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NK cells are important players in the innate immune system and constitute the first line of defense against cancer cells. Natural killer (NK) cells were defined by Herberman in 1976 as a new lymphocyte population. NK cells are distributed throughout the body, accounting for 5-20% of all lymphocytes in the blood and organs. Due to the presence of unique chemokine receptors, NK cells are distributed differently in healthy tissues, in the bone marrow, spleen, liver, lungs, skin The concentration is higher in , kidney, uterus and secondary lymphoid tissue. NK cells are derived from CD34+ common lymphoid progenitor cells and differentiate into immature and mature NK cells in the bone marrow (BM). It then distributes to lymphoid and nonlymphoid peripheral organs and tissues, including PB, spleen, lungs, liver, and uterus.

The development process of NK cells

NK cells have cytotoxic capabilities similar to CD8+ T cells that play a role in adaptive immunity but lack CD3 and T cell receptors (TCR). NK cells mainly circulate in the blood, accounting for approximately 5-10% of peripheral blood mononuclear cells (PBMC), and are present in lymphoid tissues such as bone marrow and spleen. Similar to other ILCs, NK cells originate from common lymphoid progenitor (CLP) cells in the bone marrow (Figure 1), with an average renewal period of approximately 2 weeks.

During development, a process called “education” describes the interaction of NK cells expressing the immunoreceptor tyrosine inhibitory motif (ITIM) with major histocompatibility complex-I (MHC-I), Helps NK cells gain permission and avoid attacking healthy normal cells. Interestingly, tumor cells always lack or express only low levels of MHC-I to escape CD8+ T cell-mediated cytotoxicity, whereas licensed NK cells are fully activated. However, tumor cells also express molecules that activate NK cells, such as MHC class I peptide-related sequence A (MICA) and MICB, supporting the use of NK cells as anticancer agents. In addition, unlicensed NK cells also play important roles in the body, such as eliminating murine cytomegalovirus (MCMV) infection and MHC-I+ cells.

To date, NK cell survival and development are thought to depend mainly on cytokines (especially IL-2 and IL-15) and transcription factors (Nfil3, Id2, and Tox for development, and EOMES and T-bet for maturation). GRB2-associated binding protein 3 (GAB3) is essential for IL-2 and IL-15 mediation, and its deficiency results in impaired NK cell expansion. Furthermore, targeting relevant signals is a potential option to promote NK cell-induced cancer cytotoxicity. As mentioned previously, ablation of cytokine-inducible SH2-containing protein (CIS), which negatively regulates IL-15 to limit NK cell function, prevents metastasis and enhances CTLA-4 and PD-1 blockade therapy in vivo.

NK cell surface molecules

Due to the variable expression of NK cell surface markers, it is difficult to accurately identify this cell type and, more importantly, their functional status using one or two simple molecules or traditional immunohistochemistry. However, in human clinical and research settings, CD3-CD56+ cells are generally considered NK cells and can be further divided into CD56bright and CD56dim subpopulations. CD56 is not only a marker but also plays an important role in the terminal differentiation of NK cells, as its blockade by monoclonal antibodies significantly inhibits the transition from CD56bright to CD56dim, thereby limiting the cytotoxic capacity.

Consistently, CD3-NK1.1+ and CD3-CD49b+ cells were defined as NK cells in mice. In recent studies, based on the consensus of adding more functional proteins rather than surface molecules to the classification, it was proposed that natural cytotoxic receptor 46 (NKp46), which belongs to the natural cytotoxic receptors (NCR), should also be included in this Conceptual NK cell system in panel.

Activating and inhibitory signals in NK cells

As the main effector cell type in innate immunity, NK cells are able to kill tumor cells and virus-infected cells at a very early stage. Lacking abundantly produced receptors to specifically distinguish the incalculable number of antigens in the body, they rely on “missing self” and “induced self” modes to recognize target cells by maintaining a precise balance between activating costimulation and inhibition. Signaling (mainly through functional receptors). These interacting signals ultimately determine the activation and functional status of NK cells.

Activating signals include cytokine-binding receptors, integrins, killer receptors (CD16, NKp40, NKp30, and NKp44), receptors that recognize non-self antigens (Ly49H), and other receptors (e.g., NKp80, SLAM, CD18, CD2, and TLR3/9). In general, NK cell activating receptors can be divided into at least three types according to their respective ligands, including MHC-I specific receptors, MHC-I related receptors, and MHC-I non-related receptors (Table 1 ).

It should be emphasized that NCRs belonging to the third group include three molecules (NKp30, NKp44, and NKp46), and NKp30 has been shown to recognize B7-H6 expressed on tumor cells and can be used as a new therapeutic option in the future.

NK cell surface receptors and ligands on tumor cells are involved in tumor recognition. NK cells express a set of stimulatory (or activating) and inhibitory receptors and recognize healthy cells and abnormal cells, such as viral infections or potentially tumorigenic cells, through the appearance of MHC-1 receptors.

Modulation of natural killer (NK) cell responses according to the “missing self” and “altered self” models. ((A) The presence of major histocompatibility complex (MHC)-I as an inhibitory NK cell receptor ligand, and the lack of stress-induced ligands on the surface of healthy cells, results in a dominant inhibitory signal to NK cells.( B) The presence of stress-inducing ligands that activate NK cell receptors and downregulation of MHC-I by tumor cells results in the primary activation signal for NK cells. [Color figure can be viewed at]

Inhibitory signals mainly include receptors that recognize MHC-I, such as Ly49s, NKG2A, and LLT1, as well as some receptors unrelated to MHC-I (Table 1). In addition, MHC-I-specific inhibitory receptors can generally be divided into three types based on structure and function: killer cell immunoglobulin-like receptors (KIRs), killer lectin-like receptors (KLRs), and leukocyte immunoglobulins like receptor (LILR).

NK cell subpopulations according to mature site

Conventional NK (cNK) cells mainly exist in peripheral blood and migrate to specific locations to function. NK cells also include tissue-resident NK (trNK) cells. The complex process of NK cell differentiation occurs in several different tissues, including bone marrow, liver, thymus, spleen, and lymph nodes, and may involve cell cycling at different stages of maturation between these tissues. In bone marrow, blood, spleen, and lungs, NK cells are fully differentiated, whereas in lymph nodes and intestines, NK cells are immature and progenitor. Single-cell transcriptome analysis of bone marrow and blood NK cells helps elucidate their characteristic changes during development. For example, high expression of TIM-3, CX3CR1 and ZEB2 represents a more mature state.

In summary, NK cells in various tissues have different characteristics, have different functions and form close relationships with other stromal cells. In the lung, trNK cells display a distinct phenotype from circulating NK cells (mainly CD56dim) and are thought to express varying levels of CD16, CD49a, and CD69, with CD56dimCD16+ cells representing the majority of the entire NK family. Notably, CD69+ cells are the predominant type of CD56brightCD16-NK cells. However, in the thymus, the majority of NK cells are CD56highCD16-CD127+, which are highly dependent on GATA3 compared with the CD56+CD16+ subset. In addition, they produce more effector molecules, including TNF-α and IFN-γ.

Similarly, hepatic trNK cells can be divided into two groups, including CD56brightCD16+/- and CD56dimCD16+, both lacking CD3 and CD19. In addition, CD49a+CD56+CD3-CD19-NK cells have been identified in liver biopsies. In addition, hepatic NK cells can generate memory for structurally diverse antigens, depending on the surface molecule CXCR6. In the uterus, the majority of NK cells are CD56brightCD16- and express high levels of KIR. For skin NK cells, it is interesting that only very few CD56+CD16+, which are commonly found in peripheral blood, can be detected. Interestingly, trNK cells differ between subcutaneous (CD56dim) and visceral (CD56bright) adipose tissue and can generally be divided into three groups based on CD49b and Eomes, showing different CD49a (CD49b+ Eomes- subgroup) and CD69 expression levels (CD49b-Eomes+ subgroup).

NK cell subpopulations classified according to functional molecules

Based on the expression of surface CD56, NK cells can be divided into CD56bright and CD56dim. CD56dim NK cells mainly exist in peripheral blood and are always CD16 positive, express high levels of KIR and LFA-1, and display cell killing ability.

CD16 is a key receptor that mediates antibody-dependent cellular cytotoxicity (ADCC) and induces immunoreceptor tyrosine activation motif (ITAM) phosphorylation. Based on time-resolved single-cell assays, NK cell cytotoxicity was inhibited through necrosis and apoptosis. Therefore, FasL/FasR interaction, forin/granzyme release, and Ca2+ influx are all important for NK cell function. However, CD56bright NK cells are similar to helper cells and mainly secrete cytokines such as IFN-γ, TNF-β, and GM-CSF. The researchers even further divided these cells into NK1 and NK2 categories, consistent with Th1 and Th2, which primarily secrete IFN-γ and IL-5, respectively.

In addition to the established cytotoxic cNK cells, NK cells have been shown to differentiate into antigen-presenting NK (AP-NK) cells, helper NK (NKh) cells, and regulatory NK (NKreg) cells, each defined by surface molecules and individual functions. A new CD8αα+ MHC-II+ phenotype with professional APC capabilities is thought to represent unusual AP-NK cells, recognizing and eliminating autoreactive T cells and ultimately killing them like cNK cells. Human plasmacytoid dendritic cells (DC) activated by the prophylactic vaccine FSME upregulated the expression of CD56 on their surface.

Invariant natural killer T cells (iNKT) constitute a subset of T cells that express NK cell markers. NKT is activated by antigen presented by CD1d and can not only secrete Th1-type cytokines, but also secrete Th2-type cytokines to participate in immunity. Th1-polarized iNKT cells exhibit a tumor-depleting phenotype, whereas Th2-polarized iNKT cells resemble polarized T cells and contribute to tumor progression. Recent studies have also highlighted new functional subtypes of iNKT cells. However, in recent years, due to their close relationship with innate immunity, iNKT cells have been potentially defined as a special subset of ILCs.

As seen from the development and function of NK cells, they play different roles in cancer biology. NK cells exert anti-tumor immunity through interactions with cancer cells, stromal cells, extracellular matrix, especially metabolites.

Different ways of NK cell-mediated tumor killing and immune system regulation: (A) NK cells can enhance antigen presentation to T cells by killing immature DCs while promoting IFN-γ and TNF-α-mediated DC maturation Presentation. (B) NK cells can specifically recognize cells that lack expression of their own MHC class I molecules (Missing-self). (C) ADCC can kill target cells. (D) The Fas/FasL pathway is a very efficient NK cell-mediated cell killing (E) The cytokine pathway can exert anti-tumor potential because cytokines (such as NK cells) secrete a variety of cytokines such as TNF- α. (F) The NK cell receptor NKG2D recognizes “inducible self” ligands that are expressed at very high rates in response to activation of tumor-related pathways. (G) Checkpoint blockade can inhibit NK cell inhibition by preventing the interaction of N cell inhibitory receptors with their ligands. (H) As a result of adoptive NK cell transfer, mismatch between donor and recipient, inhibitory KIR, NK cells eliminate allogeneic tumor cells lacking their own MHC. (I) CAR-NK cells specifically designed to target overexpressed tumor antigens can also be used to eliminate specific tumor cells. (J) Specifically designed bispecific molecules are also used to specifically eliminate tumor cells because these special molecules bind to activate NK cell receptors on one side and tumor antigens on the other side. (K) NK cells can enhance or attenuate the activity of macrophages and T cells through the production of IFN-γ and IL-10.