KIR2DS4 Antibody

What is KIR2DS4 and what role does it play in NK cell function?

KIR2DS4 (CD158i) is an activating member of the killer cell immunoglobulin-like receptor family expressed on natural killer (NK) cells. It functions as a receptor for HLA-C alleles but, unlike inhibitory KIRs, does not inhibit NK cell activity . KIR2DS4 plays a crucial role in NK cell-mediated immune responses by recognizing specific peptide-HLA complexes and triggering NK cell activation. Research has demonstrated that KIR2DS4 has a strong preference for peptides carrying a tryptophan (Trp) at position 8 of 9-mer peptides bound to HLA-C*05:01 . When engaged by appropriate peptide-HLA complexes, KIR2DS4 can potently activate NK cells to degranulate and produce cytokines such as IFN-γ and TNF-α .

The receptor exists in two major allelic variants: a full-length receptor (KIR2DS4-fl) capable of binding HLA-I, and a version with a 22-base pair deletion (KIR2DS4-del) that creates a truncated soluble protein unable to bind HLA-I . This genetic variation significantly impacts NK cell function across individuals and populations. Notably, KIR2DS4-mediated activation can override the lack of NK cell licensing, suggesting an important role in immune surveillance against pathogens and tumor cells .

What types of KIR2DS4 antibodies are available for research?

Researchers investigating KIR2DS4 have several antibody options available, varying in source, clonality, and applications:

• Rabbit polyclonal antibodies:

  • Examples include ab126128 (Abcam) and NBP2-15006 (Bio-Techne/Novus Biologicals)

  • These antibodies typically target recombinant fragments within human KIR2DS4

  • Applications include immunohistochemistry (IHC-P) and western blotting (WB)

• Mouse monoclonal antibodies:

  • Examples include NBP2-11762 (Bio-Techne/Novus Biologicals, clone MM0442-2G42)

  • Generated against human recombinant KIR2DS4/CD158i

  • Applications typically include flow cytometry and western blotting

When selecting a KIR2DS4 antibody, researchers should consider several factors including the specific experimental application, the cellular/tissue context of experiments, and whether detection of specific KIR2DS4 variants is required. For applications requiring high specificity such as distinguishing between closely related KIR family members, monoclonal antibodies may be preferred. For broader detection of KIR2DS4 in applications like IHC, polyclonal antibodies often provide stronger signal due to recognition of multiple epitopes.

What are the common applications for KIR2DS4 antibodies?

KIR2DS4 antibodies are versatile tools employed in various research applications investigating NK cell biology and immune responses:

• Immunohistochemistry (IHC):

  • Detection of KIR2DS4 in formalin-fixed paraffin-embedded (FFPE) tissues

  • Visualization of KIR2DS4 distribution in different tissues

  • Typical dilutions range from 1:100 to 1:1000

  • Example: Using antibody NBP2-15006 at 1:500 dilution for detection in breast carcinoma tissue

• Western Blotting (WB):

  • Detection and semi-quantification of KIR2DS4 protein in cell or tissue lysates

  • Confirmation of protein size (predicted molecular weight of 34 kDa)

  • Typical dilutions range from 1:500 to 1:3000

  • Example: Using antibody ab126128 at 1:1000 dilution with Raji whole cell lysate

• Flow Cytometry:

  • Analysis of KIR2DS4 expression on NK cell populations

  • Cell sorting based on KIR2DS4 expression

  • Functional studies linking receptor expression to NK cell activity

• Functional Assays:

  • Redirected antibody-mediated degranulation assays

  • Blocking studies to investigate KIR2DS4-dependent activities

  • Investigation of receptor-ligand interactions

Each application requires specific optimization for antibody concentration, incubation conditions, and detection methods to achieve optimal signal-to-noise ratio and specificity. Researchers should validate antibodies in their specific experimental systems before conducting extensive studies.

How can I optimize KIR2DS4 antibody use in western blotting experiments?

Optimizing western blotting experiments with KIR2DS4 antibodies requires careful attention to several methodological details:

Sample Preparation:

• Protein extraction: Use RIPA or NP-40 based lysis buffers with protease inhibitors

• Protein quantification: Load 20-30 μg of total protein per lane for cell lysates (as demonstrated with Raji cell lysate)

• Denaturation: Heat samples at 95°C for 5 minutes in reducing sample buffer

Gel Electrophoresis:

• Use 12% SDS-PAGE gels for optimal resolution around the 34 kDa range (predicted molecular weight of KIR2DS4)

• Include appropriate molecular weight markers covering the 25-50 kDa range

Transfer and Blocking:

• Transfer to PVDF membrane (preferable over nitrocellulose for this protein)

• Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

Antibody Incubation:

• Primary antibody dilution:

  • For ab126128: Use at 1:1000 dilution

  • For NBP2-15006: Use at 1:500-1:3000 dilution range

• Incubate overnight at 4°C with gentle agitation

• Wash 3-5 times with TBST (10 minutes each)

• Use appropriate HRP-conjugated secondary antibody

Detection and Validation:

• Develop using enhanced chemiluminescence (ECL) substrate

• Expected band: 34 kDa for full-length KIR2DS4

• Positive control: Raji cell lysate has been validated for KIR2DS4 detection

• Negative control: Use cell lines known not to express KIR2DS4

Troubleshooting Tips:

• If background is high: Increase washing time/cycles or reduce antibody concentration

• If signal is weak: Increase protein loading, increase antibody concentration, or extend exposure time

• If multiple bands appear: Validate specificity with KIR2DS4 knockdown or overexpression controls

What are the best practices for using KIR2DS4 antibodies in immunohistochemistry?

Successful immunohistochemistry (IHC) with KIR2DS4 antibodies requires optimized protocols for tissue preparation, antigen retrieval, and detection:

Tissue Preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin according to standard protocols

• Section tissues at 4-6 μm thickness

• Mount on positively charged slides

Antigen Retrieval:

• Heat-induced epitope retrieval (HIER) is recommended:

  • Citrate buffer (pH 6.0) for 20 minutes at 95-100°C

  • Allow slides to cool to room temperature for 20 minutes

• Wash in PBS or TBS (3 × 5 minutes)

Blocking and Primary Antibody:

• Block endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Block non-specific binding with 5-10% normal serum for 30 minutes

• Primary antibody dilution:

  • For ab126128: Use at 1:500 dilution for paraffin sections

  • For NBP2-15006: Use at 1:100-1:1000 dilution range

• Incubate overnight at 4°C in a humidified chamber

Detection System:

• Use biotin-free polymer detection systems to reduce background

• Apply appropriate secondary antibody for 30 minutes at room temperature

• Develop with DAB (3,3'-diaminobenzidine) for 2-10 minutes with monitoring

• Counterstain with hematoxylin, dehydrate, and mount

Controls and Validation:

• Positive tissue control: Breast carcinoma tissue has been validated for KIR2DS4 detection

• Negative controls: Omit primary antibody or use isotype control

• Validation strategies: Compare staining patterns with published literature

Optimization Tips:

• Titrate antibody concentration to determine optimal signal-to-noise ratio

• Test different antigen retrieval methods if background is high or signal is weak

• For dual staining with other NK cell markers, use sequential staining protocols

How do KIR2DS4 antibodies perform in different tissue types?

The performance of KIR2DS4 antibodies varies across different tissue types, reflecting the distribution of NK cells:

Lymphoid Tissues:

• Peripheral blood: KIR2DS4 antibodies perform well in flow cytometry of peripheral blood NK cells, where expression is typically variegated (present on a subset of NK cells)

• Lymph nodes: Antibodies detect KIR2DS4+ NK cells primarily in paracortical regions

• Spleen: Detection in red pulp and at lower levels in white pulp regions

Epithelial and Tumor Tissues:

• Breast carcinoma: Both ab126128 and NBP2-15006 have been validated for IHC in breast carcinoma tissues, showing reliable detection of KIR2DS4

• Other carcinomas: Variable staining depending on NK cell infiltration

• Epithelial tissues: Generally low background with specific staining of infiltrating NK cells

Factors Affecting Performance:

• Fixation duration: Prolonged fixation may reduce epitope accessibility

• Tissue processing: Fresh frozen tissues often yield stronger signal than FFPE samples

• Cellular density of KIR2DS4+ cells: Tissues with sparse NK cell infiltration may require signal amplification methods

• Background considerations: Some tissues (like liver) may show higher non-specific background requiring careful titration

Optimization Strategies for Challenging Tissues:

• Adjust antibody concentration based on target tissue

• Modify antigen retrieval conditions for specific tissue types

• Consider signal amplification for tissues with low KIR2DS4 expression

• Use multi-color approaches to confirm specificity

How can I characterize the functional specificity of KIR2DS4 in peptide-dependent NK cell activation?

Characterizing the functional specificity of KIR2DS4 in peptide-dependent NK cell activation requires sophisticated experimental approaches:

1. Peptide-HLA Complex Generation:

• Recombinant expression and purification of HLA-C*05:01 heavy chain and β2-microglobulin

• Refolding with synthetic peptides of interest (e.g., peptides with Trp at position 8)

• Validation of properly folded complexes using conformation-specific antibodies

• Biotinylation of complexes for bead-based assays or tetramer formation

2. Binding Assays to Determine KIR2DS4-Peptide/HLA Interactions:

• Soluble KIR2DS4-Fc fusion protein binding to peptide-loaded HLA-C*05:01

• Surface plasmon resonance (SPR) to measure binding kinetics and affinity

• ELISA-based binding assays with immobilized peptide-HLA complexes

3. Functional NK Cell Assays:

• NK cell degranulation assay:

  • Isolate primary NK cells from KIR2DS4+ donors

  • Use peptide-loaded target cells (e.g., 221-C*05:01-ICP47 cells)

  • Measure CD107a expression by flow cytometry

  • Gate on specific NK cell subsets (e.g., CD56dim, KIR2DL1/S1-, KIR2DS4+)

• Cytokine production assay:

  • Stimulate NK cells with peptide-loaded target cells

  • Perform intracellular cytokine staining for IFN-γ and TNF-α

  • Analyze by flow cytometry with appropriate gating strategies

4. Validation Using Peptide Variants:

• Create peptide libraries with systematic amino acid substitutions at position 8

• Test dose-dependent activation with different peptide concentrations (0.01-10 μM)

• Compare half-maximal activation concentrations between peptide variants

5. Inhibitory Receptor Influence Analysis:

• Gate NK cells based on KIR2DS4 and inhibitory receptor expression (e.g., KIR2DL1)

• Compare activation between NK cell subsets with different receptor combinations

• Establish hierarchical relationships between activating and inhibitory signals

Research using these approaches has revealed that KIR2DS4 binds preferentially to HLA-C*05:01 presenting peptides with Trp at position 8, and this interaction is sufficient to trigger potent NK cell activation that can override the lack of NK cell licensing .

What are the methodological considerations when studying KIR2DS4 interactions with HLA-C allotypes?

Studying KIR2DS4 interactions with HLA-C allotypes presents several methodological challenges that researchers must address:

1. HLA-C Allotype Selection and Expression:

• KIR2DS4 interacts with a subset of C1 and C2 HLA-C allotypes, unlike other KIR2D receptors that dominantly bind either C1 or C2

• Methodological approaches:

  • Generate cell lines expressing specific HLA-C allotypes (e.g., 221-C*05:01-ICP47)

  • Use TAP-deficient cells to control peptide loading

  • Verify HLA expression levels by flow cytometry

2. Peptide Repertoire Considerations:

• The sequence and diversity of peptides presented significantly influence KIR2DS4 binding

• Methodological approaches:

  • Use synthetic peptide libraries with systematic variations

  • Consider both endogenous and exogenous peptide sources

  • Focus on position 8 variations, particularly tryptophan substitutions

3. Binding Assay Optimization:

• Multiple techniques can assess KIR2DS4-HLA interactions with different advantages:

4. Functional Assay Selection:

• Different assays reveal different aspects of KIR2DS4-HLA interactions:

  • Degranulation (CD107a) assays: Measure immediate cytotoxic potential

  • Cytokine production: Assess immunomodulatory functions

  • Cytotoxicity assays: Determine target cell killing efficiency

  • Calcium flux: Evaluate immediate signaling events

5. NK Cell Subset Stratification:

• KIR2DS4+ NK cells are heterogeneous and should be stratified by:

  • Licensing status (R+ vs. R- subsets)

  • Co-expression of inhibitory receptors (especially KIR2DL1)

  • Expression levels of KIR2DS4 (high vs. low)

These methodological considerations are crucial for generating reliable data on KIR2DS4-HLA interactions and their functional consequences in NK cell biology.

How can I distinguish between KIR2DS4-fl and KIR2DS4-del in experimental settings?

Distinguishing between the full-length KIR2DS4 (KIR2DS4-fl) and the deleted variant (KIR2DS4-del) is crucial for functional studies, as these variants have different capacities for HLA binding and NK cell activation:

1. Genomic DNA Analysis:

• PCR-based genotyping:

  • Design primers flanking the 22-bp deletion region in exon 5

  • PCR amplification will generate different-sized products

  • Analyze by agarose gel electrophoresis or capillary electrophoresis

• Sequence-specific primer PCR (SSP-PCR):

  • Design primers specific to either the full-length or deleted sequence

  • Perform parallel PCR reactions with specific primer sets

  • Presence/absence of amplification indicates the variant

2. mRNA/Transcript Analysis:

• RT-PCR and fragment analysis:

  • Extract RNA from NK cells

  • Perform reverse transcription

  • PCR amplify the region spanning the deletion

  • Analyze fragment sizes by gel electrophoresis

• Quantitative RT-PCR:

  • Design primers/probes specific to each variant

  • Perform qRT-PCR to determine relative expression levels

  • Calculate the ratio of KIR2DS4-fl to KIR2DS4-del transcripts

3. Protein Detection Methods:

• Western blotting:

  • Use antibodies recognizing the N-terminal domain common to both variants

  • KIR2DS4-fl will appear at ~34 kDa

  • KIR2DS4-del will appear as a smaller protein or may not be detected depending on antibody epitope

• Flow cytometry:

  • Use antibodies against the extracellular domain

  • Combine with antibodies specific to the transmembrane/cytoplasmic domain (present only in KIR2DS4-fl)

  • Differential staining patterns will distinguish membrane-bound vs. secreted forms

4. Functional Discrimination:

• HLA binding assays:

  • KIR2DS4-fl binds specific HLA-C allotypes, while KIR2DS4-del lacks this ability

  • Use soluble HLA-C tetramers loaded with appropriate peptides

  • Only NK cells expressing KIR2DS4-fl will bind these tetramers

• Activation assays:

  • Stimulate NK cells with HLA-C*05:01 loaded with peptides like P2-AW

  • Only cells expressing KIR2DS4-fl will show activation responses

  • Measure degranulation (CD107a) or cytokine production by flow cytometry

Implementing multiple complementary approaches provides the most robust discrimination between KIR2DS4 variants and ensures accurate interpretation of functional studies.

What techniques can be employed to study the signaling pathways downstream of KIR2DS4 activation?

Investigating signaling pathways downstream of KIR2DS4 activation requires sophisticated techniques spanning from immediate receptor-proximal events to transcriptional responses:

1. Immediate Signaling Events Detection:

• Phosphorylation studies:

  • Stimulate NK cells with anti-KIR2DS4 antibodies or appropriate HLA-C/peptide complexes

  • Lyse cells at different time points (30 seconds to 30 minutes post-stimulation)

  • Perform western blotting with phospho-specific antibodies against DAP12, ZAP70/Syk kinases, LAT, PLCγ, and MAP kinases

• Calcium flux assay:

  • Load NK cells with calcium-sensitive dyes

  • Establish baseline fluorescence by flow cytometry

  • Add KIR2DS4-specific stimulus

  • Monitor real-time changes in intracellular calcium levels

2. Biochemical Analysis of Signaling Complexes:

• Immunoprecipitation and co-immunoprecipitation:

  • Stimulate NK cells with KIR2DS4-specific antibodies

  • Lyse cells in non-denaturing conditions

  • Immunoprecipitate KIR2DS4 or associated signaling molecules

  • Analyze by western blotting for interacting partners

• Proximity ligation assay (PLA):

  • Fix stimulated NK cells on slides

  • Incubate with primary antibodies against KIR2DS4 and potential interacting proteins

  • Apply PLA probes, perform ligation and amplification

  • Visualize protein-protein interactions by fluorescence microscopy

3. Transcriptional Response Analysis:

• qRT-PCR for immediate-early genes

• RNA-seq for global transcriptional changes

• ChIP-seq for transcription factor binding

4. Functional Output Measurement with Pathway Inhibition:

5. Comparative Analysis with Other NK Receptors:

• Compare signaling pathways triggered by KIR2DS4 with other receptors:

  • Other activating KIRs (KIR2DS1, KIR3DS1)

  • Natural cytotoxicity receptors (NKp46, NKp30)

  • NKG2D and 2B4

  • Assess unique vs. shared signaling components

These complementary approaches provide a comprehensive understanding of the signaling mechanisms initiated by KIR2DS4 engagement and how they integrate with other NK cell receptor pathways to determine functional outcomes.