Introduction to NK Cells and Indicator Selection

I. Introduction to NK Cells

NK cells, also known as Natural Killer cells, are lymphocytes and vital components of the immune system. Morphologically, NK cells belong to large granular lymphocytes, constituting the third major lymphocyte category alongside T cells and B cells, accounting for approximately 15% of all immune cells (white blood cells) in the bloodstream.

Positioned in the peripheral blood, liver, and spleen, NK cells, core cells of the innate immune system, exhibit rapid response capabilities without the need for antigen sensitization. They can destroy target cells without MHC restrictions, playing a crucial role in the early stages of immune responses.

NK Cell Schematic Diagram

NK cells serve as the primary "warriors" responsible for eliminating aging cells, those infected by viruses, and abnormal cells such as tumors. In the face of cancer cells, NK cells can directly recognize and kill them without the activation or sensitization of other cells, earning them the title of the body's first line of defense against cancer. A decrease in NK cell quantity or function significantly increases the risk of tumorous diseases.

■ Compared to other innate immune cells like neutrophils and macrophages, NK cells have a broader range of bacterial clearance and viral resistance.

■ In comparison to acquired immune cells like T cells, NK cells exhibit more sensitive responses to anticipating "enemy" invasions and demonstrate a broader spectrum of cytotoxicity, not limited to tumor cell destruction but also contributing to virus resistance and anti-aging.

II. Development and Classification of NK Cells

The mainstream model for NK cell development suggests that NK cells originate from CD34+CD45RA+ hematopoietic progenitor cells (HPCs) in the bone marrow. In the bone marrow, NK cells develop from HPCs through common lymphoid progenitors (CLPs) and NK cell precursors (NKPs), then migrate to peripheral blood (Conventional NK cells, cNK cells) or tissues (tissue-resident NK cells, trNK cells).

The differentiation of trNK cells occurs in various tissue locations, including the lungs, thymus, liver, uterus, skin, subcutaneous fat tissue, and kidneys. In these sites, NK cells exhibit distinct phenotypic features and functions, constituting the NK cell's circulation at different maturity stages. Despite the same organ and tissue, NK cells show high heterogeneity.

Development and Subgroups of NK Cells

NK cells are commonly defined as CD3‒CD56+ cells, typically categorized into two types based on the relative expression of surface proteins CD56 and CD16: CD56hiCD16- (immunoregulatory, cytokine production) and CD56lowCD16+ (cytotoxic). Among these, CD56lowCD16+ constitutes 90% of all NK cells, while CD56hiCD16-/low accounts for approximately 10%.

III. Selection of NK Cell Detection Indicators and Case Studies

How should one choose indicators for detecting NK cells? Based on past experimental experience, we have summarized the following:

■ Human: CD3-CD56+CD16+

■ Mouse: CD3-CD49b+/NK1.1+

Peripheral Blood NK Cell Detection Case in Humans

During the development of human NK cells, they express specific markers such as CD56, with some NK cells also expressing FcγRIIIA (CD16), and ultimately, CD57. When detecting samples from peripheral blood or other non-purified NK cells, CD3 should be included in the analysis to exclude a specific type of T cell—NKT (CD3+CD56+). The results of human peripheral blood NK cell detection are shown in the following figure:

Flow Cytometry Results of Human Peripheral Blood NK Cell Detection

① The detection criteria for NK cells are CD3-CD16+CD56+.

② It is advisable to have isotype controls for CD16 and CD56, and other indicators can be omitted if grouping is clear.

③ For human peripheral blood samples, it is recommended to stain with CD45, and using CD45 along with SSC helps gate the lymphocyte population. Utilizing a single-stained tube for CD45, running at a low speed, and setting a threshold is beneficial.

NK Cell Detection Case in C57BL/6 Mice

CD49b and NK1.1 are two markers for identifying mouse NK cells. Experimentalists should choose one based on the mouse strain; for example, BALB/c mice use CD49b (DX5), while C57BL/6 mice use NK1.1. During analysis, gate CD3-CD49b+ or CD3-NK1.1+. The results of NK cell detection in C57BL/6 mice are shown in the following figure:

Flow Cytometry Results of NK Cell Detection in C57BL/6 Mice

① Adding the CD45 indicator to peripheral blood samples helps gate the lymphocyte population directly through CD45 and SSC.

② The detection criteria for C57BL/6 mouse NK cells are CD3-NK1.1+.

③ The key factor in this experiment is red blood cell lysis; excessive or insufficient lysis can lead to unclear lymphocyte population grouping.

IV. NK Cell Detection Flow Antibody Recommendations

NK Cell Detection Flow Antibody Recommendations