BTN2A2 Antibody

What is BTN2A2 and where is it expressed in the immune system?

BTN2A2 (Butyrophilin 2A2) is an immunoregulatory protein predominantly expressed by antigen-presenting cells (APCs) that regulates T cell immunity. Flow cytometry analysis has revealed that BTN2A2 is expressed on multiple immune cell types with varying expression patterns. The protein is expressed at low levels on resting CD4+ and CD8+ T cells, with slight increases upon activation with anti-CD3 antibody . More significantly, BTN2A2 is expressed on professional APCs including CD19+ B cells, CD11c+ dendritic cells (DCs), and CD11b+ F4/80+ peritoneal macrophages . Notably, activation of these APCs with TLR ligands such as lipopolysaccharide (LPS) or Pam3CysK4 significantly upregulates BTN2A2 expression on their cell surface . This differential expression pattern suggests BTN2A2 plays an important role in APC-T cell interactions during immune responses.

How does BTN2A2 regulate T cell function?

BTN2A2 functions as a negative regulator of T cell activation through several interconnected mechanisms. The protein directly interacts with CD45RO on activated T cell surfaces, resulting in retention of CD45 phosphatase activity within the immune synapse during TCR activation . This interaction leads to enhanced CD45 phosphatase activity, which negatively regulates TCR signaling pathways.

Functional studies have demonstrated that BTN2A2 reduces T cell activation markers (CD69, CD44hiCD62Llo) in a dose-dependent manner . Treatment with recombinant BTN2A2-Ig fusion protein increases the proportion of CD44loCD62Lhi naive T cells while decreasing effector memory T cell populations . At the cellular differentiation level, BTN2A2 promotes regulatory T cell (Treg) expansion while suppressing T helper 17 (Th17) cell differentiation . This immunomodulatory effect involves upregulation of IL2Rbeta and Foxp3 (Treg-associated genes) and downregulation of IL-21 and RORγt (Th17-associated genes), shifting the Treg/Th17 balance toward immunoregulation .

What experimental methods are available to detect BTN2A2 expression?

Several complementary techniques can be employed to detect BTN2A2 expression in different experimental contexts:

• Flow cytometry: Commercial anti-BTN2A2 antibodies can be used to detect surface expression on immune cells. This approach allows for quantification of expression levels and identification of BTN2A2-expressing cell subsets .

• Western blotting/Immunoblotting: This technique can be used to detect BTN2A2 protein in cell lysates, providing information about total protein expression levels and molecular weight .

• Co-immunoprecipitation (co-IP): BTN2A2 interactions with binding partners like CD45RO can be assessed by co-IP followed by immunoblotting, as demonstrated in studies with Jurkat cells .

• Confocal microscopy: Fluorescently labeled anti-BTN2A2 antibodies can visualize BTN2A2 localization and potential colocalization with interaction partners like CD3ζ and CD45 .

• RT-PCR: This technique allows for quantification of BTN2A2 gene expression at the mRNA level, complementing protein detection methods .

What is the molecular mechanism of BTN2A2's interaction with CD45RO?

BTN2A2 functions as a physiological ligand for CD45RO on activated T cells, binding with high affinity (Kd of 63.5nM) as demonstrated by microscale thermophoresis . The molecular interaction between BTN2A2 and CD45RO has significant functional consequences for T cell signaling.

Confocal microscopy studies have confirmed that BTN2A2 prevents the exclusion of CD45 from the immune synapse during early T cell activation, maintaining colocalization between CD45 and CD3ζ . Importantly, this interaction is specific to the CD45RO isoform predominantly expressed in activated T cells, but not the CD45RA isoform. This specificity was confirmed through blocking experiments where anti-CD45RO antibodies (but not anti-CD45RA antibodies) inhibited BTN2A2-Fc binding to activated Jurkat cells .

How does BTN2A2 binding affect downstream TCR signaling pathways?

BTN2A2 binding to CD45RO has profound effects on TCR signaling cascades that ultimately influence T cell differentiation and function. By enhancing CD45 phosphatase activity proximal to the TCR complex, BTN2A2 reduces phosphorylation of key signaling intermediaries in the TCR pathway .

At the molecular level, retention of CD45 near the TCR complex leads to dephosphorylation of CD3ζ and ZAP-70, attenuating proximal TCR signaling events. This dampened TCR signaling fundamentally alters the T cell activation program, shifting the balance from effector to regulatory phenotypes .

The functional consequences of these signaling alterations are significant. Short-term exposure of CD4+ T cells to BTN2A2 results in reduced proliferation but enhanced survival, phenotypes associated with a pro-differentiation state similar to TGF-β treatment . At the transcriptional level, BTN2A2 treatment upregulates genes associated with regulatory T cell development (Foxp3, IL2Rbeta) while downregulating genes involved in inflammatory T cell programs (IL-21, RORγt, TBX21, GATA3) . These changes collectively contribute to BTN2A2's ability to promote immunoregulation and suppress inflammatory responses.

What is the therapeutic potential of BTN2A2 in autoimmune disease models?

BTN2A2 shows significant therapeutic potential in multiple autoimmune disease models through its immunoregulatory effects. In the collagen-induced arthritis (CIA) model, administration of BTN2A2-Ig fusion protein attenuated established disease compared to control treatment . This therapeutic effect was associated with reduced T cell activation (decreased CD69 expression), decreased effector memory T cell populations (CD44hiCD62Llo), and inhibited T cell proliferation as measured by both CFSE dilution and Ki67 staining .

In a nephrotoxic serum (NTS)-induced model of crescentic glomerulonephritis, administration of BTN2A2-Fc reduced disease severity as evidenced by decreased proteinuria and reduced glomerular crescent formation . Mechanistically, this therapeutic effect was associated with increased CD4+Foxp3+ gene expression, decreased CD4+RORγt gene expression, and attenuated T cell activation marker CD5 levels in lymphoid organs. BTN2A2-Fc treatment also lowered IL-17A protein expression in the kidneys compared to controls .

Additionally, BTN2A2 showed efficacy in a mouse model of immune-mediated spontaneous abortion, where treatment of pregnant dams with recombinant BTN2A2 helped animals tolerate their pregnancies, reducing fetal loss . These findings across multiple disease models highlight BTN2A2's broad therapeutic potential in conditions where pathogenic T cell responses drive tissue injury.

How do BTN2A2 knockout models compare to antibody-mediated inhibition?

Genetic knockout studies provide important complementary evidence to antibody-based approaches when studying BTN2A2 function. CRISPR-Cas9-mediated knockout of BTN2A2 in mice results in the absence of BTN2A2 expression on professional APCs, including CD11c+ dendritic cells and CD19+ B cells .

In the nephrotoxic serum (NTS)-induced glomerulonephritis model, BTN2A2 genetic deficiency significantly exacerbated kidney injury, representing the opposite effect of recombinant BTN2A2-Fc administration . This reciprocal relationship between knockout and protein treatment strongly supports BTN2A2's endogenous role in limiting inflammatory responses.

When isolated APCs from BTN2A2-/- animals were co-cultured with T cells from Foxp3+ reporter mice, the absence of BTN2A2 from both dendritic cells and B cells impaired T cell differentiation to Foxp3+ Tregs compared to wildtype APCs . This finding indicates that endogenous BTN2A2 expression on APCs is necessary for optimal regulatory T cell induction.

Together, these knockout studies complement antibody and recombinant protein approaches by revealing the physiological role of endogenous BTN2A2 in immune regulation and demonstrating that both loss-of-function and gain-of-function approaches produce consistent and opposite results.

What are the recommended protocols for using BTN2A2 antibodies in flow cytometry?

When using BTN2A2 antibodies for flow cytometry, researchers should consider the following methodological recommendations:

• Cell preparation: Single-cell suspensions should be prepared from tissues of interest (spleen, lymph nodes, peripheral blood) using standard protocols. For splenic or lymphoid tissue analysis, mechanical disruption followed by red blood cell lysis is typically sufficient .

• Antibody selection: Choose commercially available anti-BTN2A2 antibodies that have been validated for flow cytometry applications. Confirm the antibody specificity using appropriate controls (isotype control antibodies and/or BTN2A2-knockout cells when available) .

• Staining protocol:

  • Use 1-2 × 10^6 cells per staining condition

  • Block Fc receptors with anti-CD16/CD32 antibodies (10 minutes, 4°C) to prevent non-specific binding

  • Stain with anti-BTN2A2 antibody at the manufacturer's recommended concentration (typically 0.25-1 μg per test)

  • Include additional markers to identify specific cell populations (e.g., CD4, CD8, CD19, CD11c, F4/80)

  • Incubate for 30 minutes at 4°C in the dark

  • Wash twice with flow buffer (PBS + 2% FBS + 0.1% sodium azide)

  • Fix cells if not analyzed immediately (2% paraformaldehyde)

• Controls: Include fluorescence-minus-one (FMO) controls and isotype controls to set proper gates. When analyzing activated cells, include both resting and activated populations to compare expression levels .

• Analysis considerations: When analyzing BTN2A2 expression, be aware that expression levels can vary significantly between resting and activated states, particularly for APCs. Compare mean fluorescence intensity (MFI) rather than just percent positive cells to quantify expression level changes accurately .

How can BTN2A2 antibodies be used effectively in co-immunoprecipitation studies?

Co-immunoprecipitation (co-IP) is a valuable technique for studying BTN2A2 interactions with binding partners like CD45 and TCR components. The following protocol has been effective in published studies:

• Cell preparation:

  • Culture cells of interest (e.g., Jurkat T cells, primary T cells) under appropriate conditions

  • For activation studies, stimulate cells with anti-CD3 antibodies (with or without BTN2A2) for the desired time period

  • Harvest cells (typically 20-50 × 10^6 cells per condition) and wash with ice-cold PBS

• Lysis procedure:

  • Lyse cells in ice-cold lysis buffer containing:

    • 1% NP-40 or CHAPS

    • 150 mM NaCl

    • 50 mM Tris-HCl (pH 7.4)

    • Protease inhibitor cocktail

    • Phosphatase inhibitors (for phosphorylation studies)

  • Incubate on ice for 30 minutes with occasional vortexing

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Immunoprecipitation:

  • Pre-clear lysate with Protein A/G beads (1 hour, 4°C)

  • Add anti-CD45 antibody (for CD45 pull-down) or anti-BTN2A2 antibody (5 μg per sample)

  • Incubate overnight at 4°C with gentle rotation

  • Add Protein A/G beads and incubate for 2 hours at 4°C

  • Wash beads 5 times with lysis buffer

  • Elute bound proteins by boiling in SDS sample buffer

• Western blotting:

  • Separate eluted proteins by SDS-PAGE

  • Transfer to PVDF membrane

  • Block and probe with antibodies against proteins of interest (e.g., CD45RO, CD45RA, ZAP70, CD3ζ)

  • Use appropriate secondary antibodies and detection methods

Using this approach, researchers have successfully demonstrated that BTN2A2 causes CD45 to remain associated with the TCR complex (Zap70 and CD3ζ) during T cell activation, and that BTN2A2 specifically binds to the CD45RO isoform but not CD45RA .

What are the key considerations when using BTN2A2-Ig fusion proteins in functional studies?

BTN2A2-Ig fusion proteins (combining the extracellular domain of BTN2A2 with an immunoglobulin Fc region) are valuable tools for studying BTN2A2 function. When conducting experiments with these reagents, consider the following methodological aspects:

• Fusion protein design and production:

  • The fusion protein should contain the extracellular domain of BTN2A2 linked to the constant region of an immunoglobulin (typically mouse IgG2a Fc)

  • Express in appropriate mammalian expression systems (HEK293 cells are commonly used)

  • Purify using Protein A/G affinity chromatography

  • Confirm purity by SDS-PAGE and protein identity by Western blotting

  • Ensure endotoxin levels are minimal (<0.1 EU/μg protein)

• Dosage considerations:

  • For in vitro studies, a dose-response curve should be established (typically 0.1-20 μg/ml)

  • For T cell activation assays, 5-10 μg/ml is often effective

  • For in vivo treatment, doses of 100-200 μg per injection have shown efficacy in mouse models

• Control considerations:

  • Use control Ig (matched isotype Fc portion alone) at equivalent concentrations

  • Include positive controls for the biological effect being measured (e.g., known inhibitors of T cell activation)

  • Consider including a blocking anti-CD45RO antibody condition to confirm mechanism specificity

• Application-specific protocols:

  • For T cell binding assays: Use biotinylated BTN2A2-Ig and control Ig followed by streptavidin-PE detection

  • For T cell activation assays: Measure CD69 expression after 16-18 hours of culture

  • For proliferation assays: Assess Ki67 expression or CFSE dilution after 3 days of culture

  • For T cell differentiation studies: Culture for 5-7 days before analyzing Foxp3 and RORγt expression

• In vivo administration protocols:

  • For CIA model: Administer 100 μg BTN2A2-Ig intraperitoneally every other day starting after disease onset

  • For nephrotoxic serum model: Administer BTN2A2-Fc at similar doses following NTS injection

These methodological considerations will help ensure reliable and reproducible results when using BTN2A2-Ig fusion proteins to study immune regulation.

How might BTN2A2 antibodies contribute to understanding autoimmune disease mechanisms?

BTN2A2 antibodies represent valuable tools for investigating fundamental mechanisms underlying autoimmune pathogenesis. By targeting this key immunoregulatory molecule, researchers can gain insights into several critical aspects of autoimmunity:

• Treg/Th17 balance regulation: BTN2A2 antibodies can help elucidate how this balance is dysregulated in specific autoimmune conditions. By blocking or detecting endogenous BTN2A2, researchers can assess whether defective BTN2A2 expression or function contributes to inflammatory T cell predominance in diseases like rheumatoid arthritis and nephritis .

• APC-T cell interaction dynamics: Anti-BTN2A2 antibodies allow visualization of BTN2A2 localization during APC-T cell interactions, providing insights into how this protein modulates immune synapse formation and stability in health versus autoimmunity .

• Tissue-specific expression patterns: Immunohistochemistry with BTN2A2 antibodies can reveal expression patterns in affected tissues during autoimmune processes, potentially identifying new therapeutic targets .

• Biomarker development: Quantifying BTN2A2 expression levels on immune cells from patients with autoimmune disorders could identify correlations with disease activity or treatment response, potentially yielding new biomarkers .

• Checkpoint modulation strategies: Understanding BTN2A2's role as an immune checkpoint molecule could inform development of new therapeutic approaches that harness natural immunoregulatory mechanisms rather than broadly suppressing immune function .

By applying BTN2A2 antibodies in these research contexts, investigators can advance understanding of autoimmune pathogenesis and potentially identify novel therapeutic strategies.

What are the technical challenges in developing highly specific BTN2A2 antibodies?

Developing highly specific antibodies against BTN2A2 presents several technical challenges that researchers should consider:

• Homology with related butyrophilin family members: BTN2A2 shares structural similarities with other butyrophilin family proteins, which can lead to cross-reactivity. Careful epitope selection and extensive validation are required to ensure specificity .

• Post-translational modifications: BTN2A2 may undergo glycosylation and other modifications that affect antibody recognition. Expression systems used for immunogen production should maintain these modifications to generate relevant antibodies .

• Conformational epitopes: The functional binding interface between BTN2A2 and CD45RO likely involves conformational epitopes. Preserving protein structure during immunization and screening is critical for generating antibodies that recognize native BTN2A2 .

• Species cross-reactivity considerations: Researchers often need antibodies that recognize both human and mouse BTN2A2 for translational studies. The degree of sequence conservation between species affects the likelihood of generating cross-reactive antibodies .

• Validation requirements: Comprehensive validation using multiple techniques (flow cytometry, Western blotting, immunoprecipitation) and appropriate controls (BTN2A2-knockout cells, blocking experiments) is essential to confirm specificity and functionality .

Addressing these challenges requires careful immunogen design, extensive screening protocols, and rigorous validation to ensure that antibodies specifically target BTN2A2 without cross-reacting with related proteins.