A Guide to NK Cell Markers

Natural killer cells, or NK cells, are a unique group of lymphoid cells with innate cytotoxic and immune-modulating abilities. These large, granular lymphocytes comprise 5–20% of circulating lymphocytes in humans and also reside in various peripheral tissues. NK cells contribute to immunity directly by autonomously targeting foreign, infected, or cancerous cells and releasing cytotoxic granules that directly trigger cell lysis. Indirectly, NK cells also influence immune responses by producing inflammatory cytokines such as interferon gamma (IFNG) and tumor necrosis factor alpha (TNF). That NK cells play a central and diverse role in immunity justifies the considerable interest for this cell type across many research areas, including cancer biology and immunotherapy. Recent studies have broadened our understanding of the complexity and heterogeneity of the human NK cell population through the use of various phenotypic markers. Here, we provide an overview of common NK cell protein markers used in identifying various subsets. 

It has not been a simple task for scientists to define the NK cell lineage, partly because there is no known marker specific only to NK cells. They have traditionally been defined as an IFNG-producing, cytotoxic lymphocyte that is neither a T cell nor B cell. Thus, NK cells have typically been identified in flow cytometry by first excluding other lymphocyte markers such as the CD3 T cell marker. After this sorting, two markers have become well-established as standard NK cell markers, CD56 (neural cell adhesion molecule-1, NCAM1) and CD16 (low affinity Fc gamma receptor 3A, FCGR3A, FcγRIII). The differential expression of these two surface proteins define the two main subsets of conventional NK cells, CD56^brightCD16^lo/− and CD56^dimCD16+, often simplified as CD56^bright and CD56^dim, respectively. Among circulating cells in peripheral blood, CD56^bright is less abundant, estimated to comprise only 5–10% of the population. CD56^dim represents greater than 90% of NK cells. However, CD56^bright has been noted to be abundant in certain tissues, including secondary lymphoid tissues. More subpopulations of NK cells can be further described with other markers listed below. 

Markers for NK cell development

NK cells develop from common lymphoid progenitors (CLP) in the bone marrow. Here, they progress through various developmental stages, including NK cell precursors, and immature and mature NK cells. Recent findings suggest that development can also take place in secondary lymphoid tissues such as the lymph nodes and spleen. During this period, the cells become educated in MHC-I recognition in order to avoid targeting healthy normal cells. 

One of the earliest NK cell developmental markers is IL2RB (CD122), which is expressed when a CD34+ CLP commits to the NK cell fate. Subsets of NK cell precursors can be identified by differential expressions of genes such as CD34, KIT, KLRB1, CD244, and IL-15R. For immature NK cells, the expression of KLRK1, NCR1, NCR2, NCR3, and KLRB1 have been reported. NK cells reaching the mature end of the spectrum can be differentiated by their expression of KLRD1, ITGB2, KIR receptors, PRF1, IFNG, CD56, and CD16.

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The CD56^bright subset is understood to be an earlier stage of NK cell maturation. The subset features a higher proliferative capacity and is associated with the significant expression of a number of key proteins, including CCR7, CSF2, CXCR3, IL2RB, KLRC1, and SELL. It is also reported to express higher levels of IFNG. Downregulation of CD56 gives rise to the CD56^dim NK cell subset that coincides with the expression of CD16. This subset is known for its cytotoxic activity and is associated with the expression of killer Ig-like receptors (KIRs), a family of transmembrane glycoproteins comprising several genes. Other genes associated with this subset include CX3CR1, CXCR1, ITGB2, KLGR1, and PRF1.

Image: This figure highlights notable NK cell subsets, including common cell markers.

Tissue-resident NK cell markers

In contrast to the circulating conventional NK (cNK) cells in the blood, tissue-resident NK cells (trNK) are found in a number of tissues, including lymph nodes, thymus, liver, lung, uterus, and small intestine. In general, trNK cells have been reported to be less cytotoxic, while retaining the ability to produce cytokines. Although their specific functions are less understood than the conventional group, trNK can carry out distinct roles, as studies have found differing expression profiles for cytokines and KIRs.

Recent studies show that trNK cells encompass a specialized, heterogeneous population with distinct immunophenotypic profiles. For instance, human trNK cells in lymph nodes and tonsils show high expression of ICAM1. In the uterus, trNK cells are abundant for KIRs and express different splice variants of NCR2 and NCR3. Lung trNK cells have been found to express different levels of ITGA1, CD69, and CD16. Thymus trNK cells are associated with the expression of CD56 and IL7R. Liver trNK cells markedly express KLRC1 and NCR1. Across tissues, profiles of transcription factors also vary, such as with EOMES and TBX21. Our knowledge of the role of NK cells in various tissues is still at an early stage, and new findings will undoubtedly solidify more key markers for characterizing trNK subsets and functions.

Adaptive NK cell markers

A less common subset, known as adaptive NK cells, has been suggested to develop immunological memory to become specialized memory-like cells. Studies in cytomegalovirus-infected mouse and human NK cells have revealed some notable differences in functional and phenotypic profiles from conventional NK cells. In mice, the marker Ly49H has been found in a pool of long-lived NK cells that can rapidly proliferate and ramp up production of cytokines upon viral re-exposure. In humans, adaptive NK cells with previous viral exposure have been found to express the activating MHC class I-binding receptor KLRC2. Among the proteins reported to be differentially expressed are NCR1, B3GAT1, KLRC2, and LILRB1.

Table of NK Cell Markers

The table below lists human and mouse proteins characterizing various subsets of NK cells as described by recent review literature. The majority of proteins listed are membrane markers expressed on the cell surface, but also included are other defining proteins, including transcription factors and signaling proteins, such as cytokines. Accompanying each marker are links to relevant antibodies and ELISA kits, as these immunodetection tools are routinely used in cell characterization studies via flow cytometry and immunostaining. The associated products are offered by a variety of manufacturers and can serve as a useful reference for NK cell immunophenotyping.

Note: *Some proteins are protein isoforms or multi-subunit protein complexes composed of several distinct genes. Information on Protein Type, Localization, and Size (kDa) obtained from UniProt.org (for human genes only).

References

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2. Montaldo E, Del Zotto G, Della Chiesa M, et al. Human NK cell receptors/markers: a tool to analyze NK cell development, subsets and function. Cytometry A. 2013;83(8):702-713. doi:10.1002/cyto.a.22302
3. Del Zotto G, Marcenaro E, Vacca P, et al. Markers and function of human NK cells in normal and pathological conditions. Cytometry B Clin Cytom. 2017;92(2):100-114. doi:10.1002/cyto.b.21508
4. Freud AG, Mundy-Bosse BL, Yu J, Caligiuri MA. The Broad Spectrum of Human Natural Killer Cell Diversity. Immunity. 2017;47(5):820-833. doi:10.1016/j.immuni.2017.10.008
5. Abel AM, Yang C, Thakar MS, Malarkannan S. Natural Killer Cells: Development, Maturation, and Clinical Utilization. Front Immunol. 2018;9:1869. Published 2018 Aug 13. doi:10.3389/fimmu.2018.01869
6. Wu SY, Fu T, Jiang YZ, Shao ZM. Natural killer cells in cancer biology and therapy. Mol Cancer. 2020;19(1):120. Published 2020 Aug 6. doi:10.1186/s12943-020-01238-x
7. Hashemi E, Malarkannan S. Tissue-Resident NK Cells: Development, Maturation, and Clinical Relevance. Cancers (Basel). 2020;12(6):1553. Published 2020 Jun 12. doi:10.3390/cancers12061553
8. Pfefferle A, Jacobs B, Haroun-Izquierdo A, Kveberg L, Sohlberg E, Malmberg KJ. Deciphering Natural Killer Cell Homeostasis. Front Immunol. 2020;11:812. Published 2020 May 12. doi:10.3389/fimmu.2020.00812

Header image: Colorized scanning electron micrograph of a natural killer cell from a human donor (Credit: NIAID).