KIR2DL1 Antibody

What is the specificity profile of commonly used anti-KIR2DL1 antibodies?

Different anti-KIR2DL1 antibodies exhibit distinct specificity profiles that researchers must consider when designing experiments:

Clone 143211 (MAB1844) detects both KIR2DL1 and KIR2DS5 on transfected cells but does not cross-react with KIR2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS4, 3DL1, 3DL2, or 3DS1 . This antibody is particularly useful for flow cytometry applications, as demonstrated in protocols detecting KIR2DL1/KIR2DS5 in human PBMCs .

HP-DM1 exhibits higher specificity, exclusively reacting with KIR2DL1 allotypes (with exceptions of KIR2DL1022 and likely KIR2DL1020) and potentially KIR2DS1*013 . This antibody recognizes a conformational epitope including residues M44, S67, R68, and T70, with M44 being crucial for HLA-C K80 recognition .

Other common antibodies include EB6 or 11PB6 (anti-KIR2DL1/S1 and anti-KIR2DL3*005), and HP-MA4 (anti-KIR2DL1/S1/S3/S5) . Strategic combinations of these antibodies allow precise identification of distinct KIR-expressing NK cell populations.

How can I distinguish between inhibitory KIR2DL1 and activating KIR2DS1 in experimental settings?

Distinguishing between the highly homologous KIR2DL1 and KIR2DS1 requires specific methodological approaches:

A combinatorial antibody strategy provides optimal discrimination. Using HP-DM1 in combination with broader specificity antibodies like EB6 (anti-KIR2DL1/S1) allows identification of distinct populations . Cells that are EB6+/HP-DM1+ can be identified as KIR2DL1+, while cells that are EB6+/HP-DM1- likely express KIR2DS1 .

Functional discrimination methods complement antibody-based approaches. KIR2DL1+ cells show inhibition in the presence of HLA-C2 ligands, while KIR2DS1+ cells may demonstrate activation . This functional difference can help distinguish populations when antibody specificity is insufficient.

For complete characterization, researchers should combine antibody staining with genetic confirmation of KIR haplotypes, particularly in donors with complex KIR genotypes that include both KIR2DL1 and KIR2DS1.

What epitopes are recognized by different KIR2DL1 antibodies, and how does this affect experimental design?

Epitope recognition patterns significantly impact antibody application and experimental design:

HP-DM1 mAb recognizes a conformational epitope involving four amino acids: M44, S67, R68, and T70 . The M44 residue is particularly significant as it's crucial for HLA-C K80 recognition by KIR2DL1. The differential binding of clone 1127B is determined by amino acid residue T154 .

These epitope specificities create important experimental considerations:

For blocking studies, antibodies targeting epitopes involved in HLA-C recognition (like HP-DM1) can effectively block KIR2DL1 interaction with its ligand, enabling functional studies of receptor-ligand interactions .

In allotype discrimination experiments, understanding epitope specificity allows selection of antibodies that can distinguish between KIR2DL1 allotypes, such as KIR2DL1-C245 and KIR2DL1-R245 .

When investigating co-expression of multiple KIRs, antibody selection must account for potential cross-reactivity based on shared epitopes, requiring careful panel design and validation.

What are optimal protocols for detecting KIR2DL1 expression on human NK cells by flow cytometry?

Flow cytometric detection of KIR2DL1 requires specific considerations for reliable results:

Sample preparation: PBMCs are commonly used, as demonstrated in the detection protocol from R&D Systems using MAB1844 . Isolate PBMCs using density gradient centrifugation and maintain viability during staining procedures.

Antibody panel design: Include markers for NK cell identification (CD56, CD3-) alongside KIR markers. For comprehensive characterization, use a panel including HP-DM1, EB6/11PB6, 143211, and HP-MA4 to distinguish various KIR+ subsets .

Staining protocol:

• Use 1-2×10^6 PBMCs per sample

• Block with human serum (10%) to prevent non-specific binding

• Stain with anti-KIR2DL1 antibody (optimal concentration determined by titration)

• Include appropriate fluorochrome-conjugated secondary antibody if using unconjugated primary antibodies

• Add markers for NK cell identification (CD56+CD3-)

• Analyze using appropriate compensation and gating strategies

Controls: Include isotype controls (e.g., Mouse IgG1) as shown in the R&D Systems protocol . Consider including KIR genotyped control samples with known expression patterns to validate staining patterns.

How can I assess functional differences between educated and uneducated KIR2DL1+ NK cells?

Education status significantly impacts KIR2DL1+ NK cell function and requires specific experimental approaches for assessment:

Donor selection is critical: Use donors with known HLA-C1/C2 status. KIR2DL1+ NK cells from HLA-C2+ donors would be educated, while those from HLA-C2- donors would be uneducated .

Functional assay design:

• Direct stimulation: Culture NK cells with HLA-I-devoid target cells (e.g., K562) and measure activation markers

• Antibody-dependent stimulation: Use target cells coated with antibodies (e.g., HIV-1 gp140-pulsed cells with anti-HIV-1 antibodies)

• Readouts: Measure IFN-γ production, CD107a expression (degranulation), or direct cytotoxicity

Comparative analysis: Within HLA-C2+ donors, compare responses between KIR2DL1+ and KIR2DL1- NK cells to isolate the education effect . In educated donors, KIR2DL1+ NK cells should show higher IFN-γ production than KIR2DL1- NK cells upon stimulation.

Control for differentiation status: Include markers like CD57 to distinguish education effects from maturation effects . Analyzing CD57- NK cell populations helps isolate the functional advantage conferred by education from effects of differential maturation.

How does KIR2DL1 allelic variation impact antibody binding and functional studies?

KIR2DL1 allelic variation creates important considerations for antibody selection and functional interpretation:

Antibody recognition patterns: HP-DM1 recognizes most KIR2DL1 allotypes except KIR2DL1022 and likely KIR2DL1020 . Other antibodies show differential binding patterns to KIR2DL1-C245 versus KIR2DL1-R245 allotypes .

Functional implications: Cells expressing KIR2DL1-C245 exhibit lower receptor surface density and reduced missing-self reactivity compared to cells expressing KIR2DL1-R245 . This has significant implications when interpreting functional data across donors with different allotypes.

Experimental design considerations:

For accurate interpretation of results, researchers should either genotype donors for KIR2DL1 allotypes or use antibody combinations that can distinguish these variants . This is particularly important when comparing functional data across different donor populations.

How does HLA-C2 expression influence KIR2DL1 surface expression and NK cell education?

HLA-C2 expression has distinctive effects on KIR2DL1 expression and function:

Co-expression of the HLA-C2 ligand diminishes KIR2DL1 cell surface staining intensity but does not impact the frequency of KIR2DL1-expressing cells within the NK repertoire . Interestingly, HLA-C2 expression does not affect surface staining of the activating receptor KIR2DS1, highlighting differential regulation of these related receptors .

From an educational perspective, NK cells educated through KIR2DL1/HLA-C2 interactions demonstrate enhanced functional potential, including increased IFN-γ production upon both direct and antibody-dependent stimulation . This functional enhancement represents the core mechanism by which education improves NK cell responsiveness.

For experimental design, researchers must account for donor HLA-C status when studying KIR2DL1 expression and function, as this influences both receptor detection sensitivity and the functional capacity of KIR2DL1+ NK cells .

What is the impact of education through KIR2DL1/HLA-C2 on antibody-dependent NK cell functions?

Education through KIR2DL1/HLA-C2 interactions substantially enhances NK cell antibody-dependent functions:

NK cells educated through KIR2DL1/HLA-C2 interactions show significantly enhanced activation upon antibody-dependent stimulation . Higher IFN-γ production is consistently observed in KIR2DL1+ NK cells compared to KIR2DL1- NK cells in donors carrying the HLA-C2 ligand .

This functional advantage persists independently of differentiation status. When analyzing CD57- NK cells (less differentiated), educated KIR2DL1+ NK cells still demonstrate enhanced antibody-dependent responses, confirming that education effects are not merely a byproduct of differentiation .

The enhanced antibody-dependent function of educated KIR2DL1+ NK cells may have significant implications for viral control. Recent evidence suggests these cells might contribute to protection from HIV-1 infection and suppression of viral replication during primary infection , similar to the well-documented protective effects of the KIR3DL1/HLA-Bw4 receptor/ligand pair.

How can I distinguish between KIR2DL1-C245 and KIR2DL1-R245 variants in experimental settings?

Distinguishing between these functionally distinct KIR2DL1 variants requires specific methodological approaches:

Flow cytometry discrimination: Specific antibody combinations can distinguish cells expressing KIR2DL1-C245 from those expressing KIR2DL1-R245 by differential binding patterns . The informative differential binding of anti-KIR2DL1/S1 clone 1127B appears to be determined by amino acid residue T154 .

Surface density quantification: KIR2DL1-C245 exhibits consistently lower cell surface density compared to KIR2DL1-R245, which can be measured by quantitative flow cytometry using calibration beads .

Functional assessment: Missing-self reactivity differs measurably between these variants, with KIR2DL1-R245 showing higher reactivity . This functional difference can help identify the variants in experimental settings when combined with surface phenotyping.

Expression patterns: While both KIR2DL1-C245 and KIR2DL1-R245 can be co-expressed in the same cell, NK cells preferentially express one or the other variant , creating distinguishable populations in flow cytometric analysis.

What is the role of KIR2DL1+ NK cells in HIV infection control?

KIR2DL1+ NK cells exhibit important functional properties relevant to HIV control:

Recent studies suggest that educated KIR2DL1+ NK cells might play a significant role in protection from HIV-1 infection and suppression of viral replication during primary infection . This parallels the epidemiologically established protective effects of the KIR3DL1/HLA-Bw4 receptor/ligand combination.

NK cells educated through KIR2DL1/HLA-C2 interactions show enhanced activation upon antibody-dependent stimulation with HIV-1 gp140-pulsed target cells in the presence of anti-HIV-1 antibodies . This enhanced antibody-dependent activity may contribute to viral control through more efficient elimination of antibody-coated infected cells.

For HIV research applications, investigators should consider:

• Using antibody-dependent activation assays with HIV-1 gp140-pulsed target cells

• Comparing functional responses between KIR2DL1+ and KIR2DL1- NK cells in HLA-C2+ donors

• Assessing the impact of KIR2DL1 allelic variation on anti-HIV responses

• Using blocking antibodies like HP-DM1 to investigate the specific contribution of KIR2DL1

How can I design experiments to study KIR2DL1+ NK cells in HCMV infection?

Human cytomegalovirus (HCMV) infection drives specific NK cell adaptations that interact with KIR2DL1 expression:

Both KIR2DL1-C245 and KIR2DL1-R245 showed similar expansion among NKG2C+KIR2DL1+ NK cells in HCMV-seropositive individuals , suggesting HCMV-driven expansion is not substantially affected by KIR2DL1 allotypic variation.

For studying KIR2DL1+ NK cells in HCMV contexts, design experiments with:

• Antibody panels combining anti-KIR2DL1 (HP-DM1) with anti-NKG2C to identify expanded populations

• Comparative analysis between HCMV-seropositive and seronegative donors

• Assessment of KIR2DL1 allotype-specific effects on expansion and function

• Longitudinal studies tracking KIR2DL1+NKG2C+ populations during primary infection or reactivation

When analyzing HCMV-expanded NK cells, consider that both allotypic variants of KIR2DL1 appear to participate equally in the expansion process, despite their functional differences in other contexts .

What considerations are important when studying KIR2DL1+ NK cells in haplo-HSCT settings?

Hematopoietic stem cell transplantation (HSCT) research requires special considerations for KIR2DL1 analysis:

Donor characterization is crucial: Precise phenotypic analysis of KIR2DL1+ NK cell subsets is important when selecting potential donors for αβT/B-depleted haplo-HSCT . Donors should have known KIR genotypes to properly interpret expression patterns and functional capacity.

Antibody panel optimization: HP-DM1 mAb in combination with EB6/11PB6, 143211, and HP-MA4 allows accurate identification of different KIR+ NK cell subsets in potential donors . This comprehensive approach is particularly valuable when characterizing complex KIR genotypes.

Special attention for specific genotypes: Particular care should be taken when dissecting the expression pattern of various KIR2D receptors in NK cells from KIR2DL3*005+ individuals, especially if KIR2DS1 is present .

KIR2DS5 considerations: HP-DM1 mAb significantly refines NK cell phenotyping of donors carrying KIR2DS5, either in the centromeric or telomeric region . This refinement is essential for accurate characterization of functional NK cell subsets in transplantation settings.

How do blocking antibodies against KIR2DL1 affect NK cell function in experimental settings?

Blocking antibodies provide valuable tools for investigating KIR2DL1 function:

HP-DM1 mAb has been demonstrated to effectively block KIR2DL1 recognition of C2+ HLA-C . This blocking capacity enables researchers to experimentally manipulate the inhibitory signal typically delivered by KIR2DL1 upon HLA-C2 engagement.

When designing blocking experiments:

• Include appropriate isotype controls to account for non-specific antibody effects

• Perform dose-titration experiments to determine optimal blocking concentration

• Include both KIR2DL1+ and KIR2DL1- NK cell populations as internal controls

• Measure multiple functional parameters (cytotoxicity, cytokine production, degranulation)

Blocking KIR2DL1 in NK cells from HLA-C2+ donors should enhance function against target cells expressing HLA-C2, while having minimal effects on KIR2DL1- NK cells or in donors lacking HLA-C2.

How do KIR2DL1 and KIR2DS5 co-expression patterns impact antibody selection and functional studies?

Co-expression of KIR2DL1 and KIR2DS5 creates specific experimental challenges:

The MAB1844 antibody (clone 143211) detects both KIR2DL1 and KIR2DS5 on transfected cells . This cross-reactivity necessitates careful antibody selection when studying populations potentially expressing both receptors.

For accurate identification of receptor-specific populations:

• Use HP-DM1 in combination with 143211 to distinguish KIR2DL1-only from dual KIR2DL1/KIR2DS5-expressing cells

• Incorporate genetic analysis to confirm expression patterns in complex samples

• Consider functional testing, as KIR2DL1 delivers inhibitory signals while KIR2DS5 may deliver activating signals

Functional studies of donors carrying both receptors should account for potentially opposing signals delivered by these receptors when engaging their ligands, which may obscure receptor-specific effects if not properly controlled.