromatin immunoprecipitation (ChIP) to assess NFκB binding to the FCRL3 promoter in different genotypes

  • Use siRNA or CRISPR approaches to modulate FCRL3 expression levels and assess functional consequences

  • Apply RNA-seq to identify FCRL3-dependent gene expression programs in relevant cell populations

• Statistical considerations:

  • Power calculations should account for genotype frequency in the target population

  • Use multivariate analysis to control for potential confounding variables

  • Consider correction for multiple testing when assessing multiple parameters or cell types

These methodological approaches will help researchers design rigorous experiments to elucidate FCRL3 function in human samples while accounting for genetic and phenotypic heterogeneity.

What are the optimal methods for detecting FCRL3 in tissue sections?

For optimal detection of FCRL3 in tissue sections, researchers should consider the following comprehensive approach:

• Tissue preparation and fixation:

  • For formalin-fixed paraffin-embedded (FFPE) samples: Fix tissues in 10% neutral buffered formalin for 24-48 hours, followed by paraffin embedding

  • For frozen sections: Snap-freeze tissue in OCT compound using liquid nitrogen-cooled isopentane

  • Section thickness should be optimized (typically 4-5 μm for FFPE and 6-8 μm for frozen sections)

  • For synovial tissues from RA patients, consider sampling from multiple regions to account for heterogeneity in inflammatory infiltrates

• Antigen retrieval methods:

  • For FFPE sections: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval conditions (temperature, time, buffer) specifically for the anti-FCRL3 antibody being used

  • For frozen sections, fixation in acetone or 4% paraformaldehyde prior to staining may improve morphology while preserving antigenicity

• Antibody selection and validation:

  • Use well-characterized antibodies with demonstrated specificity for FCRL3

  • Polyclonal antibodies targeting amino acids 18-180 or 122-222 of FCRL3 are available for immunohistochemistry applications

  • Validate antibody specificity using appropriate positive and negative control tissues

  • Consider using multiple antibodies targeting different epitopes to confirm staining patterns

• Detection systems:

  • For chromogenic detection: Use polymer-based detection systems with DAB or AEC substrates

  • For fluorescent detection: Use tyramide signal amplification for low-abundance targets

  • For multiplex staining: Consider sequential multiplex immunohistochemistry or multiplex immunofluorescence to co-localize FCRL3 with cell type-specific markers

  • When available, use automated staining platforms to improve reproducibility

• Analysis approaches:

  • Quantify staining using digital image analysis with appropriate algorithms for membrane staining

  • For research applications, consider whole slide scanning and automated quantification

  • Score intensity on a scale (0-3+) and calculate H-scores (intensity × percentage positive cells)

  • Validate scoring between multiple observers to ensure reproducibility

These optimized methodological approaches will enhance detection sensitivity and specificity for FCRL3 in tissue sections, providing valuable insights into its expression patterns in disease states.

How can researchers resolve discrepancies in FCRL3 association studies across different populations?

Resolving discrepancies in FCRL3 association studies across different populations requires a systematic approach that addresses multiple potential sources of variation:

• Meta-analytical approaches:

  • Conduct comprehensive meta-analyses that incorporate both published and unpublished data

  • Use random-effects models to account for between-study heterogeneity

  • Perform sensitivity analyses to identify influential studies or outliers

  • Implement Forest plots to visualize effect sizes across different populations

  • For example, meta-analyses have confirmed stronger associations between FCRL3 polymorphisms and RA risk in Asian populations compared to other ethnic groups

• Population stratification considerations:

  • Use principal component analysis or other methods to control for population stratification within studies

  • Analyze linkage disequilibrium patterns across different populations, as FCRL3 variants may tag different causal variants in different ancestral groups

  • Consider haplotype-based analyses rather than single-SNP approaches

  • Account for differences in allele frequencies across populations when interpreting effect sizes

• Phenotype harmonization:

  • Standardize disease definitions and classification criteria across studies

  • Stratify analyses by autoantibody status (e.g., ACPA-positive vs. ACPA-negative RA)

  • Consider disease severity and progression as separate phenotypes

  • The discrepant findings between studies showing association with ACPA-positive versus ACPA-negative RA may reflect heterogeneity in disease classification

• Integrative genomic approaches:

  • Incorporate expression quantitative trait loci (eQTL) data to link genetic variation to gene expression

  • Use databases like ImmuNexUT that contain RNA-seq data from immune cells of patients with immune-mediated diseases

  • Perform functional genomic annotations to identify potential causal variants

  • For instance, eQTL analyses have shown effects of genetic variants on FCRL3 expression across different immune cell types in autoimmune disease patients

• Replication and validation strategies:

  • Design replication studies with adequate power based on observed effect sizes

  • Consider trans-ethnic replication to assess consistency across populations

  • When replication fails, investigate potential effect modifiers such as environmental factors or gene-gene interactions

  • Utilize two-stage study designs with discovery and validation cohorts

What are the emerging therapeutic targets based on FCRL3 biology?

Based on FCRL3 biology, several promising therapeutic targets have emerged that hold potential for treating autoimmune diseases:

• Direct FCRL3 modulation:

  • Blocking antibodies targeting specific extracellular domains of FCRL3 could modulate its signaling functions

  • Small molecule inhibitors that interfere with FCRL3's interaction with its ligands represent another approach

  • These strategies aim to normalize Treg function in patients with elevated FCRL3 expression due to the FCRL3 -169C allele

• Targeting downstream signaling pathways:

  • Selective inhibition of signaling molecules in FCRL3-activated pathways

  • Given FCRL3's association with enhanced NFκB binding and increased promoter activity , targeted NFκB pathway modulators may prove beneficial

  • Interventions targeting the interplay between FCRL3 and TLR signaling could address the disruption of B cell tolerance observed in experimental models

• B cell-targeted approaches:

  • Since FCRL3/5 upregulation contributes to autoimmune disease pathogenesis by disrupting B cell anergy , selective targeting of FCRL3-high B cells could restore tolerance

  • Therapies aimed at removing or re-establishing anergy in autoreactive B cells expressing high levels of FCRL3

  • Development of antibody-drug conjugates specifically targeting FCRL3-expressing pathogenic B cells

• Treg enhancement strategies:

  • Given that higher FcRL3 expression on Tregs correlates with impaired suppressive function , approaches to restore Treg function in FCRL3-high individuals

  • Low-dose IL-2 therapy, which preferentially expands Tregs, might be particularly beneficial in patients with the FCRL3 risk genotype

  • Ex vivo expansion and reinfusion of autologous Tregs with modified FCRL3 expression or signaling

• Personalized medicine approaches:

  • Stratification of patients based on FCRL3 genotype and expression patterns to guide therapeutic decisions

  • More aggressive treatment approaches for patients with the FCRL3 -169C allele who are at higher risk for erosive disease

  • Combination therapies targeting both FCRL3-dependent and independent disease mechanisms

These emerging therapeutic approaches leverage our growing understanding of FCRL3 biology and hold promise for more targeted treatments of autoimmune diseases with potentially fewer side effects than current broad immunosuppressive therapies.

How does FCRL3 interact with TLR signaling in autoimmune pathogenesis?

The interaction between FCRL3 and Toll-like receptor (TLR) signaling represents a critical nexus in autoimmune pathogenesis:

• Dual modulation of immune signaling:

  • While FCRL3 can inhibit B cell receptor (BCR) signaling through its immunoreceptor tyrosine-based inhibitory motifs (ITIMs), it paradoxically promotes innate TLR9 signaling

  • This dual regulatory capacity allows FCRL3 to fine-tune immune responses in a context-dependent manner

  • In autoimmune settings, dysregulated FCRL3 expression may disrupt the normal balance between inhibitory and activating signals

• Breaking of B cell tolerance:

  • Experimental evidence suggests that FCRL3/5 overexpression results in breaking B cell tolerance in model systems

  • A proposed mechanism involves cooperative signaling between TLRs and FCRL3/5 that reinforces BCR-mediated activation of anergic B cells upon self-antigen recognition

  • This suggests a signaling hierarchy involving FCRL3, TLR, and BCR, though the detailed molecular interplay remains incompletely understood

• NF-κB pathway involvement:

  • The FCRL3 -169C variant leads to enhanced NFκB binding and increased FCRL3 promoter activity

  • Simultaneous stimulation of FCRL3 and TLR9 activates B cells via the NF-κB pathway

  • This creates a potential positive feedback loop where increased FCRL3 expression enhances NF-κB signaling, which further increases FCRL3 expression

• Cell type-specific effects:

  • In B cells, FCRL3-TLR interaction may promote autoreactive B cell activation and autoantibody production

  • In T regulatory cells, which also express both FCRL3 and TLRs, this interaction may impair suppressive function

  • These cell type-specific effects collectively contribute to breaking self-tolerance

• Therapeutic implications:

  • Understanding the FCRL3-TLR signaling axis suggests targeted therapeutic approaches

  • Selective inhibition of this interaction could potentially restore B cell tolerance without broadly suppressing immune function

  • Dual targeting of both pathways might be more effective than targeting either pathway alone

This complex interplay between FCRL3 and TLR signaling represents a promising area for further research and therapeutic development in autoimmune diseases.

What are the key unresolved questions in FCRL3 research?

Despite significant progress in understanding FCRL3 biology and its role in autoimmune diseases, several key questions remain unresolved:

• Endogenous ligand identification:

  • While human FCRL3 has been reported to bind to secretory IgA, the endogenous ligands for mouse Fcrl5 remain unidentified

  • Discovering these natural ligands would provide crucial insights into FCRL3's physiological function

  • High-throughput screening approaches and protein-protein interaction studies are needed to identify potential binding partners

• Mechanistic basis of genotype-phenotype correlations:

  • The precise molecular mechanisms linking the FCRL3 -169C variant to increased disease risk require further elucidation

  • How increased FCRL3 expression specifically alters Treg and B cell function at the molecular level remains incompletely understood

  • Systems biology approaches integrating genomic, transcriptomic, and proteomic data may help resolve these questions

• Therapeutic targeting strategies:

  • The optimal approach for therapeutically targeting FCRL3 in autoimmune diseases remains undefined

  • Should interventions aim to modulate FCRL3 expression, block its function, or target downstream pathways?

  • What cell types should be targeted, and would broad FCRL3 inhibition have unintended consequences?

• Resolution of conflicting association studies:

  • The inconsistent findings regarding FCRL3 association with ACPA-positive versus ACPA-negative RA need resolution

  • Large-scale, well-powered studies with standardized phenotyping across diverse populations are required

  • Integration of genetic data with functional studies may help explain population-specific effects

• Developmental and environmental influences:

  • How environmental factors influence FCRL3 expression and function remains largely unexplored

  • The developmental regulation of FCRL3 expression during immune cell differentiation and maturation warrants investigation

  • Studies examining FCRL3 expression in response to various stimuli and in different disease states may provide important insights

Addressing these unresolved questions will require multidisciplinary approaches combining genetics, molecular biology, immunology, and clinical research to fully elucidate FCRL3's role in health and disease.

How can transcriptomic and proteomic approaches advance FCRL3 research?

Advanced transcriptomic and proteomic approaches offer powerful tools to address complex questions in FCRL3 research:

• Single-cell RNA sequencing applications:

  • Single-cell transcriptomics can reveal heterogeneity in FCRL3 expression across immune cell populations and states

  • This approach can identify novel FCRL3-expressing cell subsets with potential roles in autoimmunity

  • Trajectory analysis can track changes in FCRL3 expression during cell differentiation or activation

  • For example, single-cell RNA-seq of B cell populations could identify transitional states where FCRL3 upregulation correlates with loss of anergy

• Proteomics for signaling pathway elucidation:

  • Phosphoproteomics can map signaling networks downstream of FCRL3 activation

  • Proximity-based labeling approaches (BioID, APEX) can identify proteins that physically interact with FCRL3

  • Comparison of signaling pathways between cells with different FCRL3 genotypes (-169C vs T/T) could reveal mechanisms underlying genotype-phenotype correlations

  • Temporal proteomic profiling following FCRL3 engagement could distinguish primary from secondary signaling events

• Multi-omics integration strategies:

  • Integration of genomic, transcriptomic, and proteomic data can provide comprehensive insights

  • Expression quantitative trait loci (eQTL) analysis can link genetic variants to gene expression patterns

  • As demonstrated in the ImmuNexUT database, eQTL effects on FCRL3/5 expression in immune cells correlate with autoimmune disease status

  • Network analysis can identify key regulatory hubs and potential therapeutic targets

• Spatial transcriptomics and proteomics:

  • Spatial technologies can map FCRL3 expression within tissues like synovium in RA

  • These approaches can reveal microanatomical relationships between FCRL3-expressing cells and other immune or stromal cells

  • Understanding spatial context may explain tissue-specific manifestations of autoimmune diseases

• Functional genomics screening:

  • CRISPR screens can identify genes that modify FCRL3 expression or function

  • Pooled CRISPR activation/inhibition approaches can systematically assess the impact of modulating FCRL3-related pathways

  • Results from such screens could identify novel therapeutic targets within the FCRL3 signaling network