BAFF Human, His

What is BAFF and what is its functional significance in B cell biology?

BAFF (B-cell activating factor), also known as BLyS, TALL-1, CD257, or TNFSF13B, is a member of the TNF ligand superfamily that serves as a master regulator of peripheral B cell survival . Produced primarily by macrophages, dendritic cells, and T lymphocytes, BAFF is essential for B cell maturation and, together with IL-6, promotes immunoglobulin class-switching and plasma cell differentiation .

BAFF exerts its functions by binding to three different TNFR-like proteins expressed by B cells: BAFF-R (primary receptor), TACI, and BCMA . The binding to BAFF-R activates both classical and noncanonical NF-κB signaling pathways, leading to the expression of genes essential for B cell survival . This signaling cascade is crucial for B cell development, as evidenced by the finding that homozygous deletion of the BAFF-R gene in humans results in severe B lymphopenia due to arrested B cell development at the transitional B cell stage .

What structural forms does BAFF adopt, and how do they differ functionally?

Human BAFF exists in multiple structural configurations that affect its biological properties:

• Trimeric form: Like most TNF family ligands, BAFF can exist as a trimer .

• 60-mer form: Uniquely among TNF family members, BAFF can also assemble into a higher-order 60-mer structure composed of 20 trimers .

Under reducing conditions (SDS-PAGE), recombinant BAFF typically migrates at approximately 19 kDa, while under native conditions, the 60-mer structure has a molecular weight of approximately 1100 kDa . These structural differences are not merely academic distinctions but have functional implications, potentially affecting receptor clustering efficiency, signaling intensity, and tissue distribution patterns.

How do expression systems affect BAFF Human, His production and characteristics?

The choice of expression system significantly impacts the characteristics of recombinant BAFF Human, His:

When expressed in human 293 cells (HEK293), the protein migrates as 55-64 kDa under reducing conditions due to glycosylation, despite a calculated molecular weight of 53.8 kDa . This glycosylation may influence the protein's stability, half-life, and potentially its biological activity. Researchers should select the expression system based on their specific experimental requirements, particularly when studying receptor interactions or in vivo functions where glycosylation may be important.

What are the optimal reconstitution and storage conditions for BAFF Human, His?

Proper handling of BAFF Human, His is critical for maintaining biological activity:

Reconstitution:

• Lyophilized protein should be reconstituted with 100μl sterile water to achieve a concentration of 0.1mg/ml .

• For specific products, follow the reconstitution protocol provided in the Certificate of Analysis for optimal performance .

Storage Conditions:

• Store lyophilized product at -20°C or lower for long-term storage .

• Avoid repeated freeze-thaw cycles to maintain protein integrity and activity .

• Working aliquots should be prepared after reconstitution to minimize freeze-thaw cycles.

Formulation Considerations:

• BAFF Human, His is typically lyophilized from filtered solutions containing buffer components such as 25 mM MES, 150 mM NaCl, pH 6.0 with trehalose as a protectant .

• The buffer composition is optimized for protein stability and activity maintenance.

How can researchers verify the functional activity of BAFF Human, His?

Multiple complementary approaches can verify BAFF functionality:

• B cell survival/proliferation assays: BAFF increases B cell survival and proliferation in dose-dependent manner . This can be quantified using viability assays with primary B cells or appropriate B cell lines.

• Surface marker modulation: BAFF treatment increases CD21/CD23 expression on B cells, which can be measured by flow cytometry as a functional readout .

• Receptor binding assays: Functional BAFF should bind its receptors with expected affinity. For example:

  • Immobilized Human BCMA binds BAFF with a linear range of 1-16 ng/mL

  • Immobilized Human TACI binds BAFF with a linear range of 1-31 ng/mL

• Mitogenic activity: The specific activity of BAFF can be determined by measuring its dose-dependent mitogenic effect on human RPMI 8226 cells. High-quality preparations show an ED50 < 20 ng/ml, corresponding to a specific activity of >5.0 x 10^4 units/mg .

• NF-κB signaling activation: Since BAFF activates NF-κB pathways through BAFF-R, reporter assays for NF-κB can serve as functional readouts.

What quality control parameters should be assessed for BAFF Human, His?

Rigorous quality control is essential for experimental reproducibility:

Complete quality assessment requires multiple analytical techniques and should be documented in the Certificate of Analysis accompanying high-quality research reagents.

How does BAFF concentration correlate with B cell populations in different physiological and pathological states?

Research has revealed a fascinating inverse relationship between soluble BAFF levels and peripheral B cell numbers:

Patients with primary antibody deficiencies (PAD) exhibit significantly higher BAFF levels compared to:

• Healthy donors

• Asplenic individuals

• Patients following anti-CD20 B cell depletion therapy

• Chronic lymphocytic leukemia patients

This inverse correlation has been corroborated in mouse models constitutively expressing human BAFF, which show higher BAFF concentrations in the absence of B cells than in their presence . These findings support the hypothesis that BAFF steady-state concentrations primarily depend on:

• The absolute number of B cells present in the organism

• The expression levels of BAFF-binding receptors on these cells

What role does BAFF play in autoimmune diseases versus immunodeficiencies?

BAFF's pivotal role in B cell biology places it at the intersection of seemingly opposing immunological disorders:

Autoimmune Diseases:

• Increased concentrations of soluble BAFF are observed in multiple autoimmune conditions

• BAFF regulates class switch recombination and selection of autoreactive B cells

• Elevated BAFF may promote survival of autoreactive B cells that would normally be eliminated

Immunodeficiencies:

• Genetic defects in BAFF pathway components cause distinct immunodeficiency phenotypes:

  • BAFF-R deficiency: Severe B lymphopenia due to arrested B cell development at the transitional B cell stage

  • TACI deficiency: Associated with certain primary antibody deficiencies

• Paradoxically, patients with PAD show elevated BAFF levels that cannot compensate for underlying developmental defects

This dual role highlights BAFF as a critical checkpoint in immune regulation, where both insufficient and excessive signaling can contribute to pathology. These insights have therapeutic implications, with BAFF-targeting therapies being developed for autoimmune conditions, while approaches to enhance BAFF signaling might benefit certain immunodeficiencies.

How do BAFF humanized mouse models contribute to our understanding of human B cell development?

Humanized mouse models have provided valuable insights into human B cell development and the role of BAFF, though with some limitations:

One study investigated whether inefficient human B cell maturation in humanized mice (hu-mice) might result from suboptimal interaction between human B cells and mouse BAFF. Researchers created a genetically engineered mouse strain in which the mouse BAFF gene was replaced with human BAFF cDNA .

Contrary to expectations, expression of human BAFF in place of mouse BAFF did not improve human B cell development in these models. In fact:

• B cells from human BAFF knock-in (hBAFFKI) hu-mice showed a more immature phenotype than those from standard hu-mice expressing mouse BAFF

• Memory B cells, plasmablasts, and plasma cells were significantly reduced

• Immunoglobulin G levels and T-cell-independent antibody responses were diminished

These findings suggested that inefficient B cell maturation in humanized mice is not primarily due to suboptimal bioactivity of mouse BAFF on human B cells, but likely involves other factors . This unexpected result highlights the complexity of cross-species signaling networks and the challenges in modeling human immune development in xenogeneic environments.

What are best practices for measuring soluble BAFF levels in research samples?

Accurate measurement of soluble BAFF requires careful consideration of multiple factors:

Antibody Selection and Assay Format:

• Use well-characterized monoclonal antibodies specifically developed for human BAFF detection

• Consider epitope specificity to ensure detection of all relevant BAFF forms (trimers and 60-mers)

• Validate assays using recombinant standards with known concentration and oligomeric state

Sample Handling Protocol:

• Collect blood in appropriate anticoagulant tubes

• Process samples consistently (centrifugation speed/time, temperature)

• Prepare multiple small-volume aliquots to avoid repeated freeze-thaw cycles

• Store at -80°C for long-term preservation

Control Samples and Standardization:

• Include internal control samples across multiple plates/runs to assess inter-assay variability

• When comparing different patient groups, process all samples simultaneously when possible

• Consider potential confounding factors (medication use, recent infections, diurnal variation)

Data Interpretation Considerations:

• BAFF levels should be interpreted in the context of B cell numbers and receptor expression

• Reference ranges should be established for specific sample types and patient populations

• Consider potential correlations with other inflammatory markers

What controls are essential for BAFF functional assays?

Robust experimental design for BAFF functional assays requires comprehensive controls:

For mitogenic activity assays on RPMI 8226 cells, the dose-response curve should demonstrate an ED50 < 20 ng/ml for high-quality BAFF preparations . Including these controls enables confident attribution of observed effects to specific BAFF-receptor interactions rather than experimental artifacts.

How can researchers distinguish between effects of different BAFF oligomeric forms?

Distinguishing between effects of BAFF trimers versus 60-mer structures requires specialized approaches:

Structural Characterization Methods:

• Size Exclusion Chromatography (SEC): Separates BAFF preparations based on molecular size

• Multi-Angle Light Scattering (MALS): Provides accurate molecular weight determination of oligomeric states

• Analytical Ultracentrifugation: Offers high-resolution analysis of protein complexes in solution

Preparation of Defined Oligomeric Forms:

• Use specific buffer conditions that favor either trimeric or 60-mer BAFF

• Employ size fractionation techniques to isolate specific oligomeric forms

• Consider structure-specific mutations that prevent higher-order oligomerization

Comparative Functional Analysis:

• Perform parallel receptor binding assays with defined oligomeric preparations

• Compare signaling pathway activation profiles (classical vs. non-canonical NF-κB)

• Analyze receptor clustering efficiency using advanced microscopy techniques

Experimental Controls:

• Include approaches to verify oligomeric state stability throughout experimental duration

• Consider receptor-specific reagents to determine if different oligomeric forms preferentially activate specific receptors

These approaches allow researchers to systematically investigate whether and how BAFF's different structural forms contribute to distinct biological activities, receptor preferences, or signaling outcomes.

How is BAFF Human, His being used in therapeutic development research?

BAFF's central role in B cell biology has made it an important target for therapeutic development:

Autoimmune Disease Applications:

• Anti-BAFF antibodies and BAFF receptor fusion proteins are being investigated for conditions like systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis

• Recombinant BAFF serves as a critical reagent for screening potential therapeutic compounds and validating their mechanism of action

Cancer Immunotherapy Research:

• BAFF modulation is being explored to enhance B cell-mediated anti-tumor responses

• BAFF pathway components may serve as biomarkers for B cell malignancies

Immunodeficiency Studies:

• Recombinant BAFF is used to investigate potential approaches for enhancing B cell recovery in immunodeficient states

• BAFF receptor agonists might benefit certain types of antibody deficiencies

In these research contexts, high-quality BAFF Human, His serves as both a tool for understanding disease mechanisms and as a control for therapeutic development assays.

What are the challenges in developing humanized mouse models for BAFF research?

Despite their potential, humanized mouse models for BAFF research face several challenges:

The replacement of mouse BAFF with human BAFF in knock-in models did not improve human B cell development as originally hypothesized . Instead, B cells from human BAFF knock-in mice showed:

• More immature phenotypes than standard humanized mice

• Reduced frequencies of memory B cells, plasmablasts, and plasma cells

• Diminished levels of immunoglobulin G

• Impaired T-cell-independent antibody responses

These unexpected findings suggest complex challenges:

• Cross-species signaling complexity: Additional factors beyond BAFF may limit human B cell development in mouse environments

• Developmental timing discrepancies: Differences in developmental timing between human and mouse B cells may affect BAFF responsiveness

• Microenvironmental factors: The murine stromal environment may lack other human factors needed for optimal B cell maturation

• Receptor expression patterns: Differences in expression patterns of BAFF receptors between developing mouse and human B cells

Future approaches may need to address multiple factors simultaneously, potentially incorporating additional human cytokines, stromal elements, or transcription factors to better recapitulate human B cell development.