BTN3A3 Antibody

Basic Research Questions

• What is BTN3A3 and how does it differ from other BTN3A isoforms?

BTN3A3 (Butyrophilin Subfamily 3 Member A3) is a type I transmembrane protein belonging to the immunoglobulin (Ig) superfamily. It is part of the BTN3A subfamily which includes three closely related isoforms: BTN3A1, BTN3A2, and BTN3A3. These molecules are expressed on the surface of various cell types, including immune cells and cancer cells .

Key differences between BTN3A3 and other isoforms:

• BTN3A3 has tumor-suppressive properties in several cancers, including ovarian cancer and non-small cell lung cancer

• Unlike BTN3A1, which is critical for phosphoantigen-induced Vγ9Vδ2 T cell activation, BTN3A3 has distinct functions in regulating cancer cell behavior

• BTN3A3 has an observed molecular weight of 65 kDa and consists of 584 amino acids

• While all three BTN3A proteins are structurally similar, they have distinct epitopes that can be targeted by specific antibodies

• What experimental techniques are commonly used to detect BTN3A3?

Multiple experimental approaches are used to study BTN3A3 expression and function:

For optimal results, researchers should:

• Validate antibody specificity using knockout/knockin cell lines

• Consider fixation methods (fresh frozen vs. FFPE) when selecting antibodies

• Perform antibody titration in each specific experimental system

• What are the known expression patterns of BTN3A3 in normal and cancer tissues?

BTN3A3 shows distinct expression patterns across various tissues and cancer types:

Normal tissues:

• Expressed by multiple immune cell populations including T cells, B cells, monocytes, dendritic cells, and NK cells

• Detected on the surface of human peripheral blood mononuclear cells (PBMCs)

Cancer tissues:

• Variable expression across cancer types including ovarian, gastric, pancreatic, breast, colorectal, and non-small cell lung cancers

• Expression is significantly lower in NSCLC tissues compared to adjacent normal tissues

• Expression levels vary across ovarian cancer cell lines (ES-2, 3AO, HO-8910, HO-8910PM, NIH:OVCAR-3, SK-OV-3)

Notably, BTN3A3 expression correlates with clinical outcomes in multiple cancer types, with higher expression generally associated with better prognosis .

• How is BTN3A3 involved in immune regulation within the tumor microenvironment?

BTN3A3 plays multiple roles in modulating immune responses within the tumor microenvironment:

• In NSCLC, the density of BTN3A3+ tumor cells positively correlates with CD8+ T cell infiltration

• Patients with low BTN3A3 expression show reduced CD8+ T cell infiltration, suggesting BTN3A3 may promote cytotoxic T cell recruitment

• BTN3A3 expression is increased by pro-inflammatory cytokines such as IFNγ and TNFα

• High BTN3A3 expression is associated with an anti-tumor immune phenotype characterized by increased CD4+/CD8+ T cell infiltration

These findings suggest BTN3A3 may function as an immune checkpoint molecule that promotes rather than inhibits anti-tumor immunity, distinguishing it from many other checkpoint molecules.

• What controls should be included when working with BTN3A3 antibodies?

To ensure experimental validity when working with BTN3A3 antibodies, researchers should include:

Positive controls:

• Cell lines with confirmed BTN3A3 expression (Jurkat, U-937, HeLa, HepG2)

• Human peripheral blood mononuclear cells (PBMCs) which naturally express BTN3A3

Negative/specificity controls:

• BTN3A1/BTN3A2/BTN3A3 triple knockout cell lines

• Isotype control antibodies matched to the primary antibody's host species and isotype

• Peptide competition assays to confirm binding specificity

• siRNA/shRNA knockdown cells to validate antibody specificity

Cross-reactivity verification:

• Test for cross-reactivity with other BTN3A family members using cells transfected with individual isoforms

• Compare staining patterns with antibodies of known specificity for different BTN3A isoforms

Advanced Research Questions

• How does BTN3A3 expression correlate with patient prognosis across different cancer types?

BTN3A3 expression demonstrates significant prognostic value across multiple cancer types:

In NSCLC, mechanistic studies reveal that:

• Patients with low BTN3A3 expression showed more aggressive and invasive phenotypes

• BTN3A3 expression positively correlated with CD8+ T cell infiltration, suggesting enhanced anti-tumor immunity

• BTN3A3 expression was significantly lower in tumor tissues compared to adjacent normal tissues

These findings indicate BTN3A3 may serve as a valuable prognostic biomarker, with its expression potentially reflecting both intrinsic tumor cell properties and the status of anti-tumor immunity.

• What molecular mechanisms underlie BTN3A3's inhibition of cancer cell proliferation and invasion?

Research has revealed specific molecular pathways through which BTN3A3 suppresses cancer progression:

FGF2/ERK1/2 pathway inhibition:

• BTN3A3 directly binds to FGF2 (Fibroblast Growth Factor 2), as demonstrated through co-immunoprecipitation and mass spectrometry

• Overexpression of BTN3A3 leads to decreased FGF2 protein levels

• Reduced FGF2 results in decreased ERK1/2 phosphorylation

• The inhibition of this pathway suppresses cancer cell proliferation, migration, and invasion

Experimental evidence from functional studies in ovarian cancer cells demonstrates:

• BTN3A3 knockdown significantly enhanced colony formation, migration (wound healing assays), and invasion (transwell assays)

• BTN3A3 overexpression significantly inhibited these malignant behaviors

• Rescuing FGF2 expression in BTN3A3-overexpressing cells restored ERK1/2 phosphorylation and cell proliferation

Additionally, BTN3A3 may inhibit cancer progression through immune-mediated mechanisms:

• Higher BTN3A3 expression correlates with increased tumor infiltration by CD4+ and CD8+ T cells

• Enhanced anti-tumor immune responses contribute to better clinical outcomes

• How can researchers effectively distinguish between BTN3A3 and other BTN3A family members?

Distinguishing between highly homologous BTN3A isoforms requires strategic experimental approaches:

Antibody-based discrimination:

• Specific antibodies have been validated for individual isoforms:

  • Anti-BTN3A3 (#HPA007904) recognizes only BTN3A3

  • Anti-BTN3A2 (#TA500730) primarily recognizes BTN3A2 with weak binding to BTN3A3

  • Pan-BTN3A antibodies (clone #20.1, clone 103.2) recognize all three isoforms

Validation approaches:

• Use of knockout/knockin cell lines: BTN3A1/BTN3A2/BTN3A3 triple knockout cells transfected with individual isoforms can validate antibody specificity

• Cross-validation with multiple techniques: Combining IHC, flow cytometry, and Western blotting with different antibodies can confirm isoform-specific detection

• Tissue staining patterns: Different BTN3A isoforms show distinct staining patterns in tissues like human tonsil

Molecular characteristics:

• BTN3A3 has a specific molecular weight (65 kDa)

• BTN3A3 has unique peptide sequences that can be targeted by mass spectrometry

• What signaling pathways interact with BTN3A3 in cancer cells?

BTN3A3 intersects with several key signaling pathways in cancer cells:

FGF2/ERK1/2 pathway:

• Direct interaction: BTN3A3 physically binds to FGF2, as demonstrated through co-immunoprecipitation and immunofluorescence co-localization

• Pathway inhibition: BTN3A3 overexpression leads to decreased FGF2 protein levels and reduced ERK1/2 phosphorylation

• Functional consequence: This inhibition suppresses cancer cell proliferation, migration, and invasion

Experimental evidence for this interaction includes:

• Mass spectrometry identification of FGF2 in BTN3A3 immunoprecipitates

• Confirmation through exogenous co-immunoprecipitation with tagged proteins

• Endogenous co-immunoprecipitation in cancer cell lines

• Immunofluorescence showing co-localization of BTN3A3 and FGF2

Functional rescue experiments demonstrate that:

• Increasing FGF2 levels in BTN3A3-overexpressing cells restores ERK1/2 phosphorylation

• This rescue also restores cancer cell proliferation

These findings establish BTN3A3 as a negative regulator of the oncogenic FGF2/ERK1/2 signaling axis in cancer cells.

• What are the current challenges and future directions in BTN3A3 research?

Researchers investigating BTN3A3 face several challenges that present opportunities for future studies:

Technical challenges:

• Antibody specificity: Many commercial antibodies recognize all three BTN3A isoforms

• Tissue preparation: Different fixation methods (fresh frozen vs. FFPE) require specific antibodies for optimal detection

• Species limitations: BTN3A molecules are specific to primates and absent in rodents, limiting standard animal models

Knowledge gaps requiring further investigation:

• Mechanism of action: How BTN3A3 regulates FGF2 protein levels remains unclear

• Isoform-specific functions: Further delineation of unique functions of BTN3A3 versus BTN3A1/A2

• Therapeutic potential: Whether targeting BTN3A3 could enhance anti-tumor immunity or suppress tumor growth

• BTN3A3 regulation: Factors controlling BTN3A3 expression in different cell types

• Immune modulation: The complete spectrum of immune cell types regulated by BTN3A3

Future research directions:

• Development of isoform-specific antibodies with improved specificity

• Creation of humanized mouse models expressing BTN3A3

• Investigation of BTN3A3 in additional cancer types and immune-related diseases

• Exploration of BTN3A3 as a prognostic biomarker in clinical settings

• Structural studies of the BTN3A3-FGF2 interaction to inform therapeutic design

Addressing these challenges will advance our understanding of BTN3A3's role in cancer biology and potentially reveal new therapeutic strategies.