FCGR2B Antibody

What is FCGR2B and why is it significant in immunological research?

FCGR2B (CD32B) is the only inhibitory Fc receptor in the Fc gamma receptor family. It plays crucial roles in:

• Inhibiting functions of activating FcγRs

• Regulating antibody production by B cells

• Controlling phagocytosis of immune complexes

• Modulating inflammatory responses

The significance of FCGR2B lies in its counterbalancing role against activating Fc receptors through its Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM). This receptor is particularly important in autoimmunity studies, as defects in its expression or function are associated with conditions like systemic lupus erythematosus .

What are the key differences between FCGR2B isoforms and how should researchers account for them?

There are two major isoforms of FCGR2B that researchers must distinguish between:

Both isoforms contain the ITIM domain in their cytoplasmic regions, but their differential capacity for internalization affects experimental outcomes. When designing experiments with FCGR2B antibodies, researchers should consider:

• Cell type-specific expression patterns

• Different functional readouts depending on isoform presence

• Potential cross-reactivity with other CD32 family members (FCGR2A, FCGR2C)

How should researchers optimize antibody dilutions for different applications?

Based on commercial antibody specifications, researchers should follow these optimization guidelines:

Crucial optimization steps include:

• Perform antibody titration for each new lot and application

• Include appropriate positive controls (placenta tissue, Raji cells show high expression)

• Use antigen retrieval with TE buffer pH 9.0 for IHC applications

• Validate specificity with known FCGR2B-expressing tissues

What are the most reliable methods to distinguish FCGR2B from other closely related Fc receptors?

Due to high sequence homology among Fc receptor family members (particularly CD32 subtypes), researchers should employ multiple strategies:

• Antibody Selection Strategy:

  • Choose antibodies targeting unique epitopes (C-terminal region differences)

  • Verify specificity using FCGR2B knockout controls

  • Test for cross-reactivity with recombinant FCGR2A and FCGR2C

• Validation Approaches:

  • Western blotting under reducing conditions (FCGR2B appears at ~36 kDa)

  • Immunoprecipitation followed by mass spectrometry

  • RT-PCR using isoform-specific primers

  • Phosphorylation-specific antibodies (pTyr292) for activated FCGR2B detection

• Cell Type Controls:
  Based on differential expression patterns, use:

  • B cells (high FCGR2B1 expression)

  • Basophils (high FCGR2B2 expression)

  • Myeloid dendritic cells (co-express FCGR2A and FCGR2B)

How can researchers effectively study FCGR2B-mediated signaling in primary B cells?

To study FCGR2B inhibitory signaling:

• Experimental Approach:

  • Use Fab'2 fragments of anti-IgM antibodies for BCR stimulation

  • Compare with intact anti-IgM antibodies (engages both BCR and FCGR2B)

  • Measure activation markers (CD80, CD86, MHC class II)

• Signaling Pathway Analysis:

  • Monitor phosphorylation of:

    • SHIP1/SHIP2 (increased with FCGR2B engagement)

    • Erk1/2 (decreased phosphorylation with FCGR2B engagement)

    • Calcium flux (reduced with FCGR2B triggering)

• Technical Controls:

  • FCGR2B-knockout cells or FCGR2B-blocking antibodies

  • Titration of stimulation conditions

  • Compare responses between marginal zone B cells and follicular B cells

Research data indicates marginal zone B cells show stronger FCGR2B-mediated inhibition compared to follicular B cells, with significant effects on Erk1/2 phosphorylation and SHIP1 recruitment .

How does FCGR2B expression affect responses to therapeutic antibodies and what are the experimental approaches to study this?

FCGR2B expression has significant implications for therapeutic antibody efficacy, particularly in lymphomas:

Experimental approaches to investigate this phenomenon:

• In vitro models:

  • Compare internalization rates using fluorescently-labeled antibodies

  • Measure antibody-dependent cellular cytotoxicity (ADCC) in cells with varied FCGR2B expression

  • Assess cell death mechanisms (direct vs. immune-mediated)

• Patient-derived samples:

  • Correlate FCGR2B expression levels with clinical response data

  • Use progression-free survival (PFS) as a readout

  • Stratify by antibody type (type I vs. type II)

The research shows high FCGR2B expression is associated with significantly shorter progression-free survival in rituximab-treated diffuse large B-cell lymphoma patients, but not in obinutuzumab-treated patients .

What are the best approaches for investigating FCGR2B genetic variations and their functional consequences?

FCGR2B variants have been associated with autoimmune susceptibility and antibody responses. Researchers should consider:

• Key Genetic Variants to Study:

  • Promoter polymorphisms (-386G/C, -120T/A)

  • Coding variants (p.Ile232Thr) affecting lipid raft localization

• Functional Assessment Methods:

  • Quantitative RT-PCR for expression level differences

  • Flow cytometry to measure surface expression

  • Luciferase reporter assays for promoter activity

  • BCR co-crosslinking assays to measure inhibitory capacity

  • Lipid raft isolation and immunoblotting

  • Calcium flux measurements

• Readouts for Variant Impact:

  • B cell activation marker expression (CD80, CD86)

  • Antibody production in response to immunization

  • Signaling pathway activation (pSHIP, pErk)

  • Cell subset distribution (marginal zone vs. follicular B cells)

Research indicates the 2B.4 promoter haplotype (-386C, -120A) leads to higher transcriptional activity than the wild-type promoter, affecting inhibitory function without necessarily changing surface expression levels .

How can researchers address contradictory data regarding FCGR2B expression and function in different B cell subsets?

Contradictory findings regarding FCGR2B expression effects have been reported. To address these discrepancies, researchers should:

• Standardized Experimental Approaches:

  • Clearly define B cell subpopulations (using comprehensive marker panels)

  • Account for activation state (resting vs. activated)

  • Control for genetic background in mouse models

  • Consider temporal aspects of signaling events

• Reconciling Discrepancies:

  • The effect of 2B.4 promoter on B cell expression shows varied results:

    • Some studies report increased expression

    • Others suggest no effect or decreased expression

    • These differences may be subset-specific

• Recommended Controls:

  • Include multiple B cell subsets in parallel experiments

  • Use both knockout and conditional knockout models

  • Compare results across different stimulation conditions

  • Verify findings across species (mouse vs. human)

For example, while FCGR2B deficiency increases IgG3 production after immunization, the effects on T-dependent vs. T-independent immune responses show important differences that could explain some conflicting literature reports .

What are the critical validation steps for ensuring FCGR2B antibody specificity?

Given the high sequence homology between FCGR2 family members, validation is critical:

• Recommended Validation Tests:

  • Western blot against recombinant FCGR2A, FCGR2B, and FCGR2C

  • Testing on FCGR2B-knockout tissues/cells

  • Peptide competition assays

  • Immunoprecipitation followed by mass spectrometry

  • Testing on multiple positive and negative control tissues

• Known Cross-Reactivity Issues:

  • Many antibodies show approximately 20% cross-reactivity with FCGR2A

  • Some cross-react with mouse FCGR2B (~10%)

  • Cross-species reactivity should be experimentally verified

• Application-Specific Validation:

  • For IHC: Test on tonsillitis tissue, placenta, and spleen

  • For Western blot: Use human placenta tissue or Raji cells

  • For flow cytometry: Use B cells with confirmed FCGR2B expression

  • For phospho-specific antibodies: Validate with appropriate stimulation conditions

How can researchers accurately quantify FCGR2B expression levels in complex cell populations?

For accurate quantification in heterogeneous samples:

• Flow Cytometry Approach:

  • Use multi-parameter panels to identify specific cell populations

  • Include lineage markers (CD19, CD27, CD38 for B cells)

  • Use isotype controls and FMO (fluorescence minus one)

  • Consider both percentage positive and mean fluorescence intensity

• Gene Expression Analysis:

  • Design primers specific to FCGR2B (avoiding regions of homology with related genes)

  • Use multiple housekeeping genes for normalization

  • Consider droplet digital PCR for absolute quantification

  • Validate RNA-seq findings with qRT-PCR

• Protein Quantification:

  • Use calibrated flow cytometry with antibody-binding capacity beads

  • Consider targeted mass spectrometry for absolute quantification

  • Employ Western blot with recombinant protein standards

Research shows marginal zone B cells have the highest expression of FCGR2B in both mice and humans, which correlates with their enhanced sensitivity to FCGR2B-mediated inhibition .

How should researchers design experiments to study FCGR2B's role in autoimmune diseases?

To investigate FCGR2B in autoimmunity:

• Mouse Model Selection:

  • FCGR2B-knockout mice (spontaneous autoimmunity development)

  • FCGR2B-conditional knockout (cell-specific deletion)

  • NOTCH2/FCGR2B double-knockout (to study marginal zone B cell contribution)

• Experimental Readouts:

  • Autoantibody titers (particularly IgG3)

  • B cell subset distribution

  • Extrafollicular plasma cell formation

  • Germinal center dynamics

  • Tissue-specific immune complex deposition

• Mechanistic Investigations:

  • Compare T-dependent vs. T-independent responses

  • Examine affinity maturation (NP-specific high vs. low affinity antibodies)

  • Monitor BCR signaling thresholds

  • Assess calcium flux and activation marker expression

Recent research shows that FCGR2B-conditional knockout mice develop spontaneous increases in autoantibody titers, particularly IgG3, suggesting enhanced extrafollicular responses. This effect was lost in mice lacking marginal zone B cells (NOTCH2-deficient) .

What approaches should be used to investigate the role of FCGR2B in cancer therapeutic resistance?

To study FCGR2B-mediated resistance to antibody therapy:

• Clinical Sample Analysis:

  • Measure FCGR2B mRNA and protein levels in patient biopsies

  • Correlate with treatment response and survival

  • Compare different antibody therapies (e.g., rituximab vs. obinutuzumab)

• Functional Studies:

  • Assess antibody internalization rates

  • Measure direct cell death vs. immune-mediated mechanisms

  • Test combination approaches to overcome resistance

• Genetic Approaches:

  • Look for FCGR2B gene amplifications

  • Test the impact of FCGR2B silencing on therapeutic efficacy

  • Create FCGR2B-overexpressing cell lines

Results from DLBCL patients show higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS) in rituximab-treated patients (hazard ratio >1), while this effect was not observed with obinutuzumab treatment .

How can researchers effectively compare human and mouse FCGR2B function in translational studies?

For cross-species translational research:

• Key Similarities and Differences:

  Feature	Human	Mouse	Experimental Consideration
  Isoforms	FCGR2B1, FCGR2B2	Same	Comparable expression patterns
  Expression	Highest on MZ B cells	Highest on MZ B cells	Conserved cell type distribution
  Signaling	ITIM-mediated	ITIM-mediated	Similar inhibitory mechanisms
  Genetic variants	p.Ile232Thr, promoter	Different polymorphisms	Limited direct comparison

• Translational Strategies:

  • Use matched assays across species (same readouts)

  • Account for differences in antibody affinities

  • Consider humanized mouse models

  • Validate key findings in human primary cells

• Comparable Readouts:

  • BCR-induced calcium flux

  • Phospho-flow for signaling events (pSHIP, pErk)

  • Activation marker upregulation (CD80, CD86)

  • Antibody production in response to defined antigens

Research demonstrates that both human and mouse marginal zone B cells show the highest expression of FCGR2B and exhibit stronger inhibitory effects when FCGR2B is engaged compared to other B cell subsets, suggesting conserved regulatory mechanisms across species .