CD Markers for Natural Killer (NK) Cells
Natural killer cells are identified by the expression of CD16 and/or CD56 in the absence of CD3, with the two major subsets being CD56bright CD16dim/- and CD56dim CD16+. 1
Primary NK Cell Markers
Essential Positive Markers
- CD56 is the hallmark marker for NK cells, expressed on the majority of peripheral blood NK cells, though a CD56-negative subset exists 1
- CD16 (FcγRIII) is expressed on most mature NK cells and mediates antibody-dependent cellular cytotoxicity (ADCC) 1
- CD45 is brightly expressed on all NK cells, distinguishing them from other leukocytes with their characteristic low side-scatter properties 1
Essential Negative Markers
- CD3-negative (surface CD3) is critical for distinguishing NK cells from T cells, though NK cells express cytoplasmic CD3ε 1
- CD19/CD20-negative excludes B-cell lineage 1
Major NK Cell Subsets
CD56bright CD16dim/- Subset
- Constitutes approximately 5-10% of peripheral blood NK cells but represents the majority in secondary lymphoid tissues 2
- Functions primarily as cytokine producers (IFN-γ, TNF-α) with weak cytotoxic activity before activation 2
- Expresses high levels of CD94, NKG2A, and lacks or has dim CD16 expression 2
CD56dim CD16+ Subset
- Represents approximately 90% of peripheral blood NK cells 2
- Highly cytotoxic with strong perforin and granzyme expression 2
- Expresses killer immunoglobulin-like receptors (KIRs) for HLA class I recognition 3
CD56-negative CD16+ Subset
- Constitutes approximately 5.67% of NK cells in healthy individuals but expands significantly during chronic viral infections (particularly HIV-1, increasing by mean difference of 10.69%) 4, 3
- Expresses CD16b (FCGR3B), CD32A/B (FCGR2A/B), and has distinct cytotoxic mechanisms relying on ADCC and antibody-dependent respiratory burst 4
- Shows higher expression of perforin 2 (MPEG1), HLA-DR, and PD-1, with lower expression of NKp46 (NCR1) and NKp80 (KLRF1) compared to CD56dim cells 4
Additional NK Cell Markers
Activation and Functional Markers
- CD27 distinguishes NK cell maturation states: CD27hi correlates with CD56bright phenotype, while CD27lo correlates with CD56dim phenotype 5
- CD57 marks terminally differentiated, highly cytotoxic NK cells with reduced proliferative capacity 6
- CD94/NKG2A inhibitory receptor complex is expressed on immature NK cells 1
- CD161 (NKR-P1A) is expressed on NK cells and some T cell subsets 6
Cytotoxic Granule Proteins
- Perforin, granzyme B, and TIA1 are intracellular markers confirming cytotoxic capacity 1
Natural Cytotoxicity Receptors (NCRs)
Clinical Application in Flow Cytometry Panels
Standard NK Cell Identification Panel
- Minimum panel: CD3/CD16/CD56 to identify CD3-CD56+ and/or CD3-CD16+ populations 1
- Three-color panel: CD3/CD19/CD16 and/or CD56 to account for all lymphocyte lineages 1
- Four-color panel: CD45/CD3/CD19/CD16 and/or CD56 for comprehensive lymphocyte gating 1
Important Technical Considerations
- NK cells have bright CD45 fluorescence but slightly more side-scattering properties than the majority of lymphocytes, requiring careful gating to ensure inclusion 1
- CD45/side scatter gates for lymphocytes should contain >95% lymphocytes to avoid contamination with monocytes or basophils 1
- Strict flow cytometry gating strategy excluding monocytes is essential when identifying CD56-negative NK cells to avoid false-positive results 4
Quality Control
- Monoclonal antibodies labeling T-cells (CD3), B-cells (CD19/CD20), and NK-cells (CD16/CD56) should account for all lymphocytes in the specimen 1
- Validation of lymphocyte gates using T-, B-, and NK-cell markers is recommended to ensure proper inclusion of all NK cell subsets 1
Disease-Specific Considerations
NK/T-Cell Lymphomas
- Typical NK-cell immunophenotype: CD20-, CD2+, cytoplasmic CD3ε+ (surface CD3-), CD4-, CD5-, CD7-/+, CD8-/+, CD43+, CD45RO+, CD56+, TCRαβ-, TCRγδ-, with EBV-EBER positivity 1
- Cytotoxic granule proteins (TIA1, perforin, granzyme B) are usually expressed 1
- TCR and immunoglobulin genes remain in germline configuration, confirming NK lineage 1