KIR2DS2 Antibody
Description
Definition and Development of KIR2DS2 Antibodies
KIR2DS2 antibodies target the extracellular domains of the KIR2DS2 receptor, which distinguishes it from inhibitory KIRs (e.g., KIR2DL2, KIR2DL3). Historically, antibody specificity was limited due to the high sequence homology between KIR2DS2 and inhibitory KIRs. Recent advancements include:
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Novel Antibody Combinations: CH-L (anti-KIR2DS2) and REA147 (anti-KIR2DL2/DL3) enable discrimination of KIR2DS2+ NK cells from KIR2DL2/DL3+ populations .
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Epitope-Specific Antibodies: Polyclonal antibodies targeting regions such as AA 39–65 (e.g., ABIN651951) provide high specificity for KIR2DS2 .
Table 2: Functional Advantages of KIR2DS2+ NK Cells
| Parameter | KIR2DS2+ NK Cells | KIR2DL2/DL3+ NK Cells |
|---|---|---|
| CD16 Expression | High | Moderate/Low |
| ADCC Response | Enhanced | Lower |
| Spontaneous Cytotoxicity | Increased | Basal |
| Target Specificity | Malignant B cells, hepatocellular carcinoma, CLL | Variable |
Cancer Immunotherapy
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NK Cell Selection: KIR2DS2+ NK cells are enriched in KIR B haplotype donors, associated with reduced relapse in hematological malignancies .
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Anti-CD20 Therapy: KIR2DS2+ NK cells show superior activation against rituximab/obinutuzumab-coated lymphoma cells, even in HLA-C–negative environments .
Experimental Tools
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Reporter Cell Lines: KIR2DS2 tetramers enable live-cell binding assays to study ligand interactions .
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Flow Cytometry Panels: CH-L/REA147 combinations enable precise gating of KIR2DS2+ NK cells in primary samples .
Challenges and Future Directions
Q&A
What is KIR2DS2 and what role does it play in NK cell function?
KIR2DS2 is an activating killer cell immunoglobulin-like receptor expressed on natural killer cells. Unlike inhibitory KIRs that detect HLA-C downregulation and are modulated by changes in peptide content of HLA-C, KIR2DS2 functions as an activation receptor . This receptor has been implicated in protective immune responses against both viral infections and cancers .
The specific challenge with KIR2DS2 has been defining its ligands and distinguishing it from inhibitory receptors due to high sequence homology with KIR2DL2 and KIR2DL3 . This homology has historically made it difficult to develop antibodies that specifically detect NK cells expressing KIR2DS2 .
Why has KIR2DS2 been difficult to study in primary human samples?
The primary challenge in studying KIR2DS2 stems from the receptor's high sequence homology with inhibitory KIRs (KIR2DL2 and KIR2DL3), making antibody discrimination difficult . Previously available antibodies such as CH-L, DX27, and GL-183 detect KIR2DS2 but also bind to KIR2DL2 and KIR2DL3, preventing specific identification .
Some antibodies like clone 1F12 can distinguish KIR2DS2 from KIR2DL2 but still bind KIR2DL3, limiting their use to donors with specific KIR genotypes (KIR2DL2/KIR2DS2 homozygous who lack KIR2DL3) . These technical limitations have restricted the ability to study KIR2DS2-specific functions in primary human NK cells, especially in individuals with heterozygous KIR genotypes .
What evidence exists for KIR2DS2's role in cancer and viral infections?
KIR2DS2 has demonstrated significant protective associations in multiple disease contexts:
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Reduced incidence of relapse following bone marrow and cord blood transplantation for hematological malignancies
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Direct recognition of viral helicase peptides in the context of HLA-C
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Enhanced activation against malignant B cell lines, liver cancer cell lines, and primary chronic lymphocytic leukemia cells
Recent research indicates that NK cells with high KIR2DS2 expression demonstrate enhanced spontaneous activation against multiple cancer cell types and show improved response to therapeutic antibodies such as anti-CD20 antibodies, making this receptor an increasingly important target for immunotherapy development .
What antibody combinations effectively identify KIR2DS2-expressing NK cells?
A novel approach utilizing specific antibody combinations has been developed to identify KIR2DS2-expressing NK cells. This method employs:
| Antibody Clone | Manufacturer | Binding Specificity | Role in Identification |
|---|---|---|---|
| CH-L | BD Biosciences | KIR2DL2, KIR2DL3, KIR2DS2 | Positive marker for all three KIRs |
| REA147 | Miltenyi Biotech | KIR2DL2, KIR2DL3 | Does not bind KIR2DS2 |
Using this antibody combination, researchers can identify:
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KIR2DL3/L2high NK cells: CH-L positive and REA147 positive
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KIR2DS2high NK cells: CH-L positive and REA147 negative
This strategy effectively identifies a population of NK cells with relatively high expression of KIR2DS2, allowing for functional studies of KIR2DS2-mediated activation .
How can researchers validate the specificity of antibody combinations for KIR2DS2 detection?
Validation of KIR2DS2 antibody specificity requires a multi-step approach:
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Cell line validation: Using NKL cell lines transfected with individual KIRs (KIR2DL2, KIR2DL3, or KIR2DS2) to confirm antibody binding patterns .
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Functional validation: Testing response to KIR2DS2-specific ligands such as viral helicase peptides LNPSVAATL (from HCV) or IVDLMCHATF (from dengue virus), which specifically activate KIR2DS2 . When testing these peptides, KIR2DS2high NK cells (CH-L+/REA147-) show increased degranulation while KIR2DL3/L2high NK cells show no response .
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Controls: NK cells from KIR2DL3 homozygous donors (lacking KIR2DS2) should show no significant change in degranulation in response to KIR2DS2-specific ligands .
This comprehensive validation approach confirms that the antibody combination genuinely identifies functionally distinct KIR2DS2high NK cells .
What flow cytometry strategies are optimal for identifying and analyzing KIR2DS2high NK cells?
An optimal flow cytometry strategy for identifying KIR2DS2high NK cells includes:
| Step | Markers/Parameters | Purpose |
|---|---|---|
| 1 | FSC/SSC | Identify lymphocyte population |
| 2 | CD56+/CD3- | Identify NK cell population |
| 3 | CH-L vs. REA147 | Discriminate KIR subpopulations |
| 4 | CD107a, IFN-γ, TNF-α | Functional readouts |
| 5 | CD16 | ADCC potential assessment |
The critical gating strategy involves:
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Identifying the CD56+CD3- NK cell population
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Creating a plot of CH-L vs. REA147 staining
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Defining populations as:
This approach allows researchers to study KIR2DS2-related functions in donors with heterozygous KIR genotypes (KIR2DL3/L2/S2), expanding research beyond the previously required KIR2DL2/KIR2DS2 homozygous donors .
How does KIR2DS2 expression correlate with NK cell anticancer activity?
Research demonstrates that KIR2DS2high NK cells possess enhanced anticancer activities across multiple models:
| Cancer Cell Type | KIR2DS2high NK Cell Response | Comparison to Other NK Subsets |
|---|---|---|
| DOHH2 (B cell lymphoma) | Increased degranulation and cytokine production (IFN-γ, TNF-α) | Superior to KIR2DL3/L2high NK cells |
| REH (acute lymphoblastic leukemia) | Significantly increased IFN-γ (p<0.01) and TNF-α (p=0.001) production | Higher than both KIR2DL3/L2/S2- and KIR2DL3/L2high NK cells |
| Primary chronic lymphocytic leukemia | Enhanced activation | Superior to other NK subsets |
| Liver cancer cell lines | Enhanced anticancer activity | Superior to other NK subsets |
| 721.221 (HLA-C-null) | Increased IFN-γ expression | Higher than other NK subsets |
These observations indicate that KIR2DS2high NK cells have inherently increased anticancer activity independent of specific ligand interactions, suggesting they represent a population of NK cells primed for anticancer responses .
What is the relationship between KIR2DS2 expression and antibody-dependent cellular cytotoxicity (ADCC)?
KIR2DS2high NK cells display significantly enhanced ADCC capabilities compared to other NK cell subsets. This enhanced function correlates with:
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Increased CD16 expression: KIR2DS2high NK cells show significantly higher surface expression of CD16 (FcγRIIIA) compared to both KIR2DL3/L2high and KIR2DL3/L2/S2- NK cells, measured by both mean fluorescence intensity and percentage of CD16+ cells .
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Enhanced response to anti-CD20 antibodies: Against rituximab-coated and obinutuzumab-coated DOHH2 lymphoma cells, KIR2DS2high NK cells demonstrate significantly higher activation compared to other NK cell subsets .
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Compensatory effect for reduced natural cytotoxicity: Against MAVER-1 cells, where KIR2DS2high NK cells had lower spontaneous activation than KIR2DL3/L2/S2- cells, the addition of anti-CD20 antibodies led to superior activation of KIR2DS2high NK cells compared to KIR2DL3/L2high NK cells .
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HLA-independent ADCC enhancement: Using HLA-C-null cell line 721.221, KIR2DS2high NK cells maintained significantly increased activation with rituximab compared to both other NK cell subsets .
These findings suggest KIR2DS2 expression identifies NK cells with enhanced responsiveness to CD16 ligation, making them particularly effective for antibody-based cancer therapies .
How might KIR2DS2 be leveraged in developing NK cell-based cancer immunotherapies?
KIR2DS2 offers several promising strategies for enhancing NK-based cancer immunotherapies:
| Approach | Methodology | Potential Advantage |
|---|---|---|
| Selection/enrichment | Flow cytometry with CH-L/REA147 antibody combination | Isolate NK cells with enhanced anticancer activity |
| Genetic screening | Identify donors with KIR2DS2 genes | Optimize allogeneic NK therapy |
| Combination with therapeutic antibodies | Pair KIR2DS2-focused NK cells with monoclonal antibodies | Leverage enhanced ADCC capability |
| Biomarker development | Monitor KIR2DS2high NK populations | Predict immunotherapy response |
| Transcriptional targeting | Target pathways identified in KIR2DS2high transcriptome | Enhance NK cytotoxicity |
The inherently enhanced anticancer activity of KIR2DS2high NK cells, particularly in combination with therapeutic antibodies, makes this receptor an attractive target for developing next-generation NK cell therapies . Transcriptomic analysis shows KIR2DS2high NK cells have upregulation of NK-mediated cytotoxicity, translation, and FCGR gene pathways, providing molecular targets for therapeutic development .
What transcriptomic differences characterize KIR2DS2high NK cells?
Bulk RNA sequencing and single-cell RNA sequencing have revealed distinct transcriptional profiles in KIR2DS2high NK cells:
| Upregulated Pathway | Functional Implication | Technique |
|---|---|---|
| NK cell-mediated cytotoxicity | Enhanced killing capacity | Bulk RNA-seq |
| Translation pathways | Increased protein synthesis capability | Bulk RNA-seq |
| FCGR gene pathways | Enhanced ADCC responses | Bulk RNA-seq |
| Cytotoxicity gene signatures | Confirmed association with enhanced NK cytotoxicity | Custom scRNA-seq |
These transcriptomic differences provide a molecular basis for understanding why KIR2DS2high NK cells demonstrate enhanced effector functions against malignant cells, both in terms of natural cytotoxicity and antibody-dependent responses .
What is known about KIR2DS2-specific ligands and their research applications?
Recent advances have identified specific ligands for KIR2DS2 with significant research implications:
| Ligand | Source | HLA Context | Validation Method |
|---|---|---|---|
| LNPSVAATL (LNP) | Hepatitis C virus helicase | HLA-C | NK degranulation assays |
| IVDLMCHATF (IVD) | Dengue virus helicase | HLA-C | NK degranulation assays |
Functional validation demonstrates that NK cells identified as KIR2DS2high using the CH-L/REA147 antibody combination specifically respond to these peptides with increased degranulation, while KIR2DL3/L2high NK cells show no response . This confirms that KIR2DS2 functions as an antigen-specific receptor for conserved flaviviral helicase peptides .
These findings provide proof-of-concept data that KIR2DS2-positive NK cells can recognize specific viral peptides, explaining the protective role of KIR2DS2 in viral infections and potentially informing approaches to enhance NK cell activity against both viral infections and cancer .
How can single-cell approaches advance our understanding of KIR2DS2 function?
Single-cell technologies offer powerful new approaches to understand KIR2DS2 biology:
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Novel scRNA-seq techniques: A customized single-cell RNA sequencing approach has been developed specifically to identify KIR2DS2+ NK cells, confirming that KIR2DS2 expression associates with enhanced NK cell-mediated cytotoxicity at the single-cell level .
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Functional heterogeneity analysis: Single-cell approaches can reveal functional differences within the KIR2DS2high NK cell population, potentially identifying specialized subsets with enhanced anticancer or antiviral activity .
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Trajectory analysis: Single-cell data can map developmental relationships between different NK subsets, providing insights into how KIR2DS2high cells develop and are maintained.
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Integration with proteomics: Combining scRNA-seq with single-cell protein analysis can reveal post-transcriptional regulation of KIR2DS2 and associated molecules.
These single-cell approaches offer unprecedented resolution for understanding KIR2DS2 biology, potentially identifying new targets for therapeutic intervention and biomarker development .
