KIR2DL5B Antibody

Basic Research Questions

• What is KIR2DL5B and how does it differ from KIR2DL5A?

KIR2DL5B is a killer cell immunoglobulin-like receptor with two Ig domains (D0-D2 type) and a long cytoplasmic tail that functions as an inhibitory receptor on NK cells. The KIR2DL5 gene underwent duplication approximately 1.7 million years ago in an ancestor of modern humans, resulting in two distinct loci: KIR2DL5A and KIR2DL5B . The primary structural difference is a single polymorphism in exon 1 that distinguishes all KIR2DL5A from all KIR2DL5B alleles . While both encode structurally similar proteins, they differ in genomic location, with KIR2DL5B positioned in the centromeric region of the KIR gene complex, whereas KIR2DL5A is found in the telomeric region . Additionally, certain KIR2DL5B alleles (notably those with type II promoters) are epigenetically silenced due to distinctive substitutions in RUNX binding sites within their promoters .

• What applications are KIR2DL5B antibodies commonly used for in research?

KIR2DL5B antibodies are utilized across multiple research applications:

• Western Blot (WB): For detecting KIR2DL5B protein in cell lysates

• ELISA: For quantitative measurement of KIR2DL5B in biological samples

• Flow Cytometry (FCM): For identifying and analyzing KIR2DL5B-expressing cell populations

• Immunohistochemistry (IHC): For detecting KIR2DL5B in tissue sections

• Immunocytochemistry (ICC): For cellular localization studies

Different antibody formats are available, including unconjugated forms and conjugated versions such as TotalSeq™-A for specialized applications . When selecting antibodies, researchers should prioritize those validated for their specific application, with documented reactivity to human samples and appropriate clonality (monoclonal or polyclonal) based on experimental requirements.

• How do I detect surface expression of KIR2DL5B on NK cells?

Surface expression of KIR2DL5B on NK cells can be assessed using flow cytometry with specific anti-KIR2DL5 antibodies. The methodology involves:

• Isolate peripheral blood mononuclear cells (PBMCs) using density gradient centrifugation

• Stain with anti-CD56 and anti-CD3 to identify NK cells (CD56+CD3-)

• Co-stain with anti-KIR2DL5 antibody (such as clone UP-R1 or newer clones like F8B30)

• Include appropriate isotype controls

• Analyze using flow cytometry focusing on the CD56dim NK cell population

Note that KIR2DL5 expression follows a variegated pattern and is typically found on less than 10% of NK cells in most healthy individuals . The receptor density on the cell surface (assessed by median fluorescence intensity) is generally lower than for several other KIRs in resting lymphocytes but increases upon expansion in the presence of IL-2 and lymphoblastoid cell lines .

• What is the relationship between KIR2DL5B and PVR (poliovirus receptor)?

Recent research has identified PVR (poliovirus receptor) as a binding partner for KIR2DL5 . The interaction between KIR2DL5 on NK cells and PVR on target cells induces inhibitory synapse formation, leading to suppression of NK cell cytotoxicity . Importantly, KIR2DL5 binds to PVR without competition with the other three known PVR receptors (DNAM-1, TIGIT, and CD96), suggesting that it binds to a distinct site on PVR . This interaction has significant implications for tumor immunity, as cancer cells often upregulate PVR expression, potentially exploiting the KIR2DL5/PVR pathway for immune evasion . Development of monoclonal antibodies that block the KIR2DL5-PVR interaction has shown potential for enhancing NK cell-mediated anti-tumor responses .

• What signaling pathways does KIR2DL5B employ to inhibit NK cell function?

KIR2DL5B inhibits NK cell function primarily through the following signaling pathway:

• Upon ligand binding, the canonical immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM)-like sequence in the cytoplasmic domain of KIR2DL5 undergo tyrosine phosphorylation

• The phosphorylated receptor preferentially recruits the Src homology region 2-containing protein tyrosine phosphatase 2 (SHP-2) over SHP-1, distinguishing it from other inhibitory KIRs

• This initiates an inhibitory cascade via ITIM/SHP-1/SHP-2 and ITSM/SHP-1 pathways

• The downstream effect is downregulation of the Vav1/ERK1/2/p90RSK/NF-κB signaling, ultimately suppressing NK cell activation and cytotoxicity

Cross-linking studies have shown that naturally expressed KIR2DL5 inhibits NK cell cytotoxicity against antibody-coated target cells to an extent comparable to the classical inhibitory receptor KIR3DL1 .

Advanced Research Questions

• How can I determine whether my subject expresses functional KIR2DL5B alleles versus non-transcribed variants?

Determining functional versus non-transcribed KIR2DL5B alleles requires a multi-level analysis approach:

Step 1: Genotyping

• Perform sequence-based typing (SBT) of KIR2DL5 genes

• Analyze the promoter region, particularly focusing on the RUNX binding site, as substitutions in this region correlate with transcriptional silencing

• Identify the promoter type (I, II, or III), as type II promoters (found in KIR2DL5B*002 alleles) are generally non-transcribed

Step 2: Expression Analysis

• Extract RNA from NK cells and perform reverse transcription PCR (RT-PCR) with KIR2DL5-specific primers

• Use quantitative PCR to assess transcript levels

• Perform flow cytometry with specific anti-KIR2DL5 antibodies such as UP-R1 or newer clones like F8B30

Step 3: Allele-Specific Analysis

• Note that certain KIR2DL5B alleles (particularly 2DL5B003 and 2DL5B00602) with intact RUNX binding sites are typically transcribed and expressed

• For 2DL5A*005 alleles, which are weakly expressed on the cell surface, newer antibodies like F8B30 are more effective than UP-R1 for detection

This comprehensive analysis helps distinguish between functional and non-transcribed KIR2DL5B variants, which is crucial for interpreting experimental results and clinical associations.

• What are the current challenges in developing antibodies that specifically distinguish between KIR2DL5A and KIR2DL5B proteins?

Developing antibodies that specifically distinguish between KIR2DL5A and KIR2DL5B presents several technical challenges:

• High sequence homology: The proteins encoded by KIR2DL5A and KIR2DL5B share extensive sequence similarity, with only minimal amino acid differences, making it difficult to generate antibodies that recognize truly distinctive epitopes.

• Domain-specific recognition requirements: Many antibodies, like UP-R1, require recognition of epitopes spanning multiple domains (both D0 and D2) for binding, limiting the potential target regions for isoform-specific recognition .

• Conformational similarities: The three-dimensional structures of KIR2DL5A and KIR2DL5B proteins are predicted to be nearly identical, further complicating the generation of isoform-specific antibodies.

• Limited expression: Many KIR2DL5B alleles are not expressed due to promoter variations, making validation of B-specific antibodies challenging.

Strategies to overcome these challenges include:

• Developing antibodies against the few amino acid differences in the extracellular domains

• Targeting differences in post-translational modifications if they exist

• Using recombinant proteins with carefully designed mutations to screen for isoform-specific recognition

• Employing phage display technology with negative selection strategies to identify rare clones with the desired specificity

• How can I evaluate KIR2DL5B-mediated inhibition of NK cell function in an experimental setting?

To evaluate KIR2DL5B-mediated inhibition of NK cell function, several experimental approaches can be employed:

Method 1: Redirected Cytotoxicity Assay

• Isolate and expand NK cells expressing KIR2DL5B (identified by flow cytometry)

• Use the murine FcR+ cell line P815 as target cells

• Add anti-CD16 antibody to trigger NK cell activation

• Add anti-KIR2DL5 antibody or isotype control

• Measure cytotoxicity (using 51Cr release or alternative non-radioactive methods)

• Compare cytotoxicity between anti-KIR2DL5 and isotype control conditions

• Include anti-CD56 as another control for non-inhibitory receptor engagement

Method 2: Cytokine Production Analysis

• Co-culture KIR2DL5B+ NK cells with target cells expressing the KIR2DL5 ligand (PVR)

• Measure cytokine production (IFN-γ, TNF-α) by ELISA or intracellular cytokine staining

• Compare cytokine production in the presence or absence of blocking anti-KIR2DL5 antibodies

Method 3: Degranulation Assay

• Co-culture KIR2DL5B+ NK cells with target cells

• Measure CD107a expression as a marker of degranulation

• Compare CD107a expression between KIR2DL5+ and KIR2DL5- NK cells from the same donor

• Test the effect of anti-KIR2DL5 blocking antibodies

Method 4: Signaling Analysis

• Stimulate KIR2DL5B+ NK cells with anti-KIR2DL5 antibodies

• Analyze tyrosine phosphorylation of the receptor

• Assess recruitment of SHP-2 and SHP-1 phosphatases by immunoprecipitation

• Examine downstream signaling events including Vav1/ERK1/2/p90RSK/NF-κB pathway inhibition

• What is the significance of KIR2DL5B polymorphism in disease susceptibility studies?

KIR2DL5B polymorphism has significant implications for disease susceptibility studies:

Disease Associations:

• Dengue virus infection: KIR2DL5B has been associated with increased susceptibility to dengue virus infection with an odds ratio of 11.43 (95% CI: 4.42-29; P < 0.001)

• Cervical cancer: One study found that KIR2DL5B frequency was significantly higher in healthy controls than in women with HPV infection (p = 0.02), suggesting a potential protective role

• Cancer immunotherapy: KIR2DL5/PVR pathway has been implicated in tumor immune evasion, with blockade showing therapeutic potential

Methodological Considerations:

• Comprehensive genotyping: Studies should employ sequence-based typing rather than simple presence/absence genotyping

• Allele-specific analysis: Different alleles may have distinct functional consequences (expressed vs. non-expressed)

• Haplotype consideration: KIR2DL5B is in linkage disequilibrium with other KIR genes, so haplotype analysis is essential

• Expression validation: Confirming functional expression (not just genetic presence) is crucial

• Ligand interaction: Consider PVR expression on relevant cells in the disease context

These considerations emphasize the importance of detailed KIR2DL5B characterization beyond simple presence/absence genotyping in disease association studies.

• How do KIR2DL5B's structure-function relationships differ from other inhibitory KIRs?

KIR2DL5B exhibits several unique structure-function relationships that distinguish it from other inhibitory KIRs:

Domain Architecture:

• Contains D0-D2 Ig-like domains rather than the D1-D2 configuration found in KIR2DL1-3

• Belongs to an ancestral lineage of KIR shared only with KIR2DL4, which is an HLA-G receptor

• The unique domain architecture may contribute to its distinct ligand specificity (PVR rather than HLA class I molecules)

Signaling Mechanisms:

• Preferentially recruits SHP-2 over SHP-1, unlike most other inhibitory KIRs

• Contains both a canonical ITIM and an ITSM-like motif in its cytoplasmic tail

• Mutation studies suggest the canonical ITIM is essential for inhibitory capacity, while the role of the ITSM-like motif remains less clear

Ligand Interaction:

• Binds to PVR (poliovirus receptor) rather than classical HLA class I molecules

• Does not compete with other PVR receptors (DNAM-1, TIGIT, CD96) for binding, suggesting it recognizes a distinct epitope on PVR

Expression Pattern:

• Displays variegated expression on NK cells similar to classical KIRs but unlike KIR2DL4

• Expression is clonal on both NK and T cells, resembling other KIRs that recognize classical HLA class I molecules rather than KIR2DL4

Understanding these unique structural and functional properties is essential for developing specific targeting strategies and interpreting experimental results involving KIR2DL5B.

• What are the recommended approaches for studying KIR2DL5B in tumor immunology?

For studying KIR2DL5B in tumor immunology, the following comprehensive approaches are recommended:

Expression Analysis:

• Characterize KIR2DL5B expression on tumor-infiltrating lymphocytes (TILs) using flow cytometry

• Assess PVR expression on tumor cells by immunohistochemistry and flow cytometry

• Correlate expression patterns with clinical outcomes and treatment responses

Functional Studies:

• Isolate KIR2DL5B+ NK cells from cancer patients and healthy donors

• Compare cytotoxicity against autologous or allogeneic tumor cells

• Evaluate the effect of KIR2DL5B blockade on NK cell function against tumor targets

• Assess cytokine production (using multiplex assays) to capture the broad spectrum of cytokines/chemokines affected by KIR2DL5B engagement, including IL-13, IL-18, IL-25, IL-27, eotaxin, EGF, GM-CSF, M-CSF, RANTES, MIP-1α, MIP-1β, CXCL-9

Genetic Analysis:

• Genotype KIR2DL5B in cancer patients and controls

• Correlate specific alleles with disease progression and treatment outcomes

• Perform haplotype analysis to account for linkage disequilibrium with other KIR genes

Therapeutic Targeting:

• Test anti-KIR2DL5B blocking antibodies in preclinical models

• Develop humanized mouse models with KIR2DL5B+ NK cells to evaluate anti-tumor responses

• Investigate combination approaches with existing immunotherapies (e.g., anti-PD-1/PD-L1 antibodies)

• Explore the potential of KIR2DL5B blockade to overcome resistance to current NK cell-based immunotherapies

• How can I differentiate between the effects of KIR2DL5B and other inhibitory receptors in NK cell functional assays?

Differentiating between KIR2DL5B and other inhibitory receptor effects requires careful experimental design:

Receptor-Specific Isolation:

• Use flow cytometry-based cell sorting to isolate NK cell subsets based on their receptor expression patterns:

  • KIR2DL5B+ only (lacking other major inhibitory receptors)

  • KIR2DL5B- but expressing other specific inhibitory receptors

  • NK cells expressing multiple inhibitory receptors

Genetic Manipulation Approaches:

• Use CRISPR/Cas9 to specifically knockout KIR2DL5B while leaving other inhibitory receptors intact

• Create NK cell lines with controlled expression of KIR2DL5B and other inhibitory receptors

Blocking Antibody Strategy:

• Compare the effects of specific blocking antibodies:

  • Anti-KIR2DL5B alone

  • Antibodies against other inhibitory receptors alone

  • Combinations of blocking antibodies

• Measure additive or synergistic effects to determine relative contributions

Ligand Manipulation:

• Use target cells expressing different ligands:

  • Cells expressing only PVR (KIR2DL5B ligand)

  • Cells expressing only HLA class I molecules (ligands for other inhibitory KIRs)

  • Cells expressing both ligand types

• Create ligand-deficient cells using CRISPR/Cas9 to eliminate specific interactions

Signaling Studies:

• Examine phosphorylation of SHP-2 vs. SHP-1 as KIR2DL5B preferentially recruits SHP-2

• Analyze downstream signaling events specific to different inhibitory pathways

• Use phospho-flow cytometry to analyze signaling events at the single-cell level

This multifaceted approach enables researchers to dissect the specific contribution of KIR2DL5B to NK cell inhibition relative to other inhibitory receptors.

• What considerations are important when designing experiments to identify novel KIR2DL5B ligands beyond PVR?

When designing experiments to identify novel KIR2DL5B ligands beyond PVR, consider these critical methodological aspects:

Protein Interaction Screening Approaches:

• Create KIR2DL5B-Fc fusion proteins with proper folding and glycosylation in mammalian expression systems

• Use these fusion proteins in:

  • Cell-based binding assays with systematic screening of cell lines from different tissue origins

  • Protein microarray screening against libraries of recombinant proteins

  • Co-immunoprecipitation followed by mass spectrometry to identify binding partners

Functional Validation Strategies:

• Confirm interaction using multiple complementary techniques:

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Biolayer interferometry to assess binding affinity

  • ELISA-based binding assays with recombinant proteins

• Validate functional relevance through:

  • Reporter cell lines expressing KIR2DL5B

  • Competitive binding assays with known ligands

Genetic Approaches:

• CRISPR/Cas9 screens to identify genes affecting KIR2DL5B-mediated inhibition

• Gain-of-function screens by expressing candidate ligands in cells normally resistant to KIR2DL5B-mediated inhibition

Structural Considerations:

• Focus on molecules structurally similar to PVR

• Consider molecules from the same protein family or sharing structural features

• Use computational modeling to predict potential interaction partners based on the KIR2DL5B structure

Critical Controls:

• Include positive controls (PVR-expressing cells) and negative controls (cells lacking any KIR2DL5B ligands)

• Use blocking antibodies against KIR2DL5B to confirm specificity

• Address potential low signal-to-noise ratios observed in previous studies

• Use multiple batches of fusion proteins to ensure consistency

Previous attempts to identify KIR2DL5 ligands using fusion proteins faced challenges with variable signal-to-noise ratios and inconsistent behavior of different protein batches , emphasizing the importance of rigorous methodology and controls in these experiments.

• How does alternative splicing affect KIR2DL5B function and antibody recognition?

Alternative splicing can significantly impact both KIR2DL5B function and antibody recognition in experimental settings:

Known Splice Variants:
The KIR2DL5B gene can undergo alternative splicing, potentially generating variants with:

• Altered extracellular domain structure

• Modified transmembrane regions

• Truncated cytoplasmic tails affecting signaling capacity

Functional Consequences:

• Splice variants lacking critical ITIM/ITSM motifs may lose inhibitory function

• Variants with altered extracellular domains may show different ligand binding properties

• Some variants may act as decoy receptors or have dominant-negative effects

Antibody Recognition Implications:

• Epitope availability: Antibodies requiring both D0 and D2 domains (like UP-R1) will not recognize splice variants lacking either domain

• Domain-specific antibodies: Newer antibodies targeting just the D0 domain (like F8B30) may detect a broader range of splice variants

• Quantification challenges: Flow cytometry or Western blot quantification may be affected by differential recognition of splice variants

Experimental Considerations:

• RT-PCR analysis with primers spanning different exon junctions to identify and quantify splice variants

• Western blotting to detect protein variants of different sizes

• Use of multiple antibodies targeting different epitopes to ensure comprehensive detection

• Functional testing of identified splice variants using recombinant expression systems

Research Implications:
The presence of alternatively spliced variants may explain some inconsistencies in studies of KIR2DL5B expression and function, highlighting the importance of using multiple detection methods and functional assays when studying this receptor.

• What are the most effective strategies for enhancing KIR2DL5B antibody specificity in complex samples?

Enhancing KIR2DL5B antibody specificity in complex samples requires multifaceted approaches:

Antibody Selection and Optimization:

• Compare multiple anti-KIR2DL5B antibodies from different sources and select those with highest specificity

• Consider newer antibody clones like F8B30 that have demonstrated improved recognition of KIR2DL5 compared to traditional antibodies like UP-R1

• Test antibodies against recombinant KIR proteins to assess cross-reactivity with other KIR family members

• For polyclonal antibodies, consider affinity purification against specific KIR2DL5B epitopes

Sample Preparation Strategies:

• Pre-clear samples with antibodies against potentially cross-reactive KIRs

• Use cell sorting to enrich for NK cell populations before analysis

• For tissue samples, optimize antigen retrieval methods for maximal epitope exposure

Assay Design Considerations:

• Flow cytometry:

  • Use multicolor panels with antibodies against other KIRs to identify true KIR2DL5B signal

  • Include Fluorescence Minus One (FMO) controls

  • Validate with KIR2DL5B-transfected cell lines as positive controls

• Western blotting:

  • Use gradient gels for optimal separation

  • Include recombinant KIR2DL5B protein as positive control

  • Consider non-reducing conditions if antibody epitope is conformation-dependent

• Immunohistochemistry/Immunocytochemistry:

  • Use antigen retrieval optimization

  • Employ detection systems with minimal background

  • Validate with known KIR2DL5B-positive and negative tissues

Genetic Validation:

• Confirm KIR2DL5B expression at the transcript level by RT-PCR

• Correlate antibody staining with known KIR2DL5B genotypes

• Use samples from individuals with KIR2DL5B gene deletions as negative controls

Innovative Approaches:

• Consider proximity ligation assays (PLA) to detect KIR2DL5B only when in proximity to known interacting partners

• Employ single-cell approaches combining protein detection with genetic analysis

• Use knockout validation in cell lines to confirm antibody specificity