gpr183b Antibody

What is GPR183 and why is it important in immunological research?

GPR183, also known as Epstein-Barr virus-induced gene 2 (EBI2), is a chemotactic receptor that belongs to the rhodopsin-like subfamily of class A transmembrane spanning (7TM) G protein-coupled receptors. It plays a crucial role in the migratory capability of cells, with oxysterol 7α-25-OHC serving as its most potent ligand . GPR183 has gained significant attention in immunological research due to its involvement in B cell positioning within secondary lymphoid organs and its expression patterns in various immune cell populations. The receptor's activity influences immune cell trafficking, organization of germinal centers, and subsequent antibody responses, making it a valuable target for investigating immune system regulation and development of targeted therapies .

What tissue distribution patterns does GPR183 exhibit and how can this inform antibody selection?

GPR183 demonstrates expression across multiple tissues and cell types, with notable presence in:

• Brain tissues: Detected in rat parietal cortex with immunoreactivity in soma and apical dendrite profiles

• Lymphoid tissues: Present in rat and mouse spleen membranes

• Immune cells: Expressed in B-lymphoblasts (Daudi cell line), T-cells (Jurkat cell line), and monocytes (THP-1 cell line)

• Additional tissues: Found in rat lung membranes

When selecting antibodies for GPR183 detection, researchers should consider this distribution pattern to optimize experimental design. For brain research, antibodies validated in neuronal tissues should be preferred, while immunological studies may benefit from antibodies tested in lymphoid tissues. Flow cytometry applications should utilize antibodies validated in relevant cell lines that match the experimental model, with consideration for the 97 commercially available flow cytometry antibodies targeting GPR183 .

How should experimental controls be designed when using GPR183 antibodies?

Proper experimental controls are essential when working with GPR183 antibodies to ensure specificity and reliability of results. Based on established methodologies, researchers should implement:

• Blocking peptide controls: Pre-incubation of the antibody with a GPR183-specific blocking peptide (e.g., GPR183 extracellular blocking peptide) should abolish specific staining, as demonstrated in immunohistochemical analyses of mouse brain sections

• Negative cell controls: Include cell lines or tissue samples known to express minimal GPR183

• Isotype controls: Particularly important for flow cytometry applications to account for non-specific binding

• Genetic controls: When possible, utilize GPR183 knockout or knockdown samples, as the depletion of this factor has been shown to impair cellular processes like ADCP initiation and cytoskeleton remodeling in B-NHL cultures

These control strategies ensure that observed signals are specifically attributable to GPR183 detection rather than non-specific interactions or background staining.

What are the optimal protocols for detecting GPR183 in different experimental settings?

The detection of GPR183 requires specialized protocols depending on the experimental technique employed:

For Western Blot Analysis:

• Use membrane preparations for brain tissue analysis

• Employ whole-cell lysates for lymphoid tissues and immune cell lines

• Western blot analysis has been successfully performed on rat brain membranes, mouse brain lysates, rat lung membranes, and human cell lines including Daudi B-lymphoblast and Jurkat T-cell leukemia

For Immunohistochemistry:

• Perfusion-fixed frozen brain sections yield optimal results

• A 1:1200 dilution of Anti-GPR183 (extracellular) antibody followed by secondary detection systems (e.g., donkey anti-rabbit-biotin and streptavidin-Cy3) provides clear visualization

• DAPI counterstaining helps localize GPR183 expression relative to cell nuclei

For Flow Cytometry:

• Live intact cell analysis is preferred for surface expression assessment

• Indirect detection methods using 2.5μg of primary antibody followed by fluorophore-conjugated secondary antibodies (e.g., goat-anti-rabbit-FITC) have proven effective

• Commercial PE and APC-conjugated anti-human GPR183 antibodies are available for direct detection

These optimized protocols enhance detection specificity and signal-to-noise ratio across different experimental platforms.

How can GPR183 antibodies be utilized in therapeutic research contexts?

GPR183 antibodies serve multiple functions in therapeutic research contexts, particularly in oncology and immunology:

In B-cell Non-Hodgkin Lymphoma (B-NHL) Research:

• GPR183 has been identified as a crucial factor in novel bispecific antibody therapies

• Transcriptomic analysis revealed GPR183 upregulation as a key event associated with synergism between TG-1801 (a CD47-CD19 bispecific antibody) and the "U2" regimen (ublituximab and umbralisib)

• Pharmacological blockade or genetic depletion of GPR183 impaired antibody-dependent cell phagocytosis (ADCP), cytoskeleton remodeling, and cell migration in B-NHL cultures

In Vaccine Development:

• The transcript ratio of MX2/GPR183 serves as a biomarker to discriminate protected from non-protected individuals after vaccination

• This ratio, measured one day after the third immunization, has shown consistent predictive power across different vaccination regimens

• When combined with anti-CSP titers, the MX2/GPR183 ratio significantly improves discrimination between protected and non-protected vaccine recipients

These applications demonstrate how GPR183 antibodies extend beyond basic research into translational and therapeutic contexts, offering potential biomarkers and mechanistic insights for novel treatment approaches.

What approaches can address epitope accessibility challenges when targeting GPR183?

As a seven-transmembrane G protein-coupled receptor, GPR183 presents unique challenges for antibody targeting due to its complex topology and limited extracellular domains. Researchers should consider:

Epitope Selection Strategies:

• Target the N-terminal extracellular domain: Antibodies targeting amino acid residues 18-31 of human GPR183 (extracellular N-terminus) have demonstrated successful detection across multiple applications

• The specific peptide sequence (C)GND(S)DLYAHHSTAR has proven effective for antibody generation

Methodological Adaptations:

• For intracellular domains: Permeabilization protocols must be optimized to maintain epitope integrity while allowing antibody access

• For membrane preparations: Appropriate detergent selection is critical to solubilize GPR183 while preserving antibody binding sites

• For live cell applications: Extracellular domain-targeting antibodies are essential to avoid permeabilization requirements

Validation Approaches:

• Cross-validate using multiple antibodies targeting different epitopes

• Confirm specificity through competitive binding assays with known GPR183 ligands

• Implement receptor internalization studies to assess functional recognition

These strategies help overcome the inherent challenges of targeting complex membrane receptors while maintaining detection specificity and sensitivity.

How should researchers interpret GPR183 expression changes in different cell populations?

Interpreting GPR183 expression changes requires consideration of cell type-specific contexts and functional implications:

In B Cells:

• Elevated GPR183 expression typically indicates active B cell positioning and migration within lymphoid tissues

• Expression changes during B cell activation may correlate with germinal center formation

• In B-NHL research, GPR183 upregulation has been associated with response to combination therapies including CD47-CD19 bispecific antibodies

In T Cells:

• GPR183 expression patterns differ from those in B cells and may indicate distinct migratory behavior

• Expression detected in Jurkat T-cell leukemia lines suggests relevance in T cell malignancies

In Myeloid Cells:

• GPR183 expression in THP-1 monocytic leukemia cells indicates potential roles in myeloid cell function

• Changes in expression may correlate with altered phagocytic activity and antigen presentation

In Clinical Contexts:

• The ratio of MX2/GPR183 transcript levels serves as a biomarker for vaccine response prediction

• This ratio demonstrates discrimination ability between protected and non-protected vaccine recipients across multiple vaccination regimens

When analyzing GPR183 expression data, researchers should contextualize findings within the specific cellular environment and functional role of the receptor in that setting.

What statistical approaches are most appropriate for analyzing GPR183 antibody-based experimental data?

The analysis of GPR183 antibody-based experimental data requires robust statistical frameworks tailored to the specific experimental design:

For Flow Cytometry Data:

• Employ fluorescence minus one (FMO) controls to establish proper gating strategies

• Apply nonparametric tests for non-normally distributed data sets

• Consider dimensionality reduction techniques (t-SNE, UMAP) for multi-parameter analysis of GPR183 in relation to other markers

For Transcriptional Analysis:

• Ratio-based approaches have proven valuable, as demonstrated by the MX2/GPR183 ratio in vaccine studies

• ROC curve analysis can assess discrimination capacity, with area under curve (AUC) serving as a performance metric

• Multiple testing correction using Benjamini-Hochberg false discovery rate (FDR) algorithm is essential for controlling false positives

For Predictive Modeling:

• Logistic regression models incorporating GPR183-related parameters with other biomarkers (e.g., anti-CSP titers) can improve predictive accuracy

• Chi-squared tests can evaluate the complementarity between different predictive factors

• Cross-validation approaches should be implemented to assess model robustness

For Therapeutic Response Assessment:

• Time-course analyses examining GPR183 expression changes in response to treatments

• Survival analysis methods (Kaplan-Meier, Cox proportional hazards) to correlate GPR183 levels with clinical outcomes

These statistical approaches ensure rigorous analysis of GPR183-related data while accounting for the complexities inherent in different experimental systems.

How can GPR183 antibodies contribute to understanding oxysterol signaling pathways in immune regulation?

GPR183 antibodies provide valuable tools for investigating the intricate relationship between oxysterol signaling and immune regulation:

Ligand-Receptor Interaction Studies:

• GPR183 antibodies can be used in competition assays with 7α-25-OHC, the most potent natural ligand for GPR183

• Such studies help map the binding domains and functional consequences of oxysterol recognition

• Immunoprecipitation approaches using GPR183 antibodies can identify co-receptors and associated signaling complexes

Spatial Regulation Analysis:

• Immunohistochemical applications of GPR183 antibodies reveal the distribution patterns of this receptor in lymphoid tissues

• These patterns can be correlated with the enzymatic machinery responsible for oxysterol production

• The resulting data inform models of how oxysterol gradients guide immune cell positioning

Signaling Pathway Dissection:

• Phospho-specific antibodies against downstream effectors can be combined with GPR183 antibodies to trace signaling cascades

• Single-cell analyses incorporating GPR183 detection help resolve heterogeneity in oxysterol responsiveness

• CRISPR-based genetic screens coupled with GPR183 antibody-based readouts can identify novel pathway components

These applications collectively enhance our understanding of how oxysterol-GPR183 signaling axes coordinate immune responses and tissue homeostasis.

What are the critical considerations when using GPR183 antibodies in multi-parameter flow cytometry studies?

Multi-parameter flow cytometry incorporating GPR183 detection requires careful experimental design:

Panel Design Considerations:

• Fluorophore selection: PE and APC-conjugated anti-human GPR183 antibodies are commercially available

• Avoid spectral overlap with markers co-expressed in GPR183-positive populations

• Include lineage markers to properly identify GPR183 expression in specific cell subsets

Optimization Parameters:

• Titration experiments are essential to determine optimal antibody concentration

• Time-dependent internalization of GPR183 following ligand exposure may affect detection efficiency

• Temperature sensitivity should be assessed, as receptor trafficking may vary between 4°C and 37°C

Analysis Approaches:

• Biexponential transformation of GPR183 expression data may better resolve positive populations

• Consider GPR183 expression as a continuous variable rather than simply positive/negative

• Correlation analyses between GPR183 and other GPCR or migratory markers may reveal functional relationships

Validation Requirements:

• Fluorescence minus one (FMO) controls are critical for accurate gating

• isotype controls matched to the specific GPR183 antibody format should be included

• Biological controls of known GPR183 expression status establish detection thresholds

These considerations ensure robust and reproducible assessment of GPR183 expression in complex cellular landscapes using flow cytometry approaches.

How can researchers troubleshoot inconsistent GPR183 antibody performance across different experimental platforms?

Inconsistent antibody performance can undermine research reproducibility. For GPR183 antibodies, consider these troubleshooting approaches:

Common Sources of Variability:

• Epitope accessibility differences: The seven-transmembrane structure of GPR183 presents differently across applications

• Fixation/permeabilization effects: Protocols that work for flow cytometry may disrupt epitopes for immunohistochemistry

• Species cross-reactivity limitations: Antibodies may perform differently across human, mouse, and rat samples

Systematic Troubleshooting Strategy:

Validation Across Platforms:

• Confirm GPR183 expression using orthogonal methods (transcript analysis, functional assays)

• When possible, use genetic approaches (siRNA knockdown, CRISPR knockout) to validate specificity

• Consider using multiple antibodies targeting different epitopes to cross-validate findings

These systematic approaches help identify and address sources of variability in GPR183 antibody performance across diverse experimental contexts.

How is GPR183 antibody research contributing to our understanding of B-cell malignancies?

GPR183 antibody research has revealed important insights into B-cell malignancies and potential therapeutic strategies:

Mechanistic Discoveries:

• Transcriptomic analysis has identified GPR183 upregulation as a crucial event associated with the synergistic effects of novel therapeutic combinations in B-cell non-Hodgkin lymphoma (B-NHL)

• Pharmacological blockade or genetic depletion of GPR183 impairs antibody-dependent cell phagocytosis (ADCP), cytoskeleton remodeling, and cell migration in B-NHL cultures exposed to combination therapy

• These findings suggest GPR183 as a key mediator in the response to targeted therapies, particularly those involving CD47-CD19 bispecific antibodies combined with PI3Kδ and CD20-targeting agents

Therapeutic Implications:

• GPR183 expression analysis may help predict response to novel therapeutic approaches, such as the TG-1801/U2 combination

• In preclinical models, this combination achieved 93% tumor growth inhibition, with 40% of animals remaining tumor-free 35 days after the last dosing

• Targeting GPR183-dependent pathways may provide a complementary approach to enhance existing therapies for B-cell malignancies

Diagnostic Applications:

• GPR183 antibodies enable the characterization of receptor expression across different B-cell malignancy subtypes

• Expression patterns may correlate with disease progression, treatment response, and clinical outcomes

• Multi-parameter analysis incorporating GPR183 could refine patient stratification strategies

These contributions highlight the importance of GPR183 in B-cell malignancy research and its potential as both a biomarker and therapeutic target.

What is the significance of the MX2/GPR183 ratio in vaccine response research?

The MX2/GPR183 transcript ratio has emerged as a significant biomarker in vaccine response research:

Discovery and Validation:

• The ratio was identified through comprehensive analysis of transcriptional signatures associated with protection following vaccination

• While many individual modules, transcripts, or module pairs showed low accuracy discrimination, the MX2/GPR183 ratio demonstrated consistent discriminatory power across multiple studies and platforms

• This ratio achieved discrimination for multiple vaccination regimens, indicating robustness as a biomarker

Statistical Performance:

• The MX2/GPR183 ratio demonstrated moderate but consistent discrimination accuracy (minimum ROC AUC > 0.65) across all studies

• When combined with anti-CSP titers, inclusion of the MX2/GPR183 ratio led to statistically significant improvements in discrimination between protected and non-protected vaccine recipients (p = 0.005 and 0.003 for standard and alternative regimens, respectively)

Biological Significance:

• MX2 is an interferon response module transcript, while GPR183 is an oxysterol receptor

• The ratio between these transcripts may reflect a balance between innate antiviral responses and lymphocyte positioning/trafficking

• This balance appears critical in determining protective immunity following vaccination

Future Applications:

• The ratio could serve as a predictive biomarker for vaccine efficacy in clinical trials

• It may inform personalized vaccination strategies based on individual immune profiles

• Further research may reveal additional contexts where this ratio provides predictive value

This research demonstrates how transcriptional analyses incorporating GPR183 can yield valuable biomarkers with clinical applications beyond direct protein detection.

What are the emerging applications of GPR183 antibodies in neuroinflammation research?

GPR183 antibodies are opening new avenues in neuroinflammation research, with several emerging applications:

Neural Expression Patterns:

• Immunohistochemical staining using GPR183 antibodies has revealed expression in rat parietal cortex, with immunoreactivity appearing in soma and apical dendrite profiles

• Western blot analysis confirms GPR183 presence in rat brain membranes and mouse brain lysates

• These expression patterns suggest potential roles for GPR183 in neural function and neuroinflammatory processes

Neuroimmune Interface Investigation:

• GPR183 antibodies enable the study of oxysterol signaling at the neuroimmune interface

• The receptor may mediate interactions between resident glial cells and infiltrating immune cells during neuroinflammatory conditions

• Co-localization studies with cell type-specific markers can map GPR183 distribution across neural and immune cell populations

Therapeutic Target Exploration:

• Targeting GPR183 may modulate neuroinflammatory responses in conditions such as multiple sclerosis, Alzheimer's disease, and stroke

• Antibodies serve as essential tools for validating GPR183 as a therapeutic target in neurological disorders

• Preclinical models incorporating GPR183 antibody-based readouts can assess the efficacy of interventions targeting this pathway

Methodological Considerations:

• Brain-specific post-translational modifications may affect antibody binding properties

• Blood-brain barrier considerations must be addressed when designing in vivo applications

• Fixation protocols must be optimized to preserve both antigenicity and neural architecture

These emerging applications highlight the expanding role of GPR183 research beyond traditional immunological contexts into neuroscience and neuroinflammatory disease research.