PVRL2 Antibody

What is PVRL2 and why is it important in immunological research?

PVRL2 is a type I transmembrane glycoprotein expressed by myelomonocytic cells, megakaryocytic cells, and CD34+ hematopoietic progenitors. It has gained significant attention as an immune checkpoint protein in cancer research. Recent studies have demonstrated that PVRL2 levels are elevated in tumor cells and tumor-derived exosomes, suggesting a critical role in tumor immune evasion. PVRL2 functions by suppressing CD8 T cells and NK cells in the tumor microenvironment, making it a potential target for cancer immunotherapy. Unlike previous assumptions that PVRL2 primarily acts through its receptor PVRIG, emerging research shows it may have additional immunosuppressive mechanisms, highlighting its importance as an independent research target .

How do I select the appropriate PVRL2 antibody for my specific application?

Selection of the appropriate PVRL2 antibody depends on your experimental approach, target species, and specific application. For flow cytometry applications, consider conjugated antibodies such as PE-conjugated monoclonal antibodies like clone R2-525, which recognizes an extracellular epitope on human CD112 . For applications requiring detection of specific amino acid regions, select antibodies targeting relevant epitopes - for instance, antibodies recognizing AA 76-353 or AA 58-212 regions . For Western blotting, unconjugated rabbit polyclonal antibodies often provide good sensitivity, while mouse monoclonal antibodies may offer higher specificity. Consider cross-reactivity requirements (human vs. mouse models) and whether your protocol involves fixed or frozen tissues. Always verify the validation data for your specific application before proceeding with experiments .

What are the recommended dilutions and sample preparation methods for PVRL2 antibody applications?

Optimal dilutions vary by application: for ELISA, starting dilutions of 1:5000-10000 are typically recommended, while flow cytometry applications generally require more concentrated antibody at approximately 1:100 dilution . For sample preparation, consider the cellular localization of PVRL2 as a transmembrane protein when designing lysis buffers and fixation protocols. For flow cytometry, gentle cell dissociation techniques are crucial to preserve the extracellular epitopes recognized by antibodies like R2-525 . For immunohistochemistry, proper antigen retrieval is essential, with citrate buffer (pH 6.0) often providing good results. Store antibodies according to manufacturer recommendations, typically at -20°C to -80°C, and avoid repeated freeze-thaw cycles. For lyophilized antibodies, proper reconstitution and subsequent aliquoting can maintain antibody performance for up to 12 months .

How can PVRL2 antibodies be used to investigate tumor immunosuppression mechanisms?

PVRL2 antibodies serve as valuable tools for investigating the multi-faceted role of PVRL2 in tumor immunosuppression. Recent research has revealed that PVRL2 functions beyond its previously understood interaction with PVRIG, suggesting more complex immunosuppressive mechanisms . To investigate these pathways, researchers can employ a multi-modal approach: use blocking antibodies in functional assays to assess the impact on immune cell activation; perform co-immunoprecipitation with PVRL2 antibodies to identify novel binding partners; conduct flow cytometric analysis to quantify PVRL2 expression on different tumor and immune cell populations; and combine with downstream signaling analysis to elucidate the molecular mechanisms. Importantly, comparing the effects of PVRL2 blockade versus PVRIG blockade can help delineate the receptor-dependent and independent functions of PVRL2. This approach has already yielded insights showing that PVRL2 deletion reduces tumor growth in an immune-dependent manner that can exceed the effects of PD-L1 deletion .

What experimental approaches can reveal the relationship between PVRL2 and other immune checkpoint molecules?

To investigate the complex interplay between PVRL2 and other immune checkpoints such as TIGIT and PD-L1, several sophisticated experimental approaches are valuable. In vitro co-culture systems using antibodies against multiple checkpoints can assess synergistic effects on T cell and NK cell activation. In vivo models comparing single checkpoint blockade versus combinatorial approaches (particularly PVRL2 with TIGIT) provide insights into potential therapeutic combinations. Recent research has demonstrated that combined TIGIT blockade with PVRL2 deletion results in greater reduction in tumor growth than either intervention alone, suggesting complementary mechanisms . Multiplex immunohistochemistry using antibodies against PVRL2, PD-L1, and other checkpoints helps visualize their co-localization in the tumor microenvironment. Flow cytometric analysis of tumor-infiltrating lymphocytes following checkpoint manipulation can determine the impact on immune cell phenotype and function. These approaches together can reveal whether these checkpoints operate through distinct or overlapping pathways, informing strategic combinations for cancer immunotherapy .

How can researchers effectively employ PVRL2 antibodies to study its role in tumor-derived exosomes?

Studying PVRL2 in tumor-derived exosomes requires specialized approaches combining exosome isolation with antibody-based detection. Begin with ultracentrifugation or size-exclusion chromatography to isolate exosomes from cell culture supernatants or patient samples. Confirm exosome isolation using nanoparticle tracking analysis and Western blotting for exosome markers (CD63, CD9). To detect PVRL2 on exosomes, perform immunoblotting with high-sensitivity PVRL2 antibodies, comparing levels with cellular PVRL2 expression. For functional studies, use flow cytometry with fluorophore-conjugated anti-PVRL2 antibodies (such as PE-conjugated clone R2-525) to analyze exosome binding to immune cells . Importantly, recent research has established a partial role for exosomal PVRL2 in promoting tumor growth, distinct from cellular PVRL2 expression, highlighting the need to investigate both pools separately . Capture-based assays using anti-PVRL2 antibodies conjugated to beads can isolate PVRL2-positive exosomes for further proteomic characterization. When designing these experiments, include appropriate controls to account for antibody specificity and potential cross-reactivity with other nectin family members.

What are the critical quality control steps for validating PVRL2 antibody specificity?

Rigorous validation of PVRL2 antibody specificity is essential for reliable research outcomes. Begin with positive and negative control samples: cell lines with known PVRL2 expression levels (positive) and PVRL2-knockout or knockdown cells (negative). When using monoclonal antibodies like R2-525, verify epitope recognition through peptide competition assays . For polyclonal antibodies, additional validation is necessary due to potential cross-reactivity. Western blot analysis should demonstrate bands of expected molecular weight (~82-85 kDa for glycosylated PVRL2). For flow cytometry applications, parallel staining with multiple antibody clones targeting different PVRL2 epitopes provides confirmation of target specificity. Consider cross-reactivity testing, particularly between human and mouse samples, as many commercially available antibodies show species-specific reactivity . If studying other nectin family members concurrently, verify antibody discrimination between PVRL2 and related proteins like PVR (CD155). Finally, include isotype controls matched to your primary antibody to distinguish specific from non-specific binding, particularly important when studying tissues with high Fc receptor expression.

How should researchers interpret discrepancies in PVRL2 detection between different antibodies and techniques?

Discrepancies in PVRL2 detection across different antibodies and techniques often reflect biological and methodological variables that require systematic investigation. First, consider epitope accessibility variations: antibodies targeting different PVRL2 domains (e.g., AA 76-353 versus AA 58-212) may show differential binding based on protein conformation or interaction with binding partners . Post-translational modifications, particularly glycosylation, can significantly impact antibody recognition and may differ between sample types. When flow cytometry and immunohistochemistry results diverge, evaluate fixation and permeabilization protocols, as these can mask or alter epitopes. For quantitative discrepancies between Western blot and ELISA, perform dilution series to ensure measurements fall within the linear range of detection. Recent research suggests PVRL2 may have context-dependent functions in different tissues and immune environments , so apparent contradictions might reflect true biological variation rather than technical issues. To resolve discrepancies, employ complementary detection methods and multiple antibody clones, ideally recognizing distinct epitopes, while documenting exact experimental conditions to facilitate reproducibility and accurate interpretation.

What optimization strategies are recommended for flow cytometric analysis of PVRL2 expression?

Optimizing flow cytometric analysis of PVRL2 requires attention to several critical parameters. Begin with antibody titration experiments to determine the optimal concentration that maximizes specific signal while minimizing background. The recommended starting dilution of 1:100 for PE-conjugated antibodies like clone R2-525 should be refined for your specific samples . Cell preparation is crucial: use gentle dissociation methods (e.g., non-enzymatic dissociation buffers or short enzyme treatment) to preserve the extracellular epitopes recognized by antibodies. For tumor samples, implement dead cell exclusion with viability dyes to prevent false-positive signals from non-specific antibody binding to necrotic cells. Include appropriate controls: fluorescence-minus-one (FMO) controls help set precise gating boundaries, while isotype controls matched to your antibody's host species and isotype (e.g., mouse IgG1 kappa for R2-525) identify non-specific binding . For multi-parameter analysis, perform compensation using single-stained controls. When analyzing heterogeneous samples like tumor tissues, use lineage markers to identify PVRL2 expression on specific cell populations. Finally, ensure consistent instrument settings across experiments by using calibration beads and standardized protocols to facilitate reliable quantitative comparisons.

How can PVRL2 antibodies be utilized to explore potential PVRIG-independent functions in tumor immunology?

Recent discoveries that PVRL2 may function independently of its known receptor PVRIG open exciting research avenues requiring specialized antibody applications . To explore these novel pathways, researchers should employ a multi-faceted approach: use domain-specific blocking antibodies that selectively disrupt PVRL2-PVRIG interactions while preserving other potential binding interfaces; perform pull-down assays with PVRL2 antibodies followed by mass spectrometry to identify novel binding partners; and conduct competitive binding assays with labeled PVRL2 antibodies in the presence of soluble PVRIG to identify PVRIG-independent binding events. Functional studies comparing PVRL2 blockade in wild-type versus PVRIG-knockout models can directly assess PVRIG-independent effects, as recent research demonstrated that PVRL2 deletion reduces tumor growth even in the absence of PVRIG . Structural biology approaches using antibody fragments can help identify distinct functional domains of PVRL2. Finally, single-cell analysis of immune populations following treatment with domain-specific antibodies can reveal cell type-specific responses to PVRIG-independent PVRL2 functions, potentially identifying novel therapeutic targets in the tumor microenvironment.

What experimental design considerations are important when using PVRL2 antibodies to evaluate potential combinatorial immunotherapy approaches?

When designing experiments to evaluate combinatorial immunotherapy approaches targeting PVRL2, several critical considerations ensure robust and translatable results. First, establish appropriate model systems: use syngeneic mouse models with intact immune systems when testing combinations of PVRL2-targeting agents with other checkpoint inhibitors like anti-TIGIT, as recent research shows these combinations produce greater tumor growth reduction than either approach alone . Consider temporal aspects of treatment: sequential versus concurrent administration of multiple antibodies may yield different outcomes, reflecting the dynamic nature of immune responses. Include comprehensive immune profiling: beyond tumor growth metrics, analyze changes in immune cell populations, activation states, and cytokine profiles using multi-parameter flow cytometry with properly validated antibody panels. When evaluating PVRL2 blockade combined with anti-TIGIT therapy, include relevant comparisons to combined deletion of PVRL2 with its paralog PVR (the primary TIGIT ligand), as these produce distinct outcomes . Incorporate pharmacodynamic biomarkers: develop assays to confirm target engagement and pathway modulation for each therapeutic component. Finally, assess potential resistance mechanisms through longitudinal sampling and analysis of escape variants, providing insights into rational triple combination strategies that might prevent therapeutic resistance.

Comparative analysis of available PVRL2 antibody formats and their optimal applications

This comprehensive guide highlights the diverse PVRL2 antibody formats available for research, with applications ranging from basic protein detection to sophisticated multi-parameter flow cytometry. Selection should be based on specific experimental requirements, including target species (human vs. mouse), application methodology, and the particular epitope of interest .