BDG2 Antibody

What is BDCA2 and which cell types express it?

Blood dendritic cell antigen 2 (BDCA2) is a C-type lectin exclusively expressed on the surface of human plasmacytoid dendritic cells (pDCs). It consists of a single extracellular carbohydrate recognition domain, a transmembrane region, and a short cytoplasmic tail that lacks signaling motifs. Despite not having intrinsic signaling capabilities, BDCA2 transmits intracellular signals by associating with transmembrane adaptor FcεRIγ, initiating a B-cell receptor (BCR)-like signaling cascade that involves SYK recruitment to the phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) of FcεRIγ. This leads to BTK and PLCγ2 activation and subsequent calcium mobilization .

pDCs are identified in flow cytometry experiments as CD14-CD20-HLA-DR+CD123+ cells. The exclusive expression of BDCA2 on pDCs makes anti-BDCA2 antibodies valuable tools for pDC identification and functional manipulation in research settings .

How does BDCA2 antibody binding affect pDC function?

Anti-BDCA2 antibody binding inhibits Toll-like receptor (TLR) 7- or TLR9-induced production of type I interferons (IFN-I) and other pro-inflammatory mediators from pDCs. This inhibition occurs through a BCR-like signaling cascade initiated by antibody-mediated ligation of BDCA2. Additionally, ligation of BDCA2 with antibodies leads to rapid receptor internalization through clathrin-mediated endocytosis .

Experimental data shows that treatment with 24F4A (a humanized monoclonal antibody against BDCA2) leads to dose-dependent inhibition of TLR9-induced IFNα production in whole blood with an average IC50 of 0.06 μg/ml. Importantly, this inhibition is specific to pDC-derived IFN-I, as 24F4A does not impact TLR3-induced IFNα production (pDCs do not express TLR3) .

What are the differences between different anti-BDCA2 antibody clones?

Different anti-BDCA2 antibody clones exhibit varying abilities to induce BDCA2 internalization and inhibit IFN-I production. For example, while the 24F4A clone demonstrates high potency in receptor internalization and inhibition of TLR9-induced IFNα, another antibody (6G6) shows limited efficacy despite achieving full receptor occupancy .

Specifically, the 24F4A antibody induces dose-dependent decrease in BDCA2 surface expression on pDCs with an average EC50 of 0.017 μg/ml. The correlation between BDCA2 internalization and IFNα inhibition (R² value of 0.68) suggests that these processes are mechanistically linked .

What is the dual mechanism of action for effector-competent anti-BDCA2 mAbs?

The effector-competent anti-BDCA2 monoclonal antibody 24F4A exhibits a dual mechanism to inhibit pDC responses:

• Primary mechanism: Engagement of BDCA2 triggers a BCR-like signaling cascade that inhibits TLR-induced IFN-I production by pDCs.

• Secondary mechanism: The Fc region of 24F4A interacts with CD32a (FcγRIIa), leading to CD32a internalization, which prevents immune complex (IC) binding and subsequent IC-induced IFN-I production .

This dual mechanism enhances the antibody's potential therapeutic efficacy, particularly in systemic lupus erythematosus (SLE) where immune complexes bind to CD32a and stimulate IFN-I secretion from pDCs. Experimental data shows that the effectorless form of 24F4A (24F4A-ef) inhibits TLR9-induced IFNα but is less effective at inhibiting IC-induced IFNα production compared to the effector-competent form .

How does anti-BDCA2 antibody affect in vivo pDC function without causing depletion?

A significant finding is that effector-competent anti-BDCA2 mAb (24F4A) induces BDCA2 internalization in vivo without leading to pDC depletion. This therapeutic approach offers potential advantages over complete pDC depletion for autoimmune disease treatment. Research shows that even partial functional inhibition of pDCs can dramatically improve lupus-like disease in mouse models of SLE, while complete pDC depletion may negatively impact anti-viral immunity .

The functional inhibition without depletion represents a unique approach that could lead to both efficacy and improved safety profiles in autoimmune diseases like SLE. This is supported by in vivo studies in cynomolgus monkeys where a single dose of 24F4A led to BDCA2 internalization within 24 hours without reducing peripheral blood pDC numbers .

What is the relationship between BDCA2 internalization and inhibition of IFN-I production?

Research demonstrates a strong correlation between BDCA2 internalization and inhibition of IFN-I production, suggesting these processes are mechanistically linked. In experiments with healthy donors, the EC50 of 24F4A-mediated BDCA2 internalization (0.017 μg/ml) correlated with the IC50 of IFNα inhibition with an R² value of 0.68 .

This correlation is further supported by comparative studies with another anti-BDCA2 mAb (6G6), which bound BDCA2 with high affinity and achieved full receptor occupancy but only led to modest BDCA2 internalization and subsequently modest inhibition of TLR9-induced IFNα. These findings indicate that the ability to induce receptor internalization may be a critical factor in selecting effective anti-BDCA2 antibodies for research or therapeutic applications .

What are the optimal protocols for measuring BDCA2 antibody effects on pDC function?

Based on experimental methodologies described in the literature, researchers can employ several protocols to evaluate BDCA2 antibody effects:

Whole Blood Assays:

• Collect whole blood in sodium heparin tubes

• Treat with increasing concentrations of anti-BDCA2 mAbs (10 to 0.0015 μg/ml) or isotype control (10 μg/ml)

• Stimulate with TLR9 ligand CpG-A (1 μM final concentration)

• Incubate for 16 hours at 37°C and 5% CO2

• Collect supernatants and measure IFNα levels using a validated ELISA kit

PBMC Assays:

• Isolate PBMCs using Ficoll gradients

• Plate at 1×10⁶ cells/well in complete RPMI media

• Follow the same treatment and stimulation protocol as for whole blood

• For specificity controls, stimulate with poly(I:C) (50 μg/ml), a TLR3 ligand not recognized by pDCs

Isolated pDC Assays:

• Isolate pDCs from PBMCs

• Plate at 1×10⁵ cells/well

• Treat with anti-BDCA2 antibodies at concentrations from 10 to 0.0001 μg/ml

• Stimulate with either CpG-A (1 μM) or R848 (5 μM)

• Incubate and collect supernatants as described above

How can researchers measure BDCA2 internalization in vitro and in vivo?

In Vitro Internalization Assays:

For whole blood:

• Treat whole blood with various concentrations of anti-BDCA2 antibody at 37°C

• Stain with anti-CD123, anti-CD20, anti-CD14, anti-HLADR (to identify pDCs) and anti-BDCA2 (clone 2D6, non-cross-blocking)

• Lyse red blood cells using commercial lyse/fix solution

• Define pDCs as CD14-CD20-HLA-DR+CD123+ cells

• Evaluate BDCA2 surface expression by flow cytometry

For isolated pDCs:

• Treat isolated pDCs with anti-BDCA2 antibodies at various concentrations

• After incubation, stain with non-competing anti-BDCA2 clone

• Analyze by flow cytometry to determine surface BDCA2 levels

In Vivo Internalization Assays:

Two complementary methods can be used:

• Direct method: Measure baseline BDCA2 expression (pre-dose) and post-dose expression using labeled anti-BDCA2 antibody

• Indirect method: Detect bound antibody using PE-labeled anti-human IgG1 mAb

  • For maximal binding assessment, "spike" pre-dose blood samples with 10 μg/ml of antibody at 4°C

  • Lyse blood and stain for pDC markers and bound antibody detection

What methodology should be used to study immune complex stimulation of pDCs?

To study immune complex (IC) stimulation of pDCs and the effects of anti-BDCA2 antibodies on this process, researchers can follow this protocol:

• Pre-form immune complexes by mixing Sm/RNP antigen (1.25 μl) with anti-RNP antibodies (2.5 μl)

• Incubate the mixture for 30 minutes at room temperature

• Dilute the mixture in media (46.75 μl) and add to 200 μl of cells in RPMI media

• Treat cells with anti-BDCA2 mAbs as described in previous protocols or with anti-CD32 antibody (10 μg/ml) as a control

• Incubate cells and measure IFNα production by ELISA

This approach is particularly relevant for SLE research, where immune complexes containing nucleic acids bind to CD32a on pDCs and trigger IFN-I production .

What is ABCG2 and how does it function in cellular biology?

ABCG2 (also known as placenta-specific ABC transporter and breast cancer resistance protein, BCRP1) is a member of the ATP-binding cassette (ABC) transporter family. These transporters play crucial roles in moving various molecules across cellular membranes using energy derived from ATP hydrolysis. ABCG2 has significant relevance in cancer research due to its involvement in conferring resistance to chemotherapeutic agents, including anthracyclines and topotecan .

Beyond its role in drug resistance, ABCG2 is a key player in stem cell biology, with widespread expression in various stem cell populations. It is responsible for the side population (SP) phenotype associated with stem cell properties. ABCG2 is abundantly expressed in placenta, liver, intestine, and stem cells, indicating its protective role in detoxifying cells and regulating stem cell differentiation .

What are the applications of ABCG2 antibody (BXP-21) in research settings?

The ABCG2 Antibody (BXP-21) is a mouse monoclonal IgG2a antibody that detects ABCG2 in mouse, rat, and human samples. It has multiple research applications:

• Western Blotting (WB): For detecting ABCG2 protein expression levels in tissue or cell lysates

• Immunoprecipitation (IP): For isolating ABCG2 protein complexes

• Immunofluorescence (IF): For visualizing ABCG2 localization in cells

• Immunohistochemistry (IHC): For detecting ABCG2 in tissue sections

The antibody is available in non-conjugated form, allowing for versatile applications across different experimental platforms. With 121 citations reported, BXP-21 has established reliability in the research community .

How can researchers use ABCG2 antibodies to study post-translational modifications?

ABCG2 undergoes various post-translational modifications that can influence its function and protein interactions. Most notably, ABCG2 may undergo N-linked glycosylation and can dimerize in vivo. Researchers can employ ABCG2 antibodies like BXP-21 to study these modifications through several approaches:

• Western blotting with and without deglycosylation enzymes to detect shifts in molecular weight

• Immunoprecipitation followed by mass spectrometry to identify specific modification sites

• Non-reducing vs. reducing gel conditions to study dimerization

• Comparison of antibody recognition before and after treatment with enzymes that remove specific modifications

Understanding these post-translational modifications is crucial for elucidating the mechanisms of ABCG2-mediated drug resistance and its roles in stem cell biology .

How do anti-BDCA2 antibodies compare with other approaches for modulating pDC function?

When comparing anti-BDCA2 antibodies with other approaches for modulating pDC function, several key distinctions emerge:

Research indicates that even partial functional inhibition of pDCs through BDCA2 ligation can dramatically improve lupus-like disease in mouse models, while complete pDC depletion may negatively impact anti-viral immunity. This suggests that the functional inhibition approach offered by anti-BDCA2 antibodies provides a unique therapeutic window that balances efficacy with safety .

What are the critical considerations for designing experiments with anti-BDCA2 antibodies?

When designing experiments with anti-BDCA2 antibodies, researchers should consider:

• Antibody Clone Selection: Different anti-BDCA2 clones exhibit varying capacities to induce BDCA2 internalization and inhibit IFN-I production. For instance, while 24F4A demonstrates high potency, other antibodies like 6G6 show limited efficacy despite achieving receptor occupancy .

• Fc Functionality: The Fc region plays a critical role in the antibody's ability to inhibit immune complex-induced IFN-I production through CD32a internalization. Researchers should select between effector-competent and effectorless antibody forms based on their experimental goals .

• Experimental Readouts: Multiple assays should be employed to comprehensively assess antibody effects:

  • Surface BDCA2 expression by flow cytometry

  • IFN-I production by ELISA

  • CD32a expression levels

  • pDC numbers and viability

• Stimulation Conditions: Different TLR ligands (CpG-A, R848) or immune complexes may reveal distinct aspects of antibody function and should be selected based on the research question .

• Time-Course Considerations: BDCA2 internalization occurs rapidly after antibody binding, while functional effects on cytokine production may require longer incubation periods .

By carefully addressing these considerations, researchers can design robust experiments that fully leverage the unique properties of anti-BDCA2 antibodies for investigating pDC biology and potential therapeutic applications.