KIR2DL5A Human

What is KIR2DL5A and how does it differ structurally from other KIR family members?

KIR2DL5A is an allele of the KIR2DL5 gene, which encodes an inhibitory killer cell immunoglobulin-like receptor. Structurally, KIR2DL5 belongs to a divergent KIR lineage conserved across primates and shares structural features with KIR2DL4. Unlike most other KIR genes, KIR2DL5 lacks the fourth exon coding for the D1 Ig-like domain and has a cytoplasmic tail 20-39 amino acids longer than other human inhibitory KIRs .

The receptor exists as a monomer of approximately 60 kDa on the cell surface . While most inhibitory KIRs recognize classical HLA class I molecules, KIR2DL5A appears to have different ligand specificity, suggesting unique functional roles in immune regulation.

Table 1. Structural, Genetic, and Functional Features of KIR2DL5 Compared to Other Human KIRs

What methodological approaches can determine KIR2DL5A inhibitory functions in immune cells?

The inhibitory function of KIR2DL5A can be investigated through several experimental approaches:

• Redirected cytotoxicity assays: Using antibody-mediated cross-linking of KIR2DL5 on NK cells against murine FcR+ P815 target cells. This method has demonstrated that KIR2DL5 cross-linking significantly inhibits target cell killing and NK cell degranulation (measured by CD107a expression) .

• Cytokine/chemokine production analysis: Multiplex cytokine arrays (e.g., 65-plex human cytokine/chemokine assays) have shown that KIR2DL5 engagement markedly decreases 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 .

• Signaling pathway analysis: Biochemical approaches can identify phosphorylation of ITIM and ITSM motifs in KIR2DL5's cytoplasmic domain following engagement, and subsequent recruitment of SHP-2 and SHP-1 phosphatases. These phosphatases downregulate the downstream Vav1/ERK1/2/p90RSK/NF-κB signaling pathway .

Which immune cell populations express KIR2DL5A and how is this expression regulated?

KIR2DL5 protein exhibits a complex expression pattern across immune cell populations:

• NK cells: KIR2DL5 displays a variegated distribution on CD56dim NK cells, unlike its structural homolog KIR2DL4 which shows ubiquitous transcription but surface expression restricted to CD56bright NK cells .

• T lymphocytes: KIR2DL5 is expressed on a variable proportion of T cells, particularly CD8+ T cells . Within CD8+ T cells, KIR2DL5 expression is highest in terminally differentiated effector memory cells (Temra) and to a lesser extent in effector memory cells, while being very low or undetectable in naive and central memory CD8+ T cells .

• Other lymphocytes: KIR2DL5 is also expressed on γδ T cells .

Expression analysis by flow cytometry and RT-PCR reveals no coordinated expression of KIR2DL5 with other KIRs, suggesting independent regulation . Importantly, a minority of NK cells express KIR2DL5 but neither other inhibitory KIRs nor NKG2A, consistent with a potential role in NK cell licensing .

What technical challenges exist in detecting KIR2DL5A expression and how can they be overcome?

Detection of KIR2DL5A expression has presented significant technical challenges:

• Antibody availability and specificity: Earlier research was limited by lack of reagents, with the first specific monoclonal antibody (UP-R1) recognizing only the most common allele, KIR2DL5A*001 . Newer antibodies like F8B30 show higher affinity (KD = 0.72 nM) and improved detection through binding the D0 domain .

• Allelic variation in expression: Only KIR2DL5A001 has been formally demonstrated to be expressed on the cell surface. Some alleles (like KIR2DL5B002) are transcribed but not detected on the surface, while others (KIR2DL5B003, KIR2DL5B004) are transcriptionally silent due to a polymorphism at the exon 2 splice site .

Strategies to overcome these challenges include:

• Using multiple detection antibodies targeting different epitopes

• Combining surface protein detection with transcript analysis

• Genotyping to determine which alleles are present before expression analysis

• Using appropriate positive controls from donors known to express KIR2DL5A*001

How does the intracellular signaling cascade of KIR2DL5A differ from other inhibitory KIRs?

KIR2DL5A employs a distinctive signaling mechanism compared to other inhibitory KIRs:

• Signaling motifs: While most inhibitory KIRs contain ITIMs, KIR2DL5A possesses both ITIM and ITSM in its cytoplasmic tail, enabling more complex signaling interactions .

• Phosphatase recruitment profile: Upon tyrosine phosphorylation, KIR2DL5A predominantly recruits SHP-2 and to a lesser extent SHP-1 . This contrasts with most inhibitory KIRs that primarily recruit SHP-1.

• Downstream pathway inhibition: Both ITIM/SHP-1/SHP-2 and ITSM/SHP-1 complexes contribute to downregulating the Vav1/ERK1/2/p90RSK/NF-κB signaling pathway .

This unique signaling profile may explain KIR2DL5A's broad inhibitory effects on cytokine/chemokine production compared to other KIRs and suggests potentially distinct roles in immune regulation.

What experimental approaches can distinguish KIR2DL5A-specific signaling from overlapping pathways?

To isolate and characterize KIR2DL5A-specific signaling:

• Receptor-specific antibody engagement: Use anti-KIR2DL5A-specific monoclonal antibodies (e.g., F8B30) to selectively engage KIR2DL5A without triggering other inhibitory receptors .

• Phospho-specific signaling analysis: Employ phospho-flow cytometry or western blotting with phospho-specific antibodies against signaling components to detect activation of specific pathways following KIR2DL5A engagement.

• Mutation studies: Generate KIR2DL5A constructs with point mutations in the ITIM or ITSM to dissect their individual contributions to downstream signaling .

• Selective phosphatase inhibition: Use selective inhibitors or siRNA knockdown of SHP-1 versus SHP-2 to determine their relative contributions to KIR2DL5A-mediated inhibition.

• Single-cell analysis: Apply single-cell RNA-seq or CyTOF to identify distinct signaling signatures in KIR2DL5A+ versus KIR2DL5A- cells within heterogeneous populations.

How does KIR2DL5A expression correlate with clinical outcomes in various malignancies?

KIR2DL5A shows significant associations with cancer progression and outcomes:

What methodological considerations should guide investigations of KIR2DL5A polymorphism in disease association studies?

When investigating KIR2DL5A polymorphism in disease contexts:

• Complex genomic context: Consider the duplication of KIR2DL5 (into KIR2DL5A and KIR2DL5B) and extensive linkage disequilibrium with other KIR genes when designing genotyping strategies .

• Allelic expression variation: Distinguish between polymorphisms affecting presence/absence, transcription, and surface expression , as illustrated in Table 2:

Table 2. KIR2DL5 Allelic Expression Patterns

• Comprehensive KIR typing: Perform complete KIR genotyping due to extensive copy number variation and presence/absence polymorphism .

• Functional validation: Complement genetic association findings with functional studies to confirm the biological impact of specific polymorphisms.

• Population stratification: Account for population-specific differences in KIR2DL5A allele frequencies when designing case-control studies.

What evidence exists regarding potential ligands for KIR2DL5A and how can novel ligands be identified?

The search for KIR2DL5A ligands remains an active area of research:

• Current evidence: Recent research suggests poliovirus receptor (PVR) may be a ligand for KIR2DL5 . Earlier investigations using KIR2DL5-Fc fusion proteins revealed dull staining of multiple human hematopoietic cell lines, independent of HLA expression .

• Methodological approaches for ligand identification:

  • Receptor-Fc fusion proteins: Generate improved KIR2DL5A-Fc constructs with enhanced sensitivity for binding studies .

  • Reporter cell assays: Develop reporter systems where KIR2DL5A engagement triggers measurable signals.

  • Genome-wide CRISPR screens: Screen target cells for genes that, when knocked out, abolish inhibition of KIR2DL5A+ effector cells.

  • Protein-protein interaction studies: Use techniques like BioID, proximity labeling, or protein microarrays to identify interacting partners.

  • Structural biology: Employ X-ray crystallography or cryo-EM to resolve KIR2DL5A structure and potential binding interfaces.

• Non-conventional ligand possibilities: Unlike other KIRs, KIR2DL5A may recognize non-HLA ligands , potentially induced under specific physiological or pathological conditions.

How can the binding affinity and specificity between KIR2DL5A and its putative ligands be accurately measured?

To characterize KIR2DL5A-ligand interactions quantitatively:

• Surface plasmon resonance (SPR): Measure real-time binding kinetics (kon and koff rates) and calculate equilibrium dissociation constants (KD) between purified KIR2DL5A and candidate ligands.

• Biolayer interferometry: Similar to SPR, this technique has been used to determine high-affinity binding of anti-KIR2DL5 antibodies (KD = 0.72 nM) and can be applied to study ligand interactions.

• Isothermal titration calorimetry (ITC): Quantify the thermodynamic parameters of binding interactions in solution.

• Cell-based binding assays: Use flow cytometry with fluorescently labeled KIR2DL5A-Fc fusion proteins to measure binding to cells expressing candidate ligands.

• Competitive binding assays: Determine specificity by measuring displacement of labeled ligand with unlabeled competitors.

• Mutagenesis studies: Identify critical binding residues by systematic mutation of amino acids in the Ig-like domains of KIR2DL5A and measure effects on ligand binding.

What cellular and animal models are most suitable for studying KIR2DL5A function in different research contexts?

For comprehensive investigation of KIR2DL5A biology:

• Cellular models:

  • Primary human NK cells: Isolated from KIR2DL5A*001-positive donors and expanded with IL-2/IL-15 .

  • NK cell lines: YT or NK-92 cells transduced with KIR2DL5A constructs.

  • Reporter cell systems: Cell lines expressing KIR2DL5A coupled to fluorescent or luminescent reporters to measure inhibitory signaling.

  • Co-culture systems: KIR2DL5A+ effector cells with target cells expressing putative ligands.

• Ex vivo models:

  • Human PBMCs: For studying KIR2DL5A in the context of other immune receptors and mixed cell populations .

  • Tumor-infiltrating lymphocytes: To examine KIR2DL5A function in the tumor microenvironment .

• In vivo models:

  • Humanized mouse models: Mice reconstituted with human immune system components including KIR2DL5A+ cells .

  • Patient-derived xenograft models: To study KIR2DL5A+ NK cell interactions with human tumors in vivo .

• Genetic models:

  • CRISPR-engineered cell lines: With KIR2DL5A knockout or specific mutations in signaling domains.

  • Transgenic expression systems: For inducible expression of KIR2DL5A in specific cell populations.

What technical considerations should be addressed when designing experiments to study KIR2DL5A regulation during immune responses?

When investigating KIR2DL5A regulation:

• Allelic specificity: Ensure experimental subjects (donors, cell lines) possess the surface-expressed KIR2DL5A*001 allele rather than non-expressed variants .

• Detection sensitivity: Use high-affinity antibodies like F8B30 that recognize specific domains (e.g., D0) for consistent detection .

• Temporal dynamics: Design experiments to capture both immediate effects (minutes to hours) and long-term adaptations (days) in KIR2DL5A expression and function.

• Microenvironmental factors: Evaluate how cytokines, metabolic conditions, and cell-cell interactions affect KIR2DL5A expression and function.

• Single-cell resolution: Apply single-cell methods to account for the variegated expression pattern of KIR2DL5A within cell populations .

• Receptor co-expression: Assess expression of other inhibitory receptors (other KIRs, NKG2A, LILRB1) that may complement or compensate for KIR2DL5A function .

• Activation state: Monitor how cell activation status affects KIR2DL5A expression and function, particularly given the unusual distribution in T cell memory subsets .