KIR2DL5A Antibody

What is KIR2DL5A and what cell types express this receptor?

KIR2DL5A (CD158f) is a 60 kDa monomeric glycoprotein with two Ig-like extracellular domains and a long cytoplasmic domain. It functions as an inhibitory receptor and belongs to the KIR (Killer-cell Immunoglobulin-like Receptor) family, which recognizes subsets of HLA-Class I alleles. KIR2DL5A is expressed on both innate and adaptive immune cells. Specifically, it is found on NK cells (particularly the CD56dim subset), γδ T cells, and a variable proportion of circulating T lymphocytes (primarily CD8+ T cells) . Within the CD8+ T cell population, KIR2DL5A expression is predominantly observed in terminally differentiated (Temra) and effector memory cell subsets, while being very low or undetectable in naive (Tn) and central memory (Tcm) CD8+ T cells . The expression pattern of KIR2DL5A is highly polymorphic between individuals, as the KIR2DL5 gene is found in only a fraction of the population .

How does KIR2DL5A signaling function in immune cells?

KIR2DL5A is a purely inhibitory receptor containing two immunoreceptor tyrosine-based inhibition motifs (ITIMs) in its cytoplasmic domain and a transmembrane region lacking charged amino acid residues. Upon ligand engagement, both intracellular ITIM and ITSM domains of KIR2DL5A undergo tyrosine phosphorylation, which is essential for its inhibitory function . These phosphorylated domains recruit protein tyrosine phosphatases, primarily SHP-2 (Src homology region 2-containing protein tyrosine phosphatase-2) and, to a lesser extent, SHP-1 . The recruitment of these phosphatases leads to the downregulation of downstream signaling pathways, particularly the Vav1/ERK1/2/p90RSK/NF-κB pathway . Functionally, KIR2DL5A engagement significantly inhibits NK cell cytotoxicity, degranulation (CD107a), and cytokine production (including IFN-γ and TNF-α) . A comprehensive cytokine analysis revealed that KIR2DL5A markedly decreases the production of numerous cytokines and chemokines, including IL-13, IL-18, IL-25, IL-27, eotaxin, EGF, GM-CSF, M-CSF, RANTES, MIP-1α, MIP-1β, and CXCL-9 .

What are the structural characteristics of KIR2DL5A that distinguish it from other KIR family members?

KIR2DL5A possesses several unique structural features that distinguish it from other KIR family members:

• Domain Organization: KIR2DL5A and KIR2DL4 are the only members of a gene lineage coding for KIRs with a D0-D2 organization of the Ig-like domains, which distinguishes them from all other KIR2Ds that have domains of the D1-D2 type .

• Inhibitory Signaling: While some KIRs have activating functions, KIR2DL5A is predicted to encode a purely inhibitory receptor based on its signaling motifs (two ITIMs and a transmembrane region lacking charged amino acid residues) .

• Genetic Representation: KIR2DL5 is represented in the human genome by two genes, KIR2DL5A and KIR2DL5B, and is found in only a fraction of individuals in the population .

• Allelic Polymorphism: KIR2DL5A exhibits significant allelic polymorphism that affects its protein expression and functionality, with some variants (like KIR2DL5A*005) being retained intracellularly rather than expressed on the cell surface .

These distinctive characteristics suggest KIR2DL5A may play specialized roles in immune regulation that differ from those of other KIR family members.

What detection methods are available for KIR2DL5A in research applications?

Several detection methods are available for KIR2DL5A research, each with specific applications:

When using antibodies for KIR2DL5A detection, it's important to consider epitope specificity. For instance, UP-R1 recognizes an epitope requiring both D0 and D2 domains, while newer antibodies like F8B30 bind through the D0 domain . Additionally, allelic polymorphisms in KIR2DL5A can affect antibody recognition, with some alleles (like KIR2DL5A*005) not being detected by certain antibodies despite being transcribed .

How do allelic polymorphisms affect KIR2DL5A expression and antibody recognition?

Allelic polymorphisms significantly impact both KIR2DL5A expression and antibody recognition, presenting a challenge for researchers studying this receptor. The KIR2DL5 genes (KIR2DL5A and KIR2DL5B) exhibit substantial polymorphism at both genetic and expression levels . Key insights into these polymorphisms include:

• Promoter Region Polymorphisms: KIR2DL5B alleles often contain a distinctive substitution in a promoter RUNX-binding site that renders them epigenetically silent, explaining why some KIR2DL5+ individuals lack detectable protein expression .

• Coding Region Polymorphisms: The transcribed allele KIR2DL5A*005 (the second most common KIR2DL5A allele) fails to confer NK cell reactivity with the UP-R1 monoclonal antibody despite being transcribed. This is due to amino acid substitutions in the coding region that affect protein trafficking and antibody recognition .

• Critical Amino Acid Substitutions: Two amino acid substitutions distinguish KIR2DL5A005 from the dominant KIR2DL5A001 allele:

  • Serine substitution for glycine-174 (conserved in most KIRs): This substitution is primarily responsible for KIR2DL5A*005 intracellular retention and also affects monoclonal antibody recognition.

  • Aspartate substitution for asparagine-152: This substitution has only a minor effect on surface expression despite destroying an otherwise conserved N-glycosylation site .

• Methodological Implications: These polymorphisms explain discrepancies between genotyping and flow cytometry studies. Researchers should employ multiple detection methods (e.g., combining genotyping with protein detection) to comprehensively assess KIR2DL5A status in study populations .

Understanding these polymorphisms is crucial when designing experiments and interpreting results in KIR2DL5A research, particularly when selecting appropriate antibodies for detection.

What are the therapeutic implications of targeting the KIR2DL5A pathway in cancer immunotherapy?

The KIR2DL5A pathway represents a promising target for cancer immunotherapy, particularly for enhancing NK cell-mediated anti-tumor responses. Recent research has revealed several key therapeutic implications:

These findings suggest that antibodies targeting KIR2DL5A could represent a novel class of immune checkpoint inhibitors for cancer immunotherapy, particularly for tumors that have developed resistance to existing immunotherapies.

What experimental approaches can be used to study KIR2DL5A signaling mechanisms?

Studying KIR2DL5A signaling mechanisms requires sophisticated experimental approaches that can dissect the complex molecular events following receptor engagement. Based on current research methodologies, the following approaches are recommended:

• Phosphorylation Analysis:

  • Western blotting with phospho-specific antibodies to detect tyrosine phosphorylation of ITIMs and ITSMs following KIR2DL5A engagement

  • Phosphoproteomics to identify downstream targets in the signaling cascade

  • Site-directed mutagenesis of tyrosine residues in ITIMs/ITSMs followed by functional assays to determine their individual contributions

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify proteins that associate with KIR2DL5A after engagement

  • Proximity ligation assays to visualize protein interactions in situ

  • FRET/BRET techniques to study dynamic interactions between KIR2DL5A and its signaling partners

• Functional Readouts:

  • NK cell redirected cytotoxicity assays using CD16-induced killing of P815 target cells

  • Degranulation assays measuring CD107a expression

  • Cytokine production assays (multiplex arrays) to assess the broad spectrum of cytokines/chemokines affected by KIR2DL5A signaling

  • Calcium flux assays to measure early signaling events

• Signaling Pathway Analysis:

  • Pharmacological inhibitors of specific signaling molecules to determine pathway dependencies

  • siRNA or CRISPR-based knockdown/knockout of candidate signaling molecules

  • Analysis of downstream effectors like Vav1, ERK1/2, p90RSK, and NF-κB activation states using western blotting or flow cytometry

• Advanced Imaging Techniques:

  • Confocal microscopy to study receptor clustering and localization

  • Super-resolution microscopy to visualize nanoscale organization of signaling complexes

  • Live-cell imaging to track signaling dynamics in real-time

These methodologies can be combined to build a comprehensive understanding of the KIR2DL5A signaling network and its role in immune cell function.

How should researchers design experiments to account for KIR2DL5A genetic and phenotypic variability?

Designing experiments that account for KIR2DL5A genetic and phenotypic variability requires careful consideration of several key factors:

• Donor Selection and Characterization:

  • Genotype donors for KIR2DL5A and KIR2DL5B presence and allelic variants

  • Screen for promoter polymorphisms, particularly in the RUNX-binding site

  • Perform flow cytometry with multiple antibody clones (e.g., UP-R1 and newer clones like F8B30) to detect surface expression

  • Consider sequencing the KIR2DL5A gene in donors to identify relevant coding polymorphisms

• Antibody Selection:

  • Use antibodies that recognize different epitopes (UP-R1 requires both D0 and D2 domains, while F8B30 binds through the D0 domain)

  • For comprehensive detection, employ antibodies like F8B30 that have been shown to outperform UP-R1 for KIR2DL5A recognition

  • Consider developing antibodies specific for different KIR2DL5A alleles

• Expression Analysis:

  • Combine surface protein detection with intracellular staining to identify retained variants

  • Use transcriptional analysis (qPCR or RNA-seq) to confirm gene expression regardless of surface detection

  • Employ western blotting to detect total KIR2DL5A protein regardless of localization

• Functional Studies:

  • Stratify donors based on KIR2DL5A expression profiles before functional analysis

  • Use cloned NK cell lines or primary NK cell clones to control for receptor heterogeneity

  • Consider transfection approaches with tagged KIR2DL5A constructs to study specific allelic variants

  • Include appropriate controls for each experiment based on expression patterns

• Data Interpretation:

  • Account for donor-to-donor variability in expression levels

  • Consider KIR2DL5A in the context of the broader KIR haplotype

  • Analyze data separately for different expression phenotypes before pooling

  • Report both genotype and phenotype data when presenting results

By implementing these design considerations, researchers can minimize confounding factors related to KIR2DL5A variability and generate more reproducible and interpretable results.