KIR2DL2 Antibody, FITC conjugated

What is KIR2DL2 and why is it important in immunological research?

KIR2DL2 (killer cell immunoglobulin-like receptor, two Ig domains and long cytoplasmic tail 2) is an inhibitory receptor expressed on natural killer (NK) cells that recognizes specific HLA-C allotypes, including HLA-Cw1, 3, 7, and 8. This receptor plays a critical role in regulating NK cell activity through inhibitory signaling pathways. The canonical human KIR2DL2 protein consists of 348 amino acid residues with a molecular mass of 38.5 kDa and is primarily localized in the cell membrane. As a member of the Immunoglobulin protein superfamily, KIR2DL2 has significant post-translational modifications, including glycosylation, which affect its function. The receptor is also known by several synonyms, including CD158b, NKAT-6, p58.2, and CD158B1 .

KIR2DL2 is particularly important in immunological research because it regulates NK cell-mediated immunity against virally infected cells and tumor cells. Studies have demonstrated that KIR2DL2 expression on NK cells impacts their ability to control viral infections such as HIV-1, making it a significant target for immunotherapy research and investigations into antiviral immune responses .

What are the primary applications for FITC-conjugated KIR2DL2 antibodies in research?

FITC-conjugated KIR2DL2 antibodies are primarily utilized in flow cytometry for identifying and characterizing KIR2DL2-expressing cells, particularly NK cells. The most common applications include:

• Flow Cytometric Analysis: FITC-conjugated KIR2DL2 antibodies enable direct visualization of KIR2DL2-expressing cells, allowing for precise quantification of receptor density and population distribution .

• Multiparameter NK Cell Phenotyping: These antibodies can be used in multicolor flow cytometry panels to characterize NK cell subsets based on their KIR expression patterns, often in combination with other NK cell markers .

• Functional NK Cell Assays: FITC-conjugated KIR2DL2 antibodies help identify specific NK cell populations in cytotoxicity assays, degranulation assays (CD107a), and cytokine production assays (IFN-γ) .

• NK Cell Education Studies: These antibodies facilitate investigations into how KIR2DL2/HLA-C interactions contribute to functional NK cell education and regulation .

While other applications such as Western Blot, ELISA, and immunohistochemistry are possible with non-conjugated variants, the FITC-conjugated antibodies are optimized specifically for flow cytometry-based applications .

How do researchers distinguish between KIR2DL2 and closely related receptors like KIR2DL3 and KIR2DS2?

Distinguishing between KIR2DL2 and its closely related receptors presents a significant challenge in research due to sequence homology and cross-reactivity of available antibodies. Effective discrimination requires a strategic approach:

Antibody Selection Strategy:
Many commercially available antibodies (such as clone GL183) recognize both KIR2DL2 and KIR2DS2, while others may cross-react with KIR2DL3. To overcome this limitation, researchers employ a combinatorial antibody approach using multiple antibodies with different specificities .

For example, researchers can combine:

• Anti-KIR2DL1/S1 antibodies (e.g., EB6 or 11PB6)

• KIR2DL2/S2-specific antibodies (e.g., GL183)

• KIR2DS2-specific antibodies (e.g., 1F12)

By analyzing the staining patterns across these antibody combinations, researchers can identify distinct KIR-expressing NK cell subsets .

Tetramer-Based Approach:
HLA-C tetramers can be used to identify KIR2DL2-expressing cells based on the receptor's binding to its HLA ligands. For instance, HLA-Cw3 tetramers can detect KIR2DL2-expressing cells when combined with antibody staining for confirmation .

Co-staining Protocol Example:

• Stain cells with HLA-Cw3 tetramer

• Co-stain with KIR2DL2/KIR2DS2-specific antibody (GL183)

• Further stain with KIR2DS2-specific antibody (1F12)

• Analyze staining patterns to identify specific populations

This approach allows researchers to precisely identify KIR2DL2+ NK cells even in individuals with complex KIR genotypes, including those with KIR2DL3*005 expression .

What are the key methodological considerations for flow cytometry experiments using FITC-conjugated KIR2DL2 antibodies?

Successful flow cytometry experiments with FITC-conjugated KIR2DL2 antibodies require careful attention to several methodological aspects:

Sample Preparation Considerations:

• Fresh PBMCs are preferred, although properly cryopreserved samples can be used with appropriate recovery protocols

• Isolation of NK cells (CD3-CD56+) prior to staining may enhance detection sensitivity

• Red blood cell lysis should be complete to avoid autofluorescence interference

Staining Protocol Optimization:

• Titrate antibody concentrations to determine optimal staining (typically 0.5-1 μg per 10^6 cells)

• Include appropriate blocking steps (Fc receptor blocking) to minimize non-specific binding

• Use compensation controls when designing multicolor panels that include FITC and other fluorochromes with spectral overlap

• Maintain consistent staining temperature and duration (typically 30 minutes at 4°C in the dark)

Panel Design Considerations:
For comprehensive NK cell analysis, include:

• Lineage markers: CD3 (negative), CD56 (positive)

• Additional KIR markers: KIR2DL1, KIR2DL3, KIR2DS1, KIR2DS2

• Functional markers: CD107a, IFN-γ, as needed for functional studies

Critical Control Samples:

• FMO (Fluorescence Minus One) control without KIR2DL2-FITC to establish proper gating

• Isotype-matched control antibody conjugated to FITC

• Positive control samples from donors with known KIR2DL2 expression

• Negative control samples from KIR2DL2-negative donors if available

Flow Cytometer Settings:

• Optimize voltages for FITC detection channel

• Set appropriate compensation for spectral overlap

• Use standardized beads for day-to-day calibration

• Consider the impact of autofluorescence in the FITC channel, particularly with fixed samples

Adhering to these methodological considerations ensures reliable identification of KIR2DL2-expressing cells and facilitates accurate comparative analyses across experiments and between laboratories.

How can researchers design experiments to investigate the functional impact of KIR2DL2 expression on NK cell activity?

Designing robust experiments to investigate KIR2DL2's functional impact requires careful consideration of NK cell biology and receptor-ligand interactions. The following experimental approaches are recommended:

NK Cell Reactivity Assays:

• NK Cell Isolation and Sorting:

  • Isolate NK cells (CD3-CD56+) from peripheral blood

  • Further sort into KIR2DL2/S2+ and KIR2DL2/S2- populations using flow cytometry

  • Ensure high purity (>95%) for conclusive functional comparisons

• Target Cell Selection:

  • Use target cell lines expressing different HLA-C allotypes (e.g., .221-C1402, .221-C1403)

  • Include HLA-C-negative control cell lines (.221)

  • Consider primary CD4+ T cells from donors with known HLA-C types for physiologically relevant assays

• Functional Readouts:

  • Cytotoxicity Assays: Measure target cell lysis using 51Cr-release or flow cytometry-based methods

  • Degranulation Assays: Measure CD107a expression on NK cell surface after stimulation

  • Cytokine Production: Quantify IFN-γ production using intracellular staining or ELISA

Experimental Design Matrix:

Receptor Blocking Studies:

• Use anti-KIR2DL2 antibodies to block receptor-ligand interactions

• Compare NK cell function before and after blocking to establish causality

• Include isotype control antibodies to control for non-specific effects

Genetic Approaches:

• Compare NK cells from donors with different KIR and HLA genotypes

• Consider siRNA knockdown of KIR2DL2 in primary NK cells

• Use CRISPR-Cas9 in NK cell lines to generate KIR2DL2 knockout models

This comprehensive experimental approach will enable researchers to determine how KIR2DL2 expression modulates NK cell function in different HLA contexts, providing insights into NK cell education and functional regulation.

How do KIR2DL2/HLA-C interactions impact NK cell-mediated control of viral infections?

KIR2DL2/HLA-C interactions significantly influence NK cell-mediated control of viral infections, particularly HIV-1, through complex mechanisms that affect NK cell activation, inhibition, and viral escape. Understanding these interactions provides insights into viral immunopathogenesis and potential therapeutic targets.

Mechanisms of NK Cell Control via KIR2DL2:
KIR2DL2+ NK cells recognize HIV-1-derived peptides presented by HLA-C molecules on infected cells. This recognition can either inhibit or activate NK cells, depending on the specific peptide-HLA-C complex and the NK cell's education status. The binding between peptide-HLA-C complexes and KIR2DL2 is sensitive to sequence variation in the bound peptide, creating a dynamic system that evolves during infection .

HIV-1 Control and KIR2DL2:
Studies of HIV-1 infection have revealed that specific KIR2DL2/HLA-C combinations can impact viral control:

• KIR2DL2/HLA-C12:02 and KIR2DL2/HLA-C14:03 Combinations:
  These specific combinations have been associated with suppression of HIV-1 replication in Japanese patients with chronic HIV-1 infection. Notably, KIR2DL2+ NK cells demonstrated stronger reactions and more effective suppression of viral replication in the presence of HLA-C*14:03 compared to other KIR/HLA combinations .

• Experimental Evidence:

  • KIR2DL2/S2+ NK cells suppressed HIV-1 replication in .221-C1403 cells at significantly higher levels than KIR2DL2/S2- NK cells

  • KIR2DL2/S2+ NK cells produced higher levels of reaction markers (IFN-γ and CD107a) when stimulated with HIV-1-infected .221-C1403 cells compared to .221-C1402 cells

  • This enhanced reactivity was confirmed using primary CD4+ T cells from HLA-C14:02 or HLA-C14:03 homozygous donors

Viral Escape Mechanisms:
HIV-1 has evolved mechanisms to escape NK cell surveillance through mutations that enhance binding of KIR2DL2 to peptide-HLA complexes, thereby inhibiting NK cell activity. Both in vitro and in vivo studies have identified viral sequence variations associated with KIR2DL2 presence or specific KIR2DL2/HLA-C1 genotypes. These mutations result in stronger KIR2DL2 binding to the peptide-HLA complex, inhibiting KIR2DL2+ NK cells and allowing infected cells to escape NK cell-mediated killing .

Methodological Approaches to Study These Interactions:

• NK Cell Reaction Assays: Measure NK cell activation markers (CD107a, IFN-γ) after stimulation with virus-infected target cells

• Viral Suppression Assays: Quantify viral replication in the presence of KIR2DL2+ vs. KIR2DL2- NK cells

• Binding Assays: Evaluate KIR2DL2 binding to different peptide-HLA-C complexes using tetramer technology

• Genetic Association Studies: Correlate KIR2DL2/HLA-C genotypes with viral control in infected populations

These findings suggest that the specific interaction between KIR2DL2 and certain HLA-C allotypes creates a permissive environment for enhanced NK cell activity against viral infections, representing a potential target for immunotherapeutic interventions.

What strategies can researchers employ to investigate the specificity spectrum of KIR2DL2 for different HLA-C ligands?

Investigating the specificity spectrum of KIR2DL2 for different HLA-C ligands requires sophisticated experimental approaches that assess receptor-ligand interactions at molecular, cellular, and functional levels. The following research strategies provide comprehensive insights:

Binding Assays to Determine Receptor-Ligand Interactions:

• Tetramer-Based Binding Studies:

  • Generate HLA-C tetramers loaded with different peptides

  • Assess binding to KIR2DL2-expressing cells by flow cytometry

  • Compare binding affinities across different HLA-C allotypes

  • Use HLA-Cw3 tetramers as positive controls based on established interactions

• Surface Plasmon Resonance (SPR):

  • Measure direct binding kinetics between purified KIR2DL2 and various HLA-C molecules

  • Determine association/dissociation constants (ka/kd) and equilibrium dissociation constants (KD)

  • Compare binding affinities between HLA-C1 and HLA-C2 group alleles

Functional Approaches to Assess Ligand Recognition:

• NK Cell Degranulation Assays:

  • Generate target cells expressing different HLA-C allotypes

  • Co-culture with KIR2DL2+ NK cells

  • Measure CD107a expression as a marker of NK cell activation

  • Compare inhibition patterns across HLA-C1 and HLA-C2 expressing targets

• Cytokine Production Assays:

  • Stimulate KIR2DL2+ NK cells with target cells expressing different HLA-C allotypes

  • Measure IFN-γ production using flow cytometry or ELISA

  • Compare inhibitory effects across HLA-C variants

Innovative Cellular Models:

• Transfected Cell Lines:

  • Generate cell lines expressing single HLA-C allotypes (.221-C1402, .221-C1403)

  • Ensure controlled expression levels of HLA-C molecules

  • Use as targets in functional assays with KIR2DL2+ NK cells

• Primary Cell Comparisons:

  • Use CD4+ T cells from donors with defined HLA-C genotypes

  • Compare NK cell responses against different homozygous donors

Comparative Analysis of KIR2D Family Members:

Research has revealed crucial differences in HLA-C recognition patterns among KIR2D family members:

• KIR2DL2+ and KIR2DL3+ NK cells appear to react similarly against HLA-C+ target cells, regardless of whether the target expresses C1 or C2 alleles

• In contrast, KIR2DL1+ NK cells specifically react against C2 alleles

• This suggests KIR2DL2 and KIR2DL3 have a broader recognition spectrum than KIR2DL1

Experimental Design Matrix:

These strategic approaches provide a comprehensive framework for investigating the specificity spectrum of KIR2DL2 for different HLA-C ligands, enhancing our understanding of NK cell education and function in various genetic contexts.

How can researchers accurately interpret data from studies examining KIR2DL2 and KIR2DS2 co-expression patterns?

Interpreting data from studies examining KIR2DL2 and KIR2DS2 co-expression presents significant challenges due to genetic linkage, antibody cross-reactivity, and complex functional interactions. Here's a comprehensive approach to accurate data interpretation:

Understanding the Genetic Basis:
KIR2DL2 and KIR2DS2 genes are in strong linkage disequilibrium, meaning they are frequently inherited together. This genetic proximity creates interpretive challenges that must be addressed through careful experimental design and analysis .

Antibody-Based Discrimination Strategies:

• Sequential Gating Approach:

  • First identify GL183+ cells (recognizing both KIR2DL2 and KIR2DS2)

  • Further discriminate using KIR2DS2-specific antibodies (e.g., 1F12)

  • Categorize as KIR2DL2+KIR2DS2-, KIR2DL2+KIR2DS2+, or KIR2DL2-KIR2DS2+

• Combinatorial Analysis:

  • Use multiple antibody combinations with overlapping specificities

  • Plot results in a correlation matrix to identify distinct populations

  • Confirm with KIR genotyping data when available

Functional Interpretation Frameworks:

Research has demonstrated that when KIR2DL2 and KIR2DS2 are co-expressed, the inhibitory signaling through KIR2DL2 typically overrides activation through KIR2DS2. This hierarchical relationship must be considered when interpreting functional data :

• KIR2DS2+KIR2DL2- NK Cells:

  • These cells typically show C1-reactivity regardless of their HLA-C environment

  • Functional assays demonstrate activation when engaging C1 targets

• KIR2DS2+KIR2DL2+ NK Cells:

  • Inhibition through KIR2DL2 typically dominates over KIR2DS2 activation

  • The functional outcome is primarily inhibitory against appropriate HLA-C targets

Data Interpretation Matrix:

Integrated Analysis Approach:

• Link Surface Expression to KIR Genotype:

  • When possible, correlate flow cytometry data with KIR genotyping

  • Consider allelic variations that may affect antibody binding

  • Account for heterogeneity in expression levels

• Functional Validation:

  • Use degranulation assays (CD107a) against appropriate targets

  • Measure cytokine production (IFN-γ) to assess functional outcomes

  • Compare responses against targets with different HLA-C allotypes

• Control Considerations:

  • Include NK cells from donors with known single KIR expression

  • Use cell lines with defined HLA-C expression

  • Consider the impact of additional inhibitory/activating receptors

How can researchers leverage FITC-conjugated KIR2DL2 antibodies to investigate NK cell education and adaptive features in viral infections?

FITC-conjugated KIR2DL2 antibodies offer powerful tools for investigating NK cell education and adaptive features during viral infections. The following methodological approaches and experimental designs can guide researchers in this emerging field:

Investigating NK Cell Education:

NK cell education (or licensing) refers to the process by which NK cells acquire functional competence through interactions between inhibitory receptors like KIR2DL2 and their cognate HLA ligands. FITC-conjugated KIR2DL2 antibodies enable researchers to:

• Stratify NK Cell Populations:

  • Identify KIR2DL2+ NK cells in individuals with different HLA-C backgrounds

  • Compare functional responses between "educated" (KIR2DL2+ in HLA-C1+ individuals) and "uneducated" (KIR2DL2+ in HLA-C1- individuals) NK cells

  • Assess how education status affects anti-viral responses

• Characterize Educational Signatures:

  • Develop multiparameter panels combining KIR2DL2-FITC with markers of education (e.g., CD57, NKG2A)

  • Correlate receptor co-expression patterns with functional readouts

  • Identify transcriptional and epigenetic markers of educated NK cells

Methodological Approaches for Adaptive NK Cells:

Recent evidence suggests NK cells can display adaptive or memory-like features following viral exposure. Researchers can investigate this phenomenon using KIR2DL2 antibodies through:

• Longitudinal Monitoring:

  • Track KIR2DL2+ NK cell frequencies before, during, and after viral infection

  • Assess phenotypic and functional changes over time

  • Compare expansion/contraction kinetics between different NK cell subsets

• Functional Evolution Assessment:

  • Compare degranulation capacity and cytokine production of KIR2DL2+ NK cells at different infection stages

  • Measure the proliferative capacity of KIR2DL2+ NK cells following stimulation

  • Assess changes in receptor expression intensity as a marker of adaptation

Experimental Design for Viral Infection Studies:

For robust investigation of KIR2DL2+ NK cells in viral infection contexts, researchers should consider:

• In Vitro Co-culture Systems:

  • Isolate KIR2DL2+ NK cells using flow cytometry

  • Co-culture with virus-infected target cells expressing different HLA-C allotypes

  • Measure viral suppression, degranulation, and cytokine production

  • Compare responses between acute and chronic stimulation conditions

• Ex Vivo Analysis of Patient Samples:

  • Collect longitudinal samples from virally infected patients

  • Perform multiparameter flow cytometry with KIR2DL2-FITC and functional markers

  • Correlate KIR2DL2+ NK cell frequency and phenotype with viral load and disease progression

  • Compare responses between different viral infections (HIV, HCV, CMV)

Advanced Research Protocol Example:

To investigate adaptations in KIR2DL2+ NK cells during HIV-1 infection:

• Isolate PBMCs from HIV+ patients at different disease stages

• Stain with:

  • KIR2DL2-FITC

  • CD3-Pacific Blue (lineage exclusion)

  • CD56-PE-Cy7 (NK cell identification)

  • CD57-APC (maturation marker)

  • NKG2C-PE (adaptation marker)

  • CD107a-BV650 (functional marker)

• Stimulate with K562 cells (HLA-negative) and HLA-C-expressing targets

• Measure:

  • Expansion of specific NK cell subsets

  • Functional capacity against different targets

  • Correlation with viral control parameters

This comprehensive approach will provide valuable insights into how KIR2DL2-expressing NK cells adapt during viral infections and contribute to immune control, potentially informing novel immunotherapeutic strategies.