CD274 Antibody

What is CD274 and what are its primary biological functions in normal and disease states?

CD274, also known as PD-L1 (Programmed Death-Ligand 1) and B7-H1, is a cell surface glycoprotein belonging to the B7 family of co-stimulatory molecules. It plays a crucial role in immune regulation through dual functions: inhibition of activated effector T cells and co-stimulation of naïve T cells .

CD274 is constitutively expressed on macrophages and dendritic cells, while its expression is induced on activated T cells, B cells, endothelial cells, and epithelial cells in response to interferons alpha, beta, and gamma . Its primary mechanism of action involves binding to the co-inhibitory molecule CD279 (PD-1) on T cells, which inhibits T cell proliferation and leads to the secretion of the regulatory cytokine interleukin-10 .

In disease states, particularly cancers, dysregulated CD274 expression contributes to immune evasion by suppressing anti-tumor immune responses. Deregulated CD274 function has been documented in chronic viral and intracellular bacterial infections, as well as in autoimmune diseases and cancers . Recent research has also identified CD274 gene amplification as a potential biomarker for predicting immune checkpoint inhibitor efficacy in solid tumors .

What are the key differences between monoclonal and polyclonal CD274 antibodies for research applications?

Both monoclonal and polyclonal CD274 antibodies have specific characteristics that make them suitable for different research applications:

The choice between monoclonal and polyclonal antibodies should be guided by the specific experimental requirements. Monoclonal antibodies like clone 29E.2A3 offer high specificity for human CD274 , while polyclonal antibodies such as 17952-1-AP provide versatility across multiple applications and species reactivity (human, mouse, rat) .

What validation methods should be employed to confirm CD274 antibody specificity before experimental use?

Proper validation of CD274 antibodies is critical for ensuring reliable and reproducible research results. Recommended validation methods include:

• Positive and negative control tissues/cells: Use tissues with known CD274 expression patterns. For example, validation data for antibody 17952-1-AP shows positive Western blot detection in human placenta tissue, A375 cells, IFN-gamma treated A549 cells, and several other tissues/cell lines .

• Knockout/knockdown validation: Use CD274 knockout or knockdown samples as negative controls. Published literature includes at least 8 studies using knockdown/knockout validation for CD274 antibodies .

• Comparative antibody testing: Test multiple antibodies targeting different CD274 epitopes to confirm consistent staining patterns.

• Cross-platform validation: Compare results across different detection methods (e.g., IHC, WB, IF) to confirm consistency.

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide to demonstrate blocking of specific binding.

• Multiple species testing: For antibodies claimed to be cross-reactive, validate across all relevant species. For example, bs-1103R has been validated for both mouse and rat samples .

• Stimulation experiments: Use interferon-gamma treatment to induce CD274 expression in cell lines as a positive control, as demonstrated with IFN gamma-treated A549 cells .

How should CD274 antibody dilutions be optimized for different detection methods?

Optimizing antibody dilutions is crucial for achieving specific signal while minimizing background. Based on validated protocols, the following dilution ranges are recommended:

For optimal results:

• Perform a dilution series experiment starting with manufacturer's recommendations

• Include appropriate positive and negative controls at each dilution

• Select the dilution that provides the best signal-to-noise ratio

• Note that "it is recommended that this reagent should be titrated in each testing system to obtain optimal results"

• Consider that optimal dilutions may be "sample-dependent" and may require adjustment for different tissue/cell types

What antigen retrieval methods are most effective for CD274 detection in formalin-fixed paraffin-embedded (FFPE) tissues?

Effective antigen retrieval is critical for exposing CD274 epitopes in FFPE tissues. Based on validated protocols, the following methods are recommended:

• Primary recommendation: TE buffer pH 9.0 has been validated for detecting CD274 in human tonsillitis tissue, mouse heart tissue, and human stomach cancer tissue .

• Alternative approach: Citrate buffer pH 6.0 may also be used as an alternative antigen retrieval method .

The choice between these methods may depend on:

• Tissue type and fixation duration

• Specific antibody clone being used

• Level of CD274 expression in the tissue

For challenging samples with low CD274 expression, extending the antigen retrieval time may improve detection. When evaluating heterogeneous samples like tumors, it's important to consider that "heterogeneity of PD-L1 expression among the different types of sample location" has been observed .

How can researchers address the challenge of CD274 expression heterogeneity in tumor samples?

CD274 expression heterogeneity presents a significant challenge in cancer research and diagnostics. Research has highlighted "the heterogeneity of PD-L1 expression among the different types of sample location" , necessitating methodological approaches to address this challenge:

• Multiple sampling strategy: Collect samples from different tumor regions to account for spatial heterogeneity. For complex cases, "a second evaluation of PD-L1 expression by IHC would be performed due to intra- and inter-observer discrepancies" .

• Paired sample analysis: When evaluating metastatic disease, compare "pairs of samples from the same patient (from primary tumor and either LN or distant metastasis)" to assess consistency or changes in CD274 expression.

• Standardized scoring system: Implement quantitative scoring methods with clear cutoffs for positive staining.

• Digital pathology and image analysis: Utilize whole-slide scanning and computational analysis to quantify CD274 expression more objectively across entire tumor sections.

• Multiplex immunostaining: Combine CD274 staining with other immune markers to provide context for its expression pattern.

• Molecular correlation: Correlate protein expression with genomic data, particularly CD274 amplification status, which has been associated with immune checkpoint inhibitor efficacy .

How does CD274 amplification status correlate with immune checkpoint inhibitor efficacy in solid tumors?

Recent comprehensive research has investigated the relationship between CD274 gene amplification and response to immune checkpoint inhibitors (ICIs) in solid tumors:

A 2024 study analyzing data from 60,155 comprehensive genomic profiling (CGP) test results compared treatment outcomes between CD274-amplified and non-amplified groups . The findings revealed:

The authors concluded that "CD274 amplification identified by CGP may therefore be a predictor of ICI efficacy for solid tumors" . This represents the largest analysis to date of patients with CD274-amplified solid tumors, suggesting potential utility of this biomarker for guiding immunotherapy decisions.

What methodological considerations are important when comparing CD274 expression across different sample types and anatomical sites?

When comparing CD274 expression across different sample types and anatomical sites, several methodological considerations are crucial:

• Standardized protocols: Use consistent fixation, processing, and staining protocols across all samples to minimize technical variability.

• Batch processing: Process and stain all comparative samples within the same batch to reduce inter-batch variability.

• Paired analysis: For samples from the same patient, perform "comparing pairs of samples from the same patient (from primary tumor and either LN or distant metastasis)" to enable direct comparison.

• Tissue-specific controls: Include appropriate positive and negative controls specific to each tissue type being evaluated.

• Documentation of microenvironment: Record and account for differences in tumor microenvironment factors that influence CD274 expression (e.g., immune cell infiltration, inflammatory status).

• Consideration of treatment effects: Document prior treatments that may alter CD274 expression patterns, particularly prior immunotherapy or radiotherapy.

• Observer standardization: Address "intra- and inter-observer discrepancies" through standardized scoring systems and potentially multiple independent evaluations.

• Integration with molecular data: Correlate protein expression patterns with genomic alterations like "CD274 amplification" to gain deeper insights into expression mechanisms.

How can multiplexed immunofluorescence be optimized for simultaneous detection of CD274 and other immune checkpoint molecules?

Multiplexed immunofluorescence enables simultaneous visualization of CD274 and other immune checkpoint molecules, providing valuable insights into their co-expression and spatial relationships. Optimization strategies include:

• Antibody panel selection: Choose CD274 antibodies validated for immunofluorescence applications. For example, antibody 17952-1-AP has been validated for IF/ICC applications at dilutions of 1:10-1:100 in HEK-293 cells , while bs-1103R has been validated at 1:50-200 dilutions .

• Sequential staining approach:

  • Begin with the lowest abundance target using the brightest fluorophore

  • Include heat or chemical stripping between rounds if using the same species antibodies

  • Validate each antibody individually before multiplexing

• Spectral unmixing: Employ spectral imaging systems to separate overlapping fluorescence signals.

• Signal amplification: For low-abundance targets, use tyramide signal amplification or other amplification methods.

• Automated image analysis: Implement computational approaches for unbiased quantification and colocalization analysis.

• Validation controls:

  • Single-stained controls for each marker

  • Fluorescence-minus-one (FMO) controls

  • Isotype controls appropriate for each primary antibody

• Cross-platform validation: Confirm key findings using alternative methods such as single-cell transcriptomics or spatial transcriptomics.

What are the methodological approaches for studying post-translational modifications of CD274 protein?

Post-translational modifications (PTMs) of CD274 can significantly impact its function, stability, and interaction with binding partners. Advanced methodological approaches include:

• Detection of glycosylation:

  • The observed molecular weight of CD274 (45-56 kDa, 65-70 kDa) compared to the calculated molecular weight (33 kDa) suggests extensive glycosylation

  • Use enzymatic deglycosylation (PNGase F, Endo H) followed by Western blotting to assess N-linked glycosylation patterns

  • Employ lectin affinity chromatography to enrich for glycosylated forms

• Phosphorylation analysis:

  • Immunoprecipitate CD274 using validated antibodies (e.g., using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate)

  • Analyze by phospho-specific antibodies or mass spectrometry

  • Use phosphatase inhibitors during sample preparation

• Ubiquitination studies:

  • Co-immunoprecipitation (CoIP) approaches, for which CD274 antibodies have been validated

  • Expression of tagged ubiquitin constructs

  • Proteasome inhibitor treatments to stabilize ubiquitinated species

• Mass spectrometry approaches:

  • Immunoprecipitate CD274 using validated antibodies

  • Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Implement targeted methodologies for specific PTM detection

• Site-directed mutagenesis:

  • Generate mutants of predicted PTM sites

  • Assess functional consequences through cellular assays

  • Combine with structural biology approaches

What approaches can be used to investigate the mechanisms of CD274-mediated immune evasion in different cancer types?

Investigating CD274-mediated immune evasion requires integrated methodological approaches:

• Genetic manipulation studies:

  • CRISPR/Cas9-mediated knockout or knockin of CD274

  • Inducible overexpression systems

  • Eight published studies have utilized knockdown/knockout approaches with verified CD274 antibodies

• Functional immune assays:

  • T cell co-culture systems measuring proliferation and cytokine production

  • Immune checkpoint blockade assays

  • Organoid or spheroid co-culture systems with immune components

• In vivo models:

  • Syngeneic mouse models with manipulated CD274 expression

  • Humanized mouse models for testing human-specific antibodies

  • Orthotopic models to recapitulate tissue-specific microenvironments

• Mechanistic signaling studies:

  • Chromatin immunoprecipitation (ChIP) assays, for which CD274 antibodies have been validated

  • Signaling pathway analysis using phospho-specific antibodies

  • Analysis of downstream transcriptional effects

• Comprehensive genomic correlations:

  • Integration with CD274 amplification status, which has been shown to correlate with "the objective response rate of 33.3% versus 18.4% in CD274-amplified versus non-amplified tumors"

  • Multi-omic approaches integrating transcriptomic, proteomic, and genomic data

  • Spatial transcriptomics to map tumor-immune interactions

These methodological approaches provide comprehensive frameworks for investigating CD274 biology in basic research and translational applications, enabling deeper understanding of its role in immune regulation and cancer progression.