KLRC1 Antibody

What is the function of KLRC1 in the immune system?

KLRC1 (NKG2A) acts as an immune inhibitory receptor involved in self-nonself discrimination. When expressed as a heterodimer with CD94 on cytotoxic and regulatory lymphocyte subsets, it recognizes non-classical major histocompatibility (MHC) class Ib molecule HLA-E loaded with self-peptides derived from the signal sequence of classical MHC class Ia molecules. This interaction enables cytotoxic cells to monitor the expression of MHC class I molecules in healthy cells and to tolerate self . In tumor microenvironments rich in HLA-E, KLRC1 acts as an immune inhibitory checkpoint that may contribute to progressive loss of effector functions of NK cells and tumor-specific T cells, a state known as cell exhaustion .

What types of anti-KLRC1 antibodies are commercially available for research?

Several types of anti-KLRC1 antibodies are available for research applications:

• Polyclonal antibodies: Such as Rabbit Anti-Human KLRC1 (N-term) Antibody, which is generated from rabbits immunized with a KLH conjugated synthetic peptide from the N-terminal region of human KLRC1 .

• Monoclonal antibodies: Including humanized monoclonal antibodies like Monalizumab that specifically target NKG2A .

• Recombinant antibodies: Multiple formats including whole IgG antibodies and Fab fragments .

• Conjugated antibodies: Available with various conjugates such as PE/Cyanine7, Allophycocyanin, and Biotin for different detection methods .

These antibodies vary in their characteristics:

How do I select the appropriate anti-KLRC1 antibody for my experiments?

Selection should be based on:

• Experimental application: Different antibodies are optimized for specific techniques. For example, the Rabbit Anti-Human KLRC1 (N-term) Antibody is recommended for Western Blotting and Immunofluorescence , while others may be specifically validated for flow cytometry .

• Recognition domain: Consider whether you need an antibody targeting the N-terminal region, C-terminal region, or a specific epitope within the KLRC1 protein.

• Clonality: Polyclonal antibodies recognize multiple epitopes and provide stronger signals but may have batch-to-batch variability. Monoclonal antibodies offer high specificity and reproducibility .

• Species reactivity: Ensure the antibody reacts with your species of interest. Many KLRC1 antibodies are human-specific, and cross-reactivity should be verified .

• Validation data: Review available validation data including Western blot results, immunofluorescence images, and flow cytometry histograms to confirm specificity and performance .

How should I design experiments to evaluate KLRC1 antibody blocking efficiency in NK cell function?

To evaluate KLRC1 antibody blocking efficiency:

• Cell preparation: Isolate NK cells from peripheral blood mononuclear cells (PBMCs) and expand them using appropriate stimuli, such as K562 cells engineered to overexpress 4-1BBL and membrane-bound human IL-21 .

• Antibody titration: Test different concentrations of the anti-KLRC1 antibody (e.g., 0.01-10 μg/ml) to determine the optimal concentration for receptor blocking. For instance, monalizumab has shown an EC50 of approximately 1.5 ± 0.78 μg/ml, while newer generation antibodies like KSQ mAb demonstrate EC50 values around 0.045 ± 0.02 μg/ml .

• Functional assays:

  • Cytotoxicity assay: Co-culture NK cells with target cells (e.g., tumor cell lines expressing HLA-E) at different effector:target ratios (e.g., 1:1) in the presence or absence of anti-KLRC1 antibodies or isotype controls .

  • After incubation (typically 24 hours at 37°C, 5% CO2), collect cells and stain with appropriate markers to differentiate NK cells from target cells (e.g., BUV395-conjugated human CD56 antibody) .

  • Analyze cell death using flow cytometry or alternative methods.

• Controls:

  • Include isotype-matched control antibodies to account for non-specific effects.

  • Include positive controls such as anti-NKG2D antibodies, which target activating receptors.

  • Include HLA-E-negative target cells as additional controls.

• Assessment methods:

  • Measure target cell death (e.g., using Annexin V/PI staining)

  • Quantify NK cell activation markers (e.g., CD69, CD107a)

  • Measure cytokine production (e.g., IFN-γ, TNF-α)

What are the key considerations for using anti-KLRC1 antibodies in flow cytometry?

When using anti-KLRC1 antibodies for flow cytometry:

• Antibody selection: Choose antibodies specifically validated for flow cytometry applications. BioLegend's purified anti-human CD159a (NKG2A) antibody (clone S19004C) is an example of a validated antibody for flow cytometry .

• Optimal concentration: Titrate the antibody to determine the optimal concentration. For example, BioLegend recommends ≤0.25 μg per million cells in 100 μL volume for their anti-CD159a antibody .

• Panel design:

  • Include markers to identify NK cells (CD56+CD3-) and relevant T cell subsets (CD8+CD3+)

  • Consider including CD94 (KLRD1) to examine the co-expression with KLRC1, as they form heterodimers

  • Include functional markers (e.g., IFN-γ, CD107a) if assessing functional responses

• Controls:

  • Fluorescence minus one (FMO) controls to set proper gates

  • Isotype controls to assess non-specific binding

  • Positive controls (e.g., NK cell line with known KLRC1 expression)

  • Negative controls (e.g., cell line lacking KLRC1 expression)

• Sample preparation:

  • Ensure proper Fc receptor blocking to prevent non-specific binding

  • Optimize fixation and permeabilization protocols if intracellular staining is required

  • Consider cell viability dyes to exclude dead cells from analysis

How can I validate KLRC1 antibody specificity in my experimental system?

Validating KLRC1 antibody specificity requires multiple approaches:

• Western blot analysis: Perform Western blotting using cell lysates from cell lines with known KLRC1 expression (e.g., MDA-MB435 cell line) . Look for a specific band at the expected molecular weight of KLRC1.

• Immunofluorescence: Conduct confocal immunofluorescent analysis with appropriate controls. For example, stain MDA-MB435 cells with the KLRC1 antibody followed by fluorophore-conjugated secondary antibody, and counterstain with DAPI for nuclear visualization and phalloidin for actin visualization .

• Flow cytometry with genetic controls:

  • Compare staining in wild-type cells versus KLRC1 knockout cells

  • Use CRISPR/Cas9 to generate KLRC1 knockout cells as negative controls

  • Overexpress KLRC1 in cells with low endogenous expression as positive controls

• Blocking peptide competition: Pre-incubate the antibody with the immunizing peptide and demonstrate reduced or abolished staining.

• Cross-reactivity assessment: Test the antibody against related proteins (e.g., other NKG2 family members) to ensure specificity.

• Reproducibility across techniques: Confirm consistent results across multiple detection methods (flow cytometry, Western blotting, immunofluorescence).

How does KLRC1 knockout compare to antibody-mediated blockade in enhancing NK cell anti-tumor activity?

KLRC1 knockout and antibody-mediated blockade show important differences in enhancing NK cell anti-tumor activity:

• Mechanistic differences:

  • KLRC1 knockout NK cells show enhanced activation, reduced suppressive signaling, and elevated expression of key transcription factors compared to antibody blockade .

  • Genetic ablation completely eliminates receptor expression, while antibody blockade may achieve only partial blocking depending on antibody affinity and concentration.

• Functional outcomes:

  • KLRC1 knockout NK cells have demonstrated superior ability to overcome inhibition from HLA-E, significantly boosting NK cell activity against solid and hematologic cancer cells .

  • This enhanced efficacy has been validated across multiple cell lines, a xenograft mouse model, and primary human leukemic cells .

• Combinatorial approaches:

  • Interestingly, combining KLRC1 knockout with antibody coating of tumor cells further enhances cytotoxicity through antibody-dependent cellular cytotoxicity (ADCC) .

• Technical aspects:

  • KLRC1 knockout using CRISPR/Cas9 ribonucleoprotein delivery has achieved up to 98% knockout efficiency in human NK cells .

  • The knockout approach eliminates variability in antibody binding and concerns about antibody half-life and tissue penetration.

• Practical considerations:

  • Antibody blockade is more readily translatable to clinical settings but may require repeated dosing.

  • Genetic knockout is better suited for adoptive cell therapy approaches but requires sophisticated cell engineering.

What are the implications of targeting KLRC1 in viral infection models versus cancer models?

The implications of targeting KLRC1 differ significantly between viral infection and cancer models:

• Viral infection context:

  • In viral infection models, NKG2A (KLRC1) plays a crucial role in sustaining virus-specific CD8+ T cells in persistent infections .

  • Klrc1-/- mice show increased susceptibility to viral infections, with marked histopathology evident in spleen sections that become highly acellular with disrupted tissue architecture compared to wild-type controls 7 days post-infection .

  • The absence of NKG2A in these models leads to a reduction in multiple leukocyte populations, including NK cells, T cells, B cells, dendritic cells, and macrophages .

• Cancer context:

  • In cancer models, NKG2A acts as an immune inhibitory checkpoint that may contribute to progressive loss of effector functions of NK cells and tumor-specific T cells .

  • Cancer cells often overexpress HLA-E (the ligand for NKG2A) to evade NK cell surveillance .

  • KLRC1 knockout or antibody blockade enhances NK cell anti-tumor activity against HLA-E+ tumors .

• Balancing immune responses:

  • These contrasting roles suggest that NKG2A functions as a regulatory switch that balances protective immunity versus immunopathology.

  • In viral infections, NKG2A may prevent excessive immune activation and associated tissue damage.

  • In cancer, NKG2A may contribute to tumor immune evasion by dampening NK and T cell responses.

• Therapeutic implications:

  • Anti-KLRC1 interventions should be carefully timed and contextualized.

  • They may be beneficial in cancer therapy but potentially detrimental during certain viral infections.

  • Combination approaches that maintain protective immunity while enhancing anti-tumor responses should be explored.

How does the binding affinity of different anti-KLRC1 antibodies correlate with their functional efficacy?

The relationship between binding affinity and functional efficacy of anti-KLRC1 antibodies shows several important patterns:

• Affinity-efficacy correlation:

  • Higher affinity antibodies generally demonstrate improved functional efficacy. For example, the KSQ mAb has approximately 830-fold higher specific monovalent affinity for NKG2A compared to monalizumab (KD of 0.058 ± 0.007 nM versus 48.1 ± 3.1 nM) .

  • This higher affinity translates to enhanced potency in in vitro antitumor cytotoxicity assays, with EC50 values of 0.045 ± 0.02 μg/ml for KSQ mAb compared to 1.5 ± 0.78 μg/ml for monalizumab .

• Pharmacokinetic parameters:

  • The relationship is not solely dependent on affinity but also influenced by pharmacokinetic properties:

• In vivo implications:

  • Semimechanistic pharmacokinetic/receptor occupancy (SM-PK/RO) modeling suggests that increased affinity may translate into substantial clinical benefits by lowering the required dose and/or reducing dosing frequency while maintaining receptor saturation .

  • The KSQ mAb is predicted to require a 10-fold lower dose than monalizumab to achieve similar receptor occupancy over a 3-week period following q3w intravenous infusion dosing .

• Epitope considerations:

  • Antibodies targeting different epitopes of KLRC1 may have varying functional consequences despite similar affinities.

  • The location of binding (N-terminal versus other regions) may impact the ability to disrupt KLRC1 interaction with HLA-E.

How do different applications of anti-KLRC1 antibodies (WB, IF, FC) compare in terms of protocol optimization?

Each application of anti-KLRC1 antibodies requires specific optimization approaches:

• Western Blotting (WB):

  • Sample preparation: For KLRC1 detection, cell lysates should be prepared with proper lysis buffers that preserve membrane proteins (e.g., using 35 μg/lane of MDA-MB435 cell line lysates) .

  • Antibody concentration: Typically higher concentrations are required compared to flow cytometry. For polyclonal antibodies, dilutions around 1:500-1:2000 are common starting points.

  • Detection systems: Secondary antibodies conjugated to HRP followed by chemiluminescent detection are commonly used.

  • Controls: Include positive control lysates from cells known to express KLRC1 and negative controls.

  • Expected results: A specific band should be visible at the molecular weight of KLRC1.

• Immunofluorescence (IF):

  • Fixation/permeabilization: Optimization of fixation (e.g., paraformaldehyde) and permeabilization (e.g., Triton X-100) conditions is crucial for maintaining KLRC1 epitope accessibility.

  • Antibody incubation: Typically overnight at 4°C for primary antibody, followed by 1-2 hours at room temperature for secondary antibody.

  • Visualization: Use appropriate secondary antibodies (e.g., Alexa Fluor 488-conjugated goat anti-rabbit IgG) and counterstain with DAPI for nuclei and phalloidin for actin filaments .

  • Controls: Include cells with known KLRC1 expression patterns and secondary-only controls.

• Flow Cytometry (FC):

  • Cell preparation: Fresh isolation or proper thawing procedures are critical for maintaining surface expression of KLRC1.

  • Antibody titration: Lower concentrations are generally required (≤0.25 μg per million cells in 100 μL volume) .

  • Incubation conditions: Typically 15-30 minutes at 4°C in the dark.

  • Washing steps: Multiple gentle washes to remove unbound antibody while preserving cell integrity.

  • Controls: Include fluorescence minus one (FMO) and isotype controls for accurate gating.

• Key differences and considerations:

  • WB detects denatured proteins and is useful for confirming molecular weight and expression levels.

  • IF preserves cellular architecture and can reveal subcellular localization patterns.

  • FC allows for quantitative assessment of KLRC1 expression at the single-cell level and enables simultaneous analysis of multiple markers.

  • Epitope accessibility may differ between applications, and antibodies validated for one application may not work for others.

What are the advantages and limitations of using recombinant versus conventional antibodies for KLRC1 research?

Recombinant and conventional antibodies each offer distinct advantages and limitations for KLRC1 research:

Recombinant Antibodies:

Advantages:

• Increased sensitivity: Recombinant technology allows for optimization of binding domains, potentially increasing detection sensitivity .

• Confirmed specificity: Sequence-defined antibodies ensure targeting of specific epitopes .

• High repeatability: The defined genetic sequence enables consistent production with minimal batch-to-batch variation .

• Sustainable supply: Once the sequence is determined, the antibody can be produced indefinitely without relying on animal immunization .

• Customizability: Formats can be easily modified (e.g., switching from whole IgG to Fab fragments) using genetic engineering techniques .

• Animal-free production: Eliminates ethical concerns associated with animal immunization .

Limitations:

• Cost: Production may be more expensive initially.

• Technical complexity: Requires specialized expertise and facilities for development.

• Limited epitope diversity: May target fewer epitopes compared to polyclonal antibodies.

Conventional Antibodies:

Advantages:

• Established protocols: Well-established production and validation methods.

• Cost-effective: Often less expensive for initial production.

• Epitope diversity: Polyclonal antibodies recognize multiple epitopes, potentially increasing detection sensitivity in certain applications.

Limitations:

• Batch-to-batch variability: Particularly significant for polyclonal antibodies, leading to inconsistent results.

• Limited sustainability: Dependent on continued animal immunization for production.

• Standardization challenges: Difficult to standardize between laboratories.

• Ethical considerations: Requires animal immunization.

Comparison Table:

For KLRC1 research, recombinant antibodies like the Hi-Affi™ portfolio offer particular advantages when consistent detection across experiments is crucial .

How do enzymatic versus genetic methods for validating KLRC1 antibody specificity compare?

Enzymatic and genetic methods for validating KLRC1 antibody specificity offer complementary approaches with distinct advantages:

Enzymatic Methods:

• Peptide competition assays:

  • Approach: Pre-incubate antibody with synthetic peptides (e.g., KLH conjugated synthetic peptide between 1-30 amino acids from the N-terminal region of human KLRC1) .

  • Advantages: Relatively simple to perform; directly tests epitope binding.

  • Limitations: Only confirms binding to linear epitopes; may not represent native protein conformation.

• Immunodepletion:

  • Approach: Deplete the antibody by passing through a column containing immobilized KLRC1 protein.

  • Advantages: Can confirm specificity using the complete protein.

  • Limitations: Requires purified protein; may give false negatives if epitope is altered during purification.

• Proteolytic treatment of samples:

  • Approach: Treat samples with specific proteases to cleave KLRC1 before antibody detection.

  • Advantages: Can confirm dependency on protein integrity.

  • Limitations: Limited specificity; affects all proteins in sample.

Genetic Methods:

• CRISPR/Cas9 knockout:

  • Approach: Generate KLRC1 knockout cells using CRISPR technology with specific gRNAs targeting KLRC1 exons (e.g., exon 2 with sequence 5′-ACTCAGACCTGAATCTGCCCC-3′) .

  • Advantages: Creates definitive negative controls; knockout efficiencies as high as 98% have been reported .

  • Limitations: Time-consuming; may have off-target effects.

• siRNA/shRNA knockdown:

  • Approach: Transiently reduce KLRC1 expression using specific siRNAs.

  • Advantages: Relatively quick; suitable for cells difficult to modify with CRISPR.

  • Limitations: Incomplete knockdown; potential off-target effects.

• Overexpression systems:

  • Approach: Express KLRC1 in cells with low endogenous expression.

  • Advantages: Creates positive controls with defined expression levels.

  • Limitations: Overexpression may alter protein localization or modification.

• Off-target analysis:

  • Approach: Use methods like CHANGE-seq to profile the entire genome for potential off-target effects of CRISPR editing, ensuring specificity of the knockout model .

  • Advantages: Comprehensive assessment of specificity; can identify unexpected interactions.

  • Limitations: Technically demanding; expensive.

Comparative Assessment:

The most robust validation combines multiple approaches: genetic manipulation to create definitive controls followed by enzymatic methods to confirm specific binding mechanisms.

What are the most common pitfalls in interpreting KLRC1 antibody staining in flow cytometry experiments?

Several common pitfalls can complicate interpretation of KLRC1 antibody staining in flow cytometry:

• False negatives due to epitope masking:

  • Problem: KLRC1 forms heterodimers with CD94 , potentially masking antibody epitopes.

  • Solution: Use antibodies targeting different epitopes; combine with CD94 staining to confirm heterodimer presence.

• Non-specific binding to Fc receptors:

  • Problem: NK cells express high levels of Fc receptors that can bind antibody Fc regions.

  • Solution: Always include proper Fc receptor blocking; use isotype controls; consider Fab fragments for problematic samples .

• Misidentification of cell populations:

  • Problem: KLRC1 is expressed on multiple immune cell subsets with varying expression levels.

  • Solution: Use comprehensive panels including lineage markers (e.g., CD56, CD3, CD8) to properly identify cell types; include NKG2 family markers to differentiate from other receptors .

• Antibody internalization:

  • Problem: Some antibodies may induce receptor internalization, leading to reduced surface staining.

  • Solution: Conduct staining at 4°C; minimize incubation time; compare surface versus intracellular staining protocols.

• Heterogeneous expression patterns:

  • Problem: KLRC1 expression varies between NK cell subsets (e.g., NK1, NK2, NK3 populations) .

  • Solution: Use additional markers like KIRs, CD161, and SIGLEC7 to identify specific NK subsets; don't rely solely on mean fluorescence intensity for population comparisons .

• Pre-analytical variables:

  • Problem: Sample handling, freezing/thawing, and processing can affect KLRC1 expression.

  • Solution: Standardize sample preparation protocols; include internal controls; consider time-dependent expression changes.

• Cross-reactivity with other NKG2 family members:

  • Problem: NKG2 family proteins share sequence similarity.

  • Solution: Validate antibody specificity using KLRC1 knockout cells ; combine with staining for other family members like NKG2C (KLRC2) .

• Context-dependent expression changes:

  • Problem: KLRC1 expression can be modulated by cytokines, viral infections, and tumor microenvironment factors.

  • Solution: Include appropriate experimental controls; consider time-course experiments; correlate with functional assays.

How can researchers troubleshoot inconsistent results between different KLRC1 antibody clones?

When encountering inconsistent results between different KLRC1 antibody clones, a systematic troubleshooting approach is essential:

• Epitope mapping and antibody characterization:

  • Analysis: Determine which epitopes are recognized by each antibody clone. Some antibodies target the N-terminal region , while others may target different domains.

  • Action: Review antibody datasheets for epitope information; request epitope data from manufacturers if unavailable.

• Clone-specific technical optimization:

  • Analysis: Different clones may require specific conditions for optimal binding.

  • Action: Perform separate titrations for each clone; optimize incubation time, temperature, and buffer conditions independently.

• Sample preparation effects:

  • Analysis: Fixation, permeabilization, or freezing/thawing may differentially affect epitope accessibility.

  • Action: Test multiple preparation methods with each clone; standardize protocols once optimal conditions are identified.

• Format and conjugation differences:

  • Analysis: Different fluorophores or modifications may affect antibody binding characteristics.

  • Action: Test the same clone with different conjugates; use secondary detection methods if primary antibodies are unconjugated.

• Species and isotype considerations:

  • Analysis: Different species origins (e.g., rabbit vs. mouse) or isotypes may contribute to discrepancies.

  • Action: Test blocking reagents specific to each antibody's species and isotype; consider species-matched controls.

• Cross-validation experiments:

  • Analysis: Determine whether differences reflect true biological variation or technical artifacts.

  • Action: Use multiple detection methods (flow cytometry, Western blot, IF) with the same clones ; correlate with mRNA expression data.

• Reference standards and controls:

  • Analysis: Establish reliable positive and negative controls for each clone.

  • Action: Use KLRC1 transfected and knockout cell lines as definitive controls ; include cells with known expression patterns.

• Batch and storage effects:

  • Analysis: Antibody stability and activity may vary between lots or with storage conditions.

  • Action: Record lot numbers; aliquot antibodies to avoid freeze-thaw cycles; store according to manufacturer recommendations (typically at -20°C for long-term storage) .

• Systematic side-by-side comparison:

  • Analysis: Direct comparison under identical conditions can reveal consistent patterns.

  • Action: Design experiments testing multiple clones simultaneously on the same samples; document all variables systematically.

How should researchers interpret contradictory findings between KLRC1 antibody data and genetic knockout models?

When facing contradictions between antibody-based data and genetic knockout models for KLRC1, consider these interpretative approaches:

• Antibody specificity reassessment:

  • Analysis: Determine if the antibody truly recognizes KLRC1 exclusively.

  • Action: Test the antibody on confirmed KLRC1 knockout cells generated using CRISPR/Cas9 ; perform off-target analysis such as CHANGE-seq to verify knockout specificity .

• Compensatory mechanisms in knockout models:

  • Analysis: Genetic knockouts may trigger compensatory expression of related receptors.

  • Action: Examine expression of other NKG2 family members (especially NKG2C/KLRC2) ; assess changes in other inhibitory receptors like KIRs or TIGIT following KLRC1 deletion.

• Timing considerations:

  • Analysis: Acute antibody blockade versus developmental genetic absence may yield different phenotypes.

  • Action: Compare acute (antibody-mediated) versus chronic (genetic) KLRC1 ablation; consider inducible knockout systems for temporal control.

• Protein-protein interaction effects:

  • Analysis: KLRC1 forms heterodimers with CD94 ; genetic deletion may affect partner proteins differently than antibody blockade.

  • Action: Assess CD94 expression and localization in both scenarios; examine effects on downstream signaling molecules.

• Context-dependent regulation:

  • Analysis: KLRC1 functions differently in various physiological contexts.

  • Action: Compare contexts systematically: viral infection models show KLRC1 is essential for sustaining virus-specific CD8+ T cells , while in cancer models, KLRC1 inhibits anti-tumor activity .

• Methodological limitations:

  • Analysis: Different assay sensitivities or experimental conditions may contribute to discrepancies.

  • Action: Standardize experimental conditions; perform dose-response studies with antibodies; verify knockout efficiency at DNA, RNA, and protein levels.

• Isoform-specific effects:

  • Analysis: Alternative splicing produces multiple KLRC1 isoforms that may be differentially affected.

  • Action: Use RNA sequencing to identify expressed isoforms; verify whether knockout affects all isoforms; confirm antibody recognition of all relevant isoforms.

• Functional outcome assessment:

  • Analysis: Phenotypic differences may be reconciled through functional testing.

  • Action: Compare functional outcomes like cytotoxicity or cytokine production between antibody blockade and genetic knockout; study downstream signaling events in both contexts .

• Integrated hypothesis development:

  • Analysis: Contradictions often reveal complex biological regulation.

  • Action: Develop integrated models explaining different outcomes; design experiments specifically testing these models; consider temporal, spatial, and context-dependent regulation of KLRC1 function.