NK Cells Markers: Revealing Subsets and Functions

Natural killer (NK) cells are a key component of the innate immune system, detecting and eliminating infected or malignant cells. Unlike T and B cells, NK cells do not require prior sensitization, making them essential for early immune responses. Their functional diversity is shaped by distinct surface markers that define specialized subsets.

Understanding NK cell markers provides insight into their classification, function, and tissue distribution. These markers are critical for identifying different NK cell populations and assessing their activity in health and disease.

Core Identification Markers

NK cells are primarily defined by the absence of CD3, a T cell marker, and the expression of CD56 in humans or NK1.1 in certain mouse strains. CD56, also known as neural cell adhesion molecule (NCAM), exists in two major forms: CD56^bright and CD56^dim. CD56^dim NK cells, which dominate peripheral blood, exhibit strong cytotoxic activity, while CD56^bright NK cells, more common in lymphoid tissues, primarily produce cytokines. This distinction is fundamental for identifying NK cell subsets and their roles in immune surveillance.

CD16 (FcγRIII) further refines NK cell classification. This low-affinity Fc receptor enables antibody-dependent cellular cytotoxicity (ADCC), a mechanism for eliminating antibody-coated targets. Most CD56^dim NK cells co-express CD16, enhancing ADCC, while CD56^bright NK cells typically lack CD16 and rely on cytokine-driven responses.

NKp46 (NCR1 in mice) is a natural cytotoxicity receptor (NCR) critical for recognizing infected or transformed cells. It is one of the most specific NK cell markers across species, expressed independently of activation status. Beyond identification, NKp46 contributes to cytotoxicity by interacting with viral hemagglutinins and tumor-associated ligands.

CD94, often in association with NKG2 receptors, further distinguishes NK cells. CD94/NKG2 heterodimers regulate NK cell activity by interacting with HLA-E, a non-classical MHC class I molecule. Inhibitory NKG2A dampens NK cell responses, while activating NKG2C enhances them, particularly in response to cytomegalovirus (CMV) infection. The expression of these receptors helps define functionally distinct NK cell subsets.

Additional Proteins for Subset Differentiation

Additional surface and intracellular proteins refine NK cell classification, distinguishing subsets based on activation status, development, and specialization. CD57, for instance, is associated with NK cell maturation and longevity. It is low on immature NK cells but increases with terminal differentiation. CD57^+ NK cells exhibit enhanced cytotoxic potential, reduced proliferative capacity, and a greater ability to mediate ADCC. These highly differentiated cells accumulate in response to chronic infections, such as CMV, contributing to long-term immune surveillance.

NKG2A, an inhibitory receptor pairing with CD94, is typically found on less mature NK cells and declines with differentiation. In contrast, activating NKG2C is more prominent in subsets that expand in response to viral infections. The balance between NKG2A and NKG2C defines functional states, with NKG2C^+ NK cells displaying heightened responsiveness to antibody-coated targets, particularly in CMV-exposed individuals.

Killer-cell Immunoglobulin-like Receptors (KIRs) further refine NK cell subsets by modulating interactions with classical HLA class I molecules. These receptors exist in both inhibitory and activating forms, shaping NK cell education and responsiveness. Inhibitory KIRs, such as KIR2DL1 and KIR3DL1, recognize specific HLA alleles and contribute to self-tolerance, while activating KIRs, including KIR2DS1 and KIR3DS1, trigger cytotoxic responses upon ligand recognition. The diversity in KIR expression influences NK cell responses to infections and malignancies.

Intracellular transcription factors also play a role in subset differentiation. T-bet and Eomes regulate NK cell development and specialization. T-bet^highEomes^low NK cells are associated with inflammatory responses and enriched in peripheral tissues, while T-bet^lowEomes^high NK cells, more common in lymphoid organs, contribute to immune regulation. The interplay between these factors dictates NK cell fate, migration, and effector functions.

Functional Roles of Marker Expression

NK cell markers are not just identifiers; they directly influence function. CD16 is central to ADCC, allowing NK cells to eliminate antibody-coated targets. This function is crucial in therapeutic settings where monoclonal antibodies, such as rituximab and trastuzumab, rely on NK cells for tumor destruction. Genetic polymorphisms affecting CD16 affinity impact ADCC effectiveness, influencing clinical outcomes in cancer therapies.

NKp46 and other NCRs contribute to tumor surveillance and pathogen recognition. NKp46 detects viral hemagglutinins and tumor-associated ligands, enabling rapid responses against infected or malignant cells. Its expression correlates with tumor control in murine models, and its downregulation in some cancers is linked to immune evasion. NKp46 engagement enhances cytotoxic granule release, reinforcing its role in immune defense.

KIR and NKG2 receptors refine NK cell functionality by balancing inhibitory and activating signals. Inhibitory KIRs engage HLA class I molecules to establish self-tolerance, preventing attacks on healthy tissues. Activating KIRs and NKG2C drive robust responses in settings where HLA expression is altered, such as viral infections or malignancy. The expansion of NKG2C^+ NK cells in CMV-exposed individuals highlights their adaptive nature, with enhanced IFN-γ production and persistence shaping long-term immune responses.

Tissue-Specific Marker Profiles

NK cell marker expression varies by tissue environment. The liver harbors a unique NK cell population characterized by high CD49a and CXCR6 expression. These liver-resident NK cells rely more on cytokine signaling than direct cytotoxicity. CD49a facilitates retention within hepatic sinusoids by interacting with collagen IV, while CXCR6 promotes interactions with stromal cells secreting CXCL16. This adaptation allows liver NK cells to maintain immune homeostasis while responding to local infections and fibrosis.

In the lungs, NK cells express CD69, a tissue residency marker that prevents recirculation. They also express CD103, which binds to E-cadherin on epithelial cells, anchoring them within pulmonary tissues. This subset helps maintain barrier integrity and responds to respiratory infections. Single-cell RNA sequencing has revealed that lung NK cells have a distinct transcriptional signature, shaped by airway-specific stimuli.

Approaches for Multi-Parameter Evaluations

The complexity of NK cell biology requires advanced techniques for assessing multiple markers simultaneously. Multi-parameter evaluations provide a comprehensive understanding of NK cell heterogeneity, revealing subtle differences relevant to disease mechanisms and therapeutic responses.

Flow cytometry is widely used for NK cell analysis. Modern instruments can measure over 20 markers simultaneously, enabling precise subset identification. Fluorochrome-conjugated antibodies targeting CD56, CD16, NKp46, and KIRs allow for detailed phenotypic characterization. Intracellular staining techniques assess cytokine production and signaling pathways, while functional assays, such as CD107a expression for degranulation, enhance the evaluation of NK cell activity. However, optimizing panel design and managing spectral overlap are crucial for accurate interpretation.

Mass cytometry (CyTOF) extends these capabilities by using metal isotope-labeled antibodies instead of fluorophores, eliminating spectral overlap and allowing for the detection of over 40 markers. This approach has identified previously unrecognized NK cell subsets, particularly in cancer immunotherapy and chronic infections. Single-cell RNA sequencing (scRNA-seq) complements cytometry by providing transcriptomic insights into NK cell diversity. By analyzing gene expression at the single-cell level, researchers can uncover novel regulatory networks and lineage relationships, shedding light on NK cell adaptation to different microenvironments. Combining these technologies with computational modeling and machine learning enhances the ability to decipher NK cell dynamics, paving the way for more targeted immunotherapies.